Cytotoxicity, antibacterial and antifungal activities of ZnO nanoparticles prepared by theArtocarpus gomezianusfruit mediated facile green combustion method

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 t[r]

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Original Article

Cytotoxicity, antibacterial and antifungal activities of ZnO

nanoparticles prepared by the Artocarpus gomezianus fruit mediated

facile green combustion method

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

Article history:

Received 27 July 2018

Received in revised form

4 November 2018

Accepted 6 November 2018

Available online 22 November 2018

Keywords:

Green synthesis

ZnO nanoparticles

Anticancer activity

MCF-7

Antibacterial

Antifungal

a b s t r a c t Spherical nanoparticles of zinc oxide (ZnO NPs) were synthesized by an eco-friendly green combustion method using citrate containing Artocarpus gomezianus fruit extract as a fuel The morphology, com-positions and structure of the product were characterized by Powder X-ray Diffraction (PXRD), Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), Fourier Transform Infra-red (FTIR), 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 TEM images demonstrate that the particles are highly uniform spherical in shape and loosely agglom-erated Scherrer's method and WeH plots were used to calculate the average crystallite sizes, yielding 39,

35, 31 and 40, 37, 32 nm for ZnO NPs prepared with 5, 10 and 15 mL of 10% Artocarpus gomezianus fruit extract, respectively These results were confirmed by the TEM observations Breast cancer cell lines (MCF-7) were subjected to in vitro anticancer activity MTT assay revealed a good anticancer activity of 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 canfind 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

Inorganic materials such as metals and metal oxides due to their

stability are more advantageous in many aspects than organic

com-pounds[1] Among the metal oxides, zinc oxide nanoparticles (ZnO

NPs) have received a special attention as an anticancer, antibacterial

and antifungal material ZnO NPs exhibit improved properties

compare to bulk materials and these novel properties are attributed

to the changes in speciﬁc characteristics such as morphology and size

of the particles[2] ZnO NPs have a wide range of applications in solar

cells, catalysts, gas sensors, luminescent devices etc.[3] Nowadays,

ZnO NPs gained also signiﬁcant attention due to their implications for cancer therapy[4] It has been found from studies that ZnO NPs cause cytotoxicity to many types of cells such as HepG2, MCF-7, HT29, Caco-2, rat C6, HeLa, THP-1 [5e8] In addition, ZnO NPs exhibit antibacterial and antifungal activity They can decrease the viability and attachment of microbes on biomedical surfaces[9] ZnO NPs can be chemically synthesized by different methods such

as, spray pyrolysis, hydrothermal treatment, sol-gel process, co-precipitation, combustionor sonochemical, etc [10e12] Generally the chemicals used in the synthesis and stabilization are toxic and 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 instance, using plants for the synthesis of ZnO NPs Hence, the green combustion synthesis is an eco-friendly alternative wet-chemical

* Corresponding author.

E-mail address: swadheshi26@gmail.com (T Ramakrishnappa).

Peer review under responsibility of Vietnam National University, Hanoi.

Contents lists available atScienceDirect Journal of Science: Advanced Materials and Devices

j o u r n a l h o m e p a g e : w w w e l s e v i e r c o m / l o c a t e / j s a m d

https://doi.org/10.1016/j.jsamd.2018.11.001

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

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method This method has proved to be an excellent technique for

preparing several grams due to its low processing temperature, short

processing time, cost effectiveness It shows good ability to achieve

high purity in making multiphase or single complex oxides[14,15]

