Biofabrication of zinc oxide nanoparticles using fruit extract of Rosa canina and their toxic potential against bacteria: A mechanistic approach

Materials Science and Engineering C 59 (2016) 296–302

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Biofabrication of zinc oxide nanoparticles using fruit extract of Rosa canina and their toxic potential against bacteria: A mechanistic approach Saeed Jafarirad a, Meysam Mehrabi a, Baharak Divband b,c, Morteza Kosari-Nasab d a

Research Institute for Fundamental Sciences (RIFS), University of Tabriz, Tabriz, Iran Research Center for Pharmaceutical Nanotechnology, Tabriz University of Medical Sciences, Tabriz, Iran Inorganic Chemistry Department, Faculty of Chemistry, University of Tabriz, Tabriz, Iran d Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran b c

a r t i c l e

i n f o

Article history: Received 23 May 2015 Received in revised form 17 September 2015 Accepted 25 September 2015 Available online 28 September 2015 Keywords: ZnO nanoparticles Plant extract Green synthesis Antibacterial effect Cytotoxicity

a b s t r a c t The use of plant extract in the biosynthesis of nanoparticles (NPs) can be an eco-friendly approach and have been suggested as a possible alternative to classic methods namely physical and chemical procedures. In this study, the biosynthesis of zinc oxide (ZnO) NPs by both “conventional heating” (CH) and “microwave irradiation” (MI) methods has been reported. Stable and spherical ZnONPs were produced using zinc nitrate and flesh extract of Rosa canina fruit (rosehip) which was used as a precursor. The flesh extract acts as a reducing and capping agent for generation of ZnONPs. The structural, morphological and colloidal properties of the as-synthesized NPs have been confirmed by X-ray Diffraction (XRD), Scanning Electron Microscopy (SEM), Energy Dispersive X-ray (EDX), Fourier transform Infrared (FT-IR) and Dynamic Light Scattering (DLS). In comparison with the CH method, the MI method has some advantages such as significantly short reaction time (within 8 min) owing to the high heating rate and thus the accelerated reaction rate. Both methods led to the synthesis of nearly identical NPs with respect to shape and size according to the results of DLS, XRD and SEM techniques. The possible mechanism for synthesis pathway has been proposed based on FT IR results, XRD patterns, potentiometric data and antioxidant activity. In addition, the antibacterial activity of as-prepared ZnONPs was investigated against several bacteria such as Listeria monocytogenes, Escherichia coli, Salmonella typhimurium. Moreover, the efficacy of ZnONPs to treat cancer cell lines were measured by means of cell viability test via MTT assay in which concentrations of 0.05 and 0.1 mg/mL of ZnONPs induced a very low toxicity. Thus, the present investigation reveals that ZnONPs have the potential for various medical and industrial applications. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Nowadays nanoscience and nanotechnology have become one of the most important research areas in physics, chemistry, engineering and biology [1]. Particles with size less than 100 nm are considered due to their special properties, such as low melting point, unique optical properties, high catalytic activity, and unusual mechanical properties [2]. Based on scientific reports, ZnO is a unique material with a wide band gap of 3.37 eV possessing numerous attractive properties [3]. In addition ZnO is considered as a potential material in the field of optoelectronics, solar cells, gas sensors, catalysts, pharmaceuticals and cosmetics [4–9]. Moreover, ZnONPs have also been used as an anti-microbial preservative for wood and food products [10,11]. In general, three kinds of methods have been applied for the generation of metal oxide NPs in solution including: (i) the “normal” synthesis under reflux conditions in the oil bath using CH, (ii) the autoclave synthesis, and (iii) the most recent synthesis using MI [12]. As it is known, the walls of the reaction flask in CH method are heated by convection or conduction. In this process the core of metal oxide NPs needs longer time to achieve the desired temperature and accordingly,

http://dx.doi.org/10.1016/j.msec.2015.09.089 0928-4931/© 2015 Elsevier B.V. All rights reserved.

