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The morphology, compositions 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.
Journal of Science: Advanced Materials and Devices (2018) 440e451 Contents lists available at ScienceDirect 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 PG Department of Biochemistry, Dayananda Sagar College, Bangalore, 560 078, India 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 b c a r t i c l e i n f o a b s t r a c t Article history: Received 27 July 2018 Received in revised form November 2018 Accepted November 2018 Available online 22 November 2018 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, compositions 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 agglomerated 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 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/) Keywords: Green synthesis ZnO nanoparticles Anticancer activity MCF-7 Antibacterial Antifungal Introduction Inorganic materials such as metals and metal oxides due to their stability are more advantageous in many aspects than organic compounds [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, * Corresponding author E-mail address: swadheshi26@gmail.com (T Ramakrishnappa) Peer review under responsibility of Vietnam National University, Hanoi 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, coprecipitation, 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 byproducts 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 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 (2018) 440e451 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, surgical devices and in nano-medicine as well [17] (iii) The advantages of using ZnO NPs as antimicrobial agents are their greater effectiveness on resistant strains of microbial pathogens, less toxicity and good heat resistance In addition, they provide mineral elements 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 containing Artocarpus gomezianus fruit source as a fuel The as-prepared ZnO NPs were used to study in detail the anticancer, antibacterial and antifungal activities 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 441 (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 Spectrometer 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 e (4,5dimethylthiazol-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 200 mL of suspension contained approximately 20,000 cells To each well of the 96 wells microtitre plate, 200 mL of the diluted cell suspension was added and incubated at 37 C and 5% CO2 atmosphere for 24 h After 24 h 200 mL of different test concentrations of test drugs were added on to the partial monolayer The plate was then incubated at 37 C and 5% CO2 atmosphere for 24 h Media containing 10% MTT reagent was then added to each well and the plate was incubated at 37 C and 5% CO2 atmosphere for 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 vialbility ¼ Test  100% Control (1) 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 fruits were washed thoroughly using double distilled water and cut into small pieces Then small pieces were dried at room temperature for 10 days under dust free conditions and subsequently grinded into a fine powder 10 g of this fine powder were boiled in 100 mL doubled distilled water to prepare a 10% crude solution, then filtered and stored in refrigerator for further usage In a typical synthesis, mL of the 10% crude solution was added to 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 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 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 (1.5418 Å) radiation The surface morphology was observe by SEM 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 CO2 incubator at 37 C for 24 h Then 180 mL of the trypsin-EDTA solution were added and the mixture was incubated at 37 C for 3e4 The tubes containing the mixture were then centrifuged for to carefully decant the supernatant The cells were resuspended in a 1X Binding Buffer 100 mL of the solution (1  105 cells) was transferred to a mL culture tube and mL of FITC (Fluorescein Isothiocyanate) Annexin V was added, slowly stirred and the incubated for 15 at room temperature (25 C) in the darkness Further, mL of Propidium Iodide (PI) and 400 mL 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 442 R Anitha et al / Journal of Science: Advanced Materials and Devices (2018) 440e451 2.6 Anticancer activity by CAM method The anticancer activity has also been checked by the Chlorioallantoic Membrane (CAM) assay Whatmann filter paper bud containing the compound ZnO NP concentration corresponding to its respective IC50 value was implanted on the chick embryo chorioallantoic membrane through a hole cut in to the shell of the egg The incubation period may range from to 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 inhibition method in Muller Hinton agar (MHA) media against GramPositive 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 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 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 was filled with Sterile Muller Hinton agar 100 mL 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 incubation 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 was filled with the steriled potato dextrose agar (PDA) media 100 mL 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 Result and discussion 3.1 Crystal structure Fig 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] d¼ kl b cos q (2) 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] bhkl cosqhkl ¼ Fig PXRD patterns of ZnO NPs prepared with (a) 5, (b) 10 and (c) 15 mL of 10% A gomezianus fruit extract kl ỵ sinqhkl D (3) where ε is the strain associated with the NPs Equation (3) represents 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 intercept (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 As the volume of the citrate containing 10% Artocarpus gomezianus fruit extract increases, the broadening of the lines also increases, indicating that the particle sizes decreases and it is in good agreement with TEM results Table Average crystallite size and strain of ZnO NPs synthesized by 5, 10 and 15 mL of Artocarpus gomezianus fruit extract Fruit extract (mL) 10 15 Strain (10À4) Average crystallite size (nm) Scherrer's method (d) WeH plots method (D) 39 35 31 40 37 32 2.42 3.17 5.36 R Anitha et al / Journal of Science: Advanced Materials and Devices (2018) 440e451 3.2 Morphological studies Fig shows 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 (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 in Fig (b), (d) and (f), respectively The EDS measurements revealed the presence of Zn and O peaks for ZnO Fig shows 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 in Fig (a-c) The absorption band near 3450 cmÀ1 is due to the hydroxyl group of H2O adsorbed on the ZnO NPs The transmittance band 443 between 1400 and 1649 cmÀ1 is due to the stretching mode of C] O The peak at 2350 cmÀ1 arises due to CO2 absorption from the atmosphere on the metallic cations The bands at 421 and 590 cmÀ1 correspond to the bonding between ZneO [20] 3.4 UVeVis