The main advantages of synthesis of ZnO NPs via the solution

combustion method towards biological activities are: (i) A larger

surface area with high porosity (as in the case of nanoparticles

fabricate by solution combustion method) ensures an increased

range of probable interaction with bio-organics present on the

viable cell surface[16] (ii) The considerable antimicrobial activities

of inorganic metal oxide nanoparticles such as ZnO NPs and their

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

of using ZnO NPs as antimicrobial agents are their greater

effec-tiveness on resistant strains of microbial pathogens, less toxicity

and good heat resistance In addition, they provide mineral

ele-ments essential to human cells and even small amounts of them

exhibit strong activity (iv) The solution combustion method is a

very simple, low-cost one, using which highly pure and highly

crystalline size nanoparticles can be obtained

Many articles have reported on the acute toxicity of ZnO NPs

However, a citrate containing A gomezianus fruit mediated spherical

ZnO NPs has not been discussed so far In this study highly uniform

spherical ZnO NPs were successfully prepared by an eco-friendly

green combustion method using different volumes of citrate

con-taining Artocarpus gomezianus fruit source as a fuel The as-prepared

ZnO NPs were used to study in detail the anticancer, antibacterial

and antifungal activities

2 Experimental

2.1 Chemicals

The chemicals used for the synthesis were of analytical grade

and were used without any further puriﬁcation Zinc nitrate was

procured from Merck The glassware used in the laboratory were

cleaned with a fresh solution of HCl/HNO3 (1:3, v/v), washed

thoroughly with double distilled water and dried Double distilled

water was used for all the experiments

2.2 Preparation of ZnO NPs

The citrate containing Artocarpus gomezianus fruit source was

collected from Mangalore, Karnataka The collected fresh, healthy

fruits were washed thoroughly using double distilled water and cut

into small pieces Then small pieces were dried at room

tempera-ture for 10 days under dust free conditions and subsequently

grinded into aﬁne powder 10 g of this ﬁne powder were boiled in

100 mL doubled distilled water to prepare a 10% crude solution,

thenﬁltered and stored in refrigerator for further usage In a typical

synthesis, 5 mL of the 10% crude solution was added to 3 g of

Zn(NO3)2.6H2O which is already dissolved in 10 mL of double

distilled water This reaction mixture was well mixed using a

magnetic stirrer for about 5e10 min and then placed in a preheated

mufﬂe furnace maintained at about 400 ± 10 C The reaction

mixture boils froths and thermally dehydrates to form foam The

whole process was completed in a few min Similar procedure was

repeated by taking 10 and 15 mL of the 10% crude sample

2.3 Characterization of ZnO NPs

PXRD data were recorded on the PANalyticalX'Pert Pro X-ray

Diffractometer with the graphite monochromatised Cu-Ka

(1.5418Å) radiation The surface morphology was observe by SEM

(JOEL JSM 840 A) with gold as contrast enhancing material covered

by the sputtering technique TEM analysis was carried out using the Hitachi H-8100 (accelerating voltage up to 200 KV, LaB6 Filament) equipped with EDS (Keney Sigma TM Quasar, USA) The FTIR studies were performed by using the Perkin Elmer Spec-trometer with KBr pellets Raman spectrum was obtained at room temperature in a back scattering geometry using a 632 nm HeNe laser with a JobinYvonLabRam HR spectrometer (LABRM-HR) The

UVeVisible absorption spectrum was obtained on the SL 159 ELICO UVeVIS Spectrometer Flow cytometry measurements were done by using the BD FACS Calibur Flow Cytometry

2.4 Anticancer activity by MTT assays The anticancer activity was checked by the 3 e (4,5-dimethylthiazol-2-yl) - 2,5 - diphenyltetrazolium bromide (MTT) assay The monolayer cell (Mammalian breast cancer ﬁbroblast cells) culture was trypsinized and the cell count was adjusted such that 200mL of suspension contained approximately 20,000 cells To each well of the 96 wells microtitre plate, 200mL of the diluted cell suspension was added and incubated at 37C and 5% CO2 atmo-sphere for 24 h After 24 h 200mL of different test concentrations of test drugs were added on to the partial monolayer The plate was then incubated at 37C and 5% CO2atmosphere for 24 h Media containing 10% MTT reagent was then added to each well and the plate was incubated at 37C and 5% CO2atmosphere for 3 h Then

100 mL of solubilization solution DMSO (DIMETHYL SULFOXIDE) was added and the plate was gently shaken to solubilize the formed formazan The absorbance was measured by microplate reader at a wavelength of 570 nm After subtracting the background and the blank, the percentage growth inhibition was calculated and the concentration of the test drug needed to inhibit the cell growth by 50% (IC50) was generated from the dose-response curve for the cell line