this leads to inhomogeneous temperature profiles within the flask. One possibility to overcome this drawback is the use of MI method, which allows the rapid and homogeneous heating of the reaction mixture to the target temperature without heating the entire oil bath. MI not only helped in employing green chemistry approach, but also led to the revolution in production of both organic and inorganic NPs [12]. As it is known, in one hand, by using classical methods for preparing NPs such as physical and chemical procedure some toxic chemical remains in products that may have adverse effects in medical application. Although, on the other hand, a lot of researches have been prepared for metal and metal oxide NPs by using green synthesis methods; however, the problem has not been solved properly. Indeed, such methods are not really green in their principle whereas extracellular biosynthesis is one of the most promised pathways of green synthesis. Recently, a variety of studies have been carried out to survey the biosynthesis of various metal ions such as Ce, Ag and Au [13–18]. Rosa canina or dog rose is a medicinal plant belonging to the family Rosaceae. Rosehip or the small fruit of Rosa canina is known for their high content of vitamin C. In addition, rosehip has a high level of antioxidants such as flavonoids and phenolic compounds. Based on reports,

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rosehip possesses preventive and curative activity against a wide range of diseases such as renal, inflammatory, gout and gastric disorders [19]. In the present research, for the first time, we describe the extracellular biosynthesis of ZnONPs, as a green technique, using rosehip extract with two methods (CH and MI). Moreover, the structural, morphological and colloidal properties and mechanistic aspects of the as-synthesized NPs have been investigated. In addition, the antibacterial activities as well as MTT assay for cell viability of as-prepared ZnONPs have been studied.

2.5. Extracellular synthesis of ZnONPs using MI method

2. Materials and methods

2.6. Characterization of ZnONPs

2.1. Materials

Crystalline structure and grain size were identified by XRD (D500, Siemens Diffractometer-Germany) Cu-Ka radiations (λ = 1.54 Ǻ) in 2θ range from 10° to 80°. FTIR spectra were measured with a TENSOR 27-Bruker Spectrometer in the 400–4000 cm−1 region. The size and shape of NPs were characterized by SEM and EDX techniques using MIRA3 FEG-SEM. In addition, the size and size distribution, polydispersity index (PDI) and zeta potential were identified using DLS Nanotrac Wave. TEM studies were carried out on a Zeiss LEO 912 Omega instrument, operating at 120 kV. TEM specimen was made by evaporating one drop of solution of the sample in ethanol onto carbon coated copper grids. Grids were blotted dry on filter paper and investigated without further treatment.

All the chemicals in this research were of analytical grade. The rosehip was collected from around Mahabad city, a county with an area of 2591 km2, approximately located at 36°46′N, 45°43′E of West Azerbaijan Province (the west northern of Iran). The fruit was washed with deionized water and dried at room temperature and then were used for further tests. 2.2. Preparation of extract The harvested fruits were washed with distillated water and kept in shadow to dry. Then seed and flesh of the fruit were separated. The flesh was powdered and weighted carefully. About 10 g of fruit powder was shaken with 100 ml deionized water for 24 h and heated in oven at 50 °C for 15 min. Light red color of mixture implied that the extraction has occurred. In order to achieve clear extraction, the mixture filtered, centrifuged and finally stored in refrigerator for further experiments. 2.3. 1-Diphenyl-1-2-picrylhydrazyl (DPPH) scavenging assay The rosehip extract and ZnONPs were screened for free radical scavenging activity by DPPH method [20]. The scavenging activity on the DPPH radical was determined by measuring the absorbance at 517 nm using a UV-spectrophotometer. Radical scavenging activity was calculated using the formula (Eq. (1)):

A typical route was applied according to CH method except that the mixture 4 times and each time for 2 min was placed under microwave irradiation at 320 W. A deep red colored suspension was created that was centrifuged at 5000 rpm. The precipitate was obtained which was centrifuged twice at 5000 rpm after washing. The obtained pale white colored precipitate was heated according to the same condition mentioned above (CH method) in air-heated furnace.