analysis Fig shows 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 characteristic 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 thin film was calculated [22]: À ahv ¼ A hv À Eg Án (4) where ahn is the photon energy, Eg is the band gap, n ¼ 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 shows the Raman spectrum of the Fig 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 (2018) 440e451 Fig TEM images of ZnO NPs prepared by (a) 5, (b) 10 and (c) 15 mL of 10% A gomezianus fruit extract Fig UVeVis spectrum of ZnO NPs prepared by 15 mL of 10% Artocarpus gomezianus fruit extract Fig 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 cmÀ1 The peak at 436 cmÀ1 relates to E2 (high), which is moved by cmÀ1 The peak at 582 cmÀ1 is set between E1 (LO) and A1 (LO), which is a great concurrence with the literature data [23] The peak at 330 cmÀ1 is because of the second-order Raman scattering The peak at 379 cmÀ1 relates to A1 (TO) and that at 410 cmÀ1 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 as a fuel were mixed in distilled water When this mixture was Fig Raman spectrum of ZnO NPs prepared by 15 mL of 10% Artocarpus gomezianus fruit extract R Anitha et al / Journal of Science: Advanced Materials and Devices (2018) 440e451 445 Fig (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 (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 selfpropagating 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 ỵ fruit extract / ZnO nanoparticles ỵ 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 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 in Fig Cells at different concentrations of ZnO NPs show different stages of cell death/necrosis [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 50 mM [26] The cytotoxic effect of ZnO NPs in MCF-7 cell lines is presented in Fig The results obtained infers an inverse relation between the drug concentration and the cell viability The percentage growth inhibition 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 (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 intensity 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 inV 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 inV 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 development 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 Fig 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 5, 10 and 15 mL fruit extract mediated ZnO NPs at 0.5 mg/100 mL 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 3.8 Antibacterial and antifungal assay Figs 13 and 14 show the photographs illustrating the antibacterial 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 The results indicate that Fig 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 (2018) 440e451 447 À O2À , 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 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 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 Zn2ỵ particles from the ZnO nanoparticles and (iv) The generation of the very responsive species of, for example, 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 mechanisms, including the release of antimicrobial ions [30], the interaction of nanoparticles with microorganisms followed subsequently 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 Zn2ỵ antimicrobial 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 Zn2ỵ ions in the medium containing the microorganisms However, the solubility of the metal oxides, such as ZnO is a function of their concentration and the time [33] Thus, low concentrations of solubilized Zn2ỵ ions can trigger a relatively high tolerance by the microorganism In the case of yeast, labile Zn2ỵ ions rapidly accumulates in dynamic vesicular compartments (vacuoles and zincosomes), 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 Zn2ỵ 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 results showed complete inhibition of E coli growth at concentrations !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 mM of ZnO showed a consistent increase in the number of colony forming units (CFU) compared to control, due to the preference of this microorganism for low concentrations of Zn2ỵ in the growth medium Conversely, S aureus showed an efflux mechanism of Zn2ỵ 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 microorganism 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 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 (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 (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 (2018) 440e451 Table Zone of inhibition of the ZnO NPs prepared by 10% Artocarpus gomezianus fruit extract against Staphylococcus aureus and Aspergillus niger Fruit extract (mL) 05 10 15 Sl No 4 Concentration (mg/100 mL) 0.5 0.05 0.005 0.0005 0.5 0.05 0.005 0.0005 0.5 0.05 0.005 0.0005 Zone of inhibition (mm) Staphylococcus aureus Aspergillus niger 16.0 ± 0.66 11.0 ± 0.33 10.5 ± 0.51 ne 10.5 ± 0.79 ne ne ne 12.25 ± 0.57 11.0 ± 0.51 ne ne 23.0 16.0 14.0 13.5 22.0 18.0 14.5 13.0 25.0 20.0 15.0 ne ± ± ± ± ± ± ± ± ± ± ± 0.57 0.88 0.66 0.33 0.33 1.20 0.51 0.33 0.79 1.15 0.51 Values are mean inhibition zone (mm) ± S.D of three replicates Note: ‘ne’ indicates no effect Fig 15 Possible mechanism of antibacterial and antifungal activities by using ZnO NPs prepared with 15 mL of 10% Artocarpus gomezianus fruit extract as a result they pointed out that the interaction between the ZnO nanoparticles and the E coli cells is caused by electrostatic forces According to Stoimenov et al [28], the global charge of bacterial cells at biological pH values is negative, due to the excess of carboxylic groups, which are dissociated and provide a negative charge to the cell surface Conversely, ZnO nanoparticles have a positive charge, with a zeta potential of ỵ24 mV, [31]) As a result, opposite charges between the bacteria and the ZnO anoparticles generate electrostatic forces, leading to a strong binding between the nanoparticles and the bacteria surface and, consequently, producing the cell membrane damage The possible 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 Conclusion We successfully synthesized the spherical ZnO NPs by the green combustion strategy utilizing the 5, 10 and 15 mL, 10% citrate containing A gomezianus solution arrangement as a fuel PXRD studies revealed that the pure hexagonal wurtzite structure was obtained The average crystallite size of the NPs was evaluated from Scherrer's and WeH plots and observed to be in the range of ~35 nm and the outcomes were additionally affirmed by the TEM experiments The SEM micrographs showed that all the samples are of agglomeration, pores and voids due to the flaming in the green combustion synthesis The TEM analysis makes it apparent 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 anticancer 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 subtracting the background and 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Materials and Devices (2018) 440e451 Fig TEM images of ZnO NPs prepared by (a) 5, (b) 10 and (c) 15 mL of 10% A gomezianus fruit extract Fig UVeVis spectrum of ZnO NPs prepared by 15 mL of 10% Artocarpus