The cell viability was expressed as follows:

Cell vialbility¼ Test

2.5 Anticancer activity by apoptosis assay The detection of the apoptosis and necrosis was done by the flow cytometry The principle of this method is that the enhancement in the intensity of the side scattered light with the intensity of the forward scattered light in the cells reveals that the enhanced granularity of cells is correlated to cellular uptake of the NPs Scattered light is defined as the laser light scattered at about a 90 angle to the axis of the laser beam and forward scattered light is the laser light scattered at narrow angles to the axis of the laser beam Cells were cultured in a 6-well plate and then incubated in a CO2incubator at 37C for 24 h Then 180mL of the trypsin-EDTA solution were added and the mixture was incubated at 37C for 3e4 min The tubes containing the mixture were then centrifuged for 5 min to carefully decant the superna-tant The cells were resuspended in a 1X Binding Buffer 100mL of the solution (1 105cells) was transferred to a 5 mL culture tube and 5 mL of FITC (Fluorescein Isothiocyanate) Annexin V was added, slowly stirred and the incubated for 15 min at room tem-perature (25C) in the darkness Further, 5mL of Propidium Iodide (PI) and 400mL of 1X Binding buffer were added to each tube and circularly shaked gently The analysis was performed by flow cytometry after the addition of PI

R Anitha et al / Journal of Science: Advanced Materials and Devices 3 (2018) 440e451 441

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2.6 Anticancer activity by CAM method

The anticancer activity has also been checked by the

Chlorio-allantoic Membrane (CAM) assay Whatmann ﬁlter paper bud

containing the compound ZnO NP concentration corresponding to

its respective IC50value was implanted on the chick embryo

chorio-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

2.7 Evaluation of the antibacterial activity

The antimicrobial activity of 15 mL of 10% Artocarpus gomezianus

fruit extract mediated ZnO NPs was examined by the zone of

in-hibition method in Muller Hinton agar (MHA) media against

Gram-Positive Staphylococcus spp

Agglomeration was prevented by the standard sonication

method, in which particles were dispersed in the media using the

sonicator The ZnO NPs obtained from 5 mL, 10 mL and 15 mL of 10%

crude samples were dissolved in Di Methyl Sulphoxide (DMSO) to

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

0.005 mg/mL 0.5 mL was prepared by the serial dilution of 0.5 mL

of the stock solution

An autoclaved petriplate wasﬁlled with Sterile Muller Hinton

agar 100mL of 24 h The test culture (Staphylococcus aureus) was

spread onto the four well bored media 100 mL of different working concentrations of the sample were loaded to four wells The plate was kept at 37 C for the incubation along with a control concerning DMSO and antibiotic (ampicillin) for a period

of 17 h The zone of inhibition was recorded following the in-cubation period

2.8 Evaluation of the antifungal activity The examination of the antifungal activity of 15 mL of 10% Artocarpus gomezianus fruit extract mediated ZnO NPs was carried out by the zone of inhibition method in potato dextrose agar (PDA) media against Aspergillus niger An autoclaved petriplate wasﬁlled with the steriled potato dextrose agar (PDA) media 100mL of spore suspension (Artocarpus niger) was spread onto the four wells

100 mL of different working concentrations of the sample were loaded to the four wells The plate was kept for incubation along with a control concerning DMSO and antifungal (ﬂuconazole) at room temperature for a period of 96 h The zone of inhibition was recorded following the incubation period

3 Result and discussion 3.1 Crystal structure

Fig 1shows 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]

d¼ kl

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]

bhklcosqhkl¼kl

whereε is the strain associated with the NPs Equation(3) repre-sents a straight line betweenbcosq(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 inTable 1 As the volume of the citrate containing 10% Artocarpus gomezianus fruit extract increases, the broadening of the lines also increases, indi-cating that the particle sizes decreases and it is in good agreement with TEM results

Fig 1 PXRD patterns of ZnO NPs prepared with (a) 5, (b) 10 and (c) 15 mL of 10%

A gomezianus fruit extract.

