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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 ficonsistent 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 the blank data The 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The antibacterial and antifungal activities of the ZnO NPs prepared with 15 mL of 10% A gomezianus fruit extract were evaluated by the zone of inhibition method against Staphylococcus aureus and. .. Krishnaiah, H.M Sathyananda, In vitro antibacterial and cytotoxicity studies of ZnO nanopowders prepared by combustion assisted facile green synthesis, Int J Mod Sci (2015) 67e77 [17] M Chandrasekhar,... 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
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