Table 1

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

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

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3.2 Morphological studies

Fig 2shows the SEM micrographs and the EDS of the ZnO

nanoparticles prepared using 5, 10 and 15 mL of citrate containing

Artocarpus gomezianus fruit extract as a fuel The combustion

product is more inﬂuenced by the type of the fuel used The nature

of the combustion differs from ﬂaming to non-ﬂaming type

Generally,ﬂaming reaction is associated with the release of large

quantity of gases The SEM micrographs (Fig 2(a), (c) and (e)) show

the agglomeration, voids and pores The pores and voids can be due

to the huge quantity of gases escaping out of the reaction mixture

during the combustion (ﬂaming)

The energy dispersive spectrometry (EDS) analysis was used to

determine the composition of ZnO NPs prepared using 5, 10 and

15 mL of citrate containing Artocarpus gomezianus fruit extract as a

fuel and results are shown inFig 2(b), (d) and (f), respectively The

EDS measurements revealed the presence of Zn and O peaks for

ZnO

Fig 3shows the TEM image of the ZnO NPs prepared using 5, 10

and 15 mL of citrate containing Artocarpus gomezianus fruit extract

as a fuel It clearly shows that the nanoparticles are of sizes in the

range 10e30 nm and spherical in shape of rather uniform

dimension

3.3 FTIR analysis

FTIR spectra of ZnO NPs prepared using 5, 10 and 15 mL of citrate

containing Artocarpus gomezianus fruit extract are shown inFig 4

(a-c) The absorption band near 3450 cm1is due to the hydroxyl

group of H2O adsorbed on the ZnO NPs The transmittance band

between 1400 and 1649 cm1is due to the stretching mode of C]

O The peak at 2350 cm1arises due to CO2absorption from the atmosphere on the metallic cations The bands at 421 and 590 cm1 correspond to the bonding between ZneO[20]

3.4 UVeVis analysis

Fig 5shows the UVeVis spectrum of the ZnO NPs prepared at room temperature using 15 mL of citrate containing A gomezianus fruit extract as a fuel At the wavelength of 367 nm, the charac-teristic absorption peak in the ZnO NPs spectrum is observed Due

to the electron transitions from the valence band to the conduction band (O2p-Zn3d), the characteristic absorption peak of the ZnO NPs can be assigned[21] From this absorption spectrum, using Tauc equation, the band-gap of the ZnO thinﬁlm was calculated[22]:

ahv ¼ A hv Eg

n

(4)

whereahnis the photon energy, Egis the band gap, n¼ 1/2 for the direct band gap transition and A is a constant which is different for different transitions The progress information gives the best linear

ﬁt in the band edge location for n ¼ 1/2 The band gap was observed

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 the size impact of the nanoparticles

3.5 Raman analysis Raman spectroscopy can give information on the vibrational properties of the ZnO NPs.Fig 6shows the Raman spectrum of the

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

The peak at 436 cm1relates to E2 (high), which is moved by

3 cm1 The peak at 582 cm1is set between E1(LO) and A1(LO),

which is a great concurrence with the literature data[23] The peak

at 330 cm1is because of the second-order Raman scattering The

peak at 379 cm1relates to A1(TO) and that at 410 cm1compared

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

as a fuel were mixed in distilled water When this mixture was

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

gomezianus fruit extract.

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 6 Raman spectrum of ZnO NPs prepared by 15 mL of 10% Artocarpus gomezianus

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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

10mM ZnO NPs, (e) MCF-7 against 50mM ZnO NPs (f) MCF-7 against 100mM ZnO NPs (g) MCF-7 against 200mM ZnO NPs (h) MCF-7 against 300mM ZnO NPs (i) MCF-7 against

500mM ZnO NPs.

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heated to 400± 10C, 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 aﬂame,

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)2.6H2Oþ 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 500mg/mL of the in vivo toxicity The

cytotoxicity results of in vitro experiments were taken after 24 h

of the incubation with different concentrations of ZnO NPs

ranging from 10 to 500 mg/mL, prepared with 15 mL of 10%

A gomezianus fruit extract and are shown inFig 7 Cells at different

concentrations of ZnO NPs show different stages of cell

death/ne-crosis[25] The drug of nano ZnO shows necrosis of the MCF-7 cells

at 100 mM indicating its toxicity is approximately near to the

standard drug camptothecin whose toxicity level is 50mM[26] The

cytotoxic effect of ZnO NPs in MCF-7 cell lines is presented inFig 8

The results obtained infers an inverse relation between the drug

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 IC50with the value of 9.3495mg/mL

Fig 9(a) and (b) show theﬂow 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 10shows a

graph of theﬂow 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 ofﬂuorochrome FITC linked to the cell.Fig 11

shows the graphical representation of counts versus FITC Annex

in-V for ZnO NPs

Fig 12shows 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 14show 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 inTable 2 The results indicate that

5, 10 and 15 mL fruit extract mediated ZnO NPs at 0.5 mg/100mL exhibited rather similar antibacterial and antifungal efﬁcacy against Gram-Positive Staphylococcus aureus and Aspergillus niger, respectively Nanoparticles provide relatively larger active surface area and thus, a higher amount of those Zn atoms that trigger a toxicity effect of ZnO towards the bacteria [27] The detailed

Fig 8 Cell viability of MCF-7 cells calculated by MTT assay Cells were incubated for

24 h with the ZnO NPs prepared with 15 mL of 10% A gomezianus fruit extract.

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reaction system of the bioactivity of ZnO is still under debate.

Numerous systems have been proposed and are identiﬁed with the

features, such as: (i) One of the conceivable mechanism depending

on the grating surface of ZnO, actually the ZnO NPs to the bacterial

surface is due to the electrostatic powers that straightforwardly

eliminate microorganisms [28], (ii) The entering ZnO NPs can

connect with the layer lipids and casues the pulverization of the cell

membrane, which prompts the lost of the membrane uprightness

and breakdown, and lastly leads to the bacterial demise[29], (iii)

The arrival of the Zn2þparticles from the ZnO nanoparticles and (iv)

The generation of the very responsive species of, for example,

O2 , H

2O2, OHwhich harm the DNA, cellﬁlms 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 Zn2þantimicrobial ions has been suggested as a reasonable hypothesis about the toxicity of ZnO against S cerevisiae[33] According to this author, the toxicity of ZnO nanoparticles could result from the solubility of the Zn2þions

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 Zn2þions can trigger a relatively high tolerance by the microorganism In the case of yeast, labile Zn2þions 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 Zn2þions, 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 results showed complete inhibition of E coli growth at concen-trations3.4 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 consistent increase in the number of colony forming units (CFU) compared to control, due to the preference of this micro-organism for low concentrations of Zn2þ in the growth me-dium Conversely, S aureus showed an efﬂux mechanism of Zn2þ during the exposure to ZnO nanoparticles in the millimolar range, indicating that the sufﬁcient ion concentration results in undesirable and potentially toxic conditions to this microor-ganism Thus, concerning the effect of ZnO against E coli at low concentrations, rather than exercising antimicrobial activities, the ZnO nanoparticles may actually increase the bacterial growth Zhang et al.[31]studied the effect of ZnO NPs on E coli cells, and

Fig 12 (a) Implantation of the drug ZnO 100mM (IC50 value) and (b) Thinning of blood vessels seen preceding the site of the drug implant.

Fig 10 Graph of Flow cytometry analysis of MCF-7 cells with Annex in-V Fluorescein

isothiocyanate (FITC) of which graph in the lower left area are the living cells.

Fig 11 Graphical representation of counts vs FITC Annex in-V for ZnO nanoparticles

prepared with 15 mL of 10% A gomezianus fruit extract.

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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/100mL) of samples 1, 2, 3, respectively.

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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/100mL) of samples 1,2,3, respectively.

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