2.7. Anti-bacterial assay of ZnONPs The ZnONPs that were synthesized using Rosa canina extract were tested for antimicrobial activity by agar disk diffusion method against Salmonella typhimurium, Staphylococcus aureus, Listeria monocytogenes and Escherichia coli. The pure cultures of bacteria were subcultured on nutrient agar medium. Each strain was put uniformly onto the individual plates using sterile cotton swabs. Filter paper disks (Whatman no. 3, 6 mm diameter) were sterilized by autoclaving. Ten milliliters of the nanoparticle's solution was loaded onto each paper disk and allowed to air dry. The dry disks were placed on the previously inoculated agar. After incubation at 37 °C for 24 h, the different levels of zone of inhibition of bacteria were measured. 2.8. MTT assay for cell viability


%I ¼ Acontrol −Asample =Acontrol  100

ð1Þ

Where, %I is % of radical scavenging activity, Acontrol is the absorbance of the control sample (DPPH solution without test sample) and Atest is the absorbance of the test sample (DPPH solution with test compound). All tests were performed in triplicate and the results were averaged. 2.4. Extracellularly synthesis of ZnONPs using CH method In order to synthesize ZnONPs, 5 ml of 5 × 10-2 M zinc nitrate solution was mixed with 10 mL of the extract. The zinc nitrate was dissolved in the extract solution under constant stirring using magnetic stirrer. After complete dissolution of the mixture, the final pH of the solution was fixed at 6.0. Then the solution was kept under vigorous stirring at 150 °C for about 5 h until color change occurs. Finally, it was allowed to cool at room temperature and the supernatant was separated. This mixture was centrifuged at 5000 rpm for 10 min and a solid precipitate was obtained. The solid product was centrifuged twice at 5000 rpm for 15 min after thorough washing and dried at 80 °C for 4 h. This precipitate was collected and dried and then heated in air heated furnace at 400 °C for 4 h. The pale white colored solid was obtained. This solid was completely powdered using a mortar that got a fine powder for further characterizations.

A549 alveolar adenocarcinoma cells (9 × 103 cells/well) were incubated in 96-well plates; each containing 200 μL of supplemented cell culture media for 24 h at 37 °C and 5% CO2. The cells were divided in 5 groups in triplicates: blank ZnONPs (different concentrations: 0.05, 0.1, 0.25, 0.5 mg/mL) were treated. After an incubation period of 24 h, the spent media were removed and the plate wells were washed with phosphate-buffered solution. Briefly, 50 μL of 2 mg/mL MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-trazolium bromide) and 150 μL of culture medium was added to each well. The cells were incubated at 37 °C and 5% CO2 for 4 h and then the media was discarded and dimethyl sulfoxide and Sorenson buffer were added to each well as solubilizer buffer. Finally, absorbance was read using an ELISA plate reader (BioTeck, Bad Friedrichshall, Germany) at 570 nm wavelength. 3. Results and discussion 3.1. Structural characterization of ZnONPs 3.1.1. XRD pattern XRD pattern of the prepared NPs have been shown in Fig. 1a. The peak positions with 2θ values of 31.76°, 34.42°, 36.25°, 47.53°, 56.59°, 62.85°, 67.94° and 69.08° are indexed as to the (100), (002), (101), (102), (110), (103), (112) and (202) planes, respectively, according to ICDD data (card No. 01-079-0206) for extracellularly biosynthesis

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phytochemicals in the extract. The weight percentages of zinc and oxygen were found to be 65.19 and 34.81, respectively.

3.2. Morphological images The SEM and TEM images of as-prepared ZnONPs by both CH and MI methods are depicted in Fig. 2a,b. These images show the spherical shape of ZnONPs with a size less than 50 nm.

3.3. Investigation on colloidal properties

Fig. 1. XRD patterns of ZnONPs prepared by a) CH and MI methods, b) EDX spectrum of bio-ZnONPs produced by CH method.

of ZnONPs using CH method. For MI method, the peak positions with 2θ values of 31.74°, 34.37°, 36.17°, 47.46°, 56.56°, 62.79°, 67.94°, 68.97° and 72.47° are indexed as to the (100), (002), (101), (102), (110), (103), (112), (201) and (004) planes, respectively, according to ICDD data (card No. 01-079-0207). All of the diffraction peaks were in adaptation to the hexagonal zinc oxide phase by comparison with the data from cards. The narrow and strong diffraction peaks indicate that the product has a well crystalline particle structure. The peaks in XRD pattern corresponding to CH method are narrower than that of MI method, which is derived from their particle size and are in accordance with the SEM and DLS observations (next sections). No characteristic peaks of any impurities were detected, suggesting that a high quality of ZnONPs was produced. The crystallite size has been estimated from the XRD pattern using the Scherrer's equation (Eq. (2)): d ¼ Kλ=β cosθ

ð2Þ

where K = 0.9 is the shape factor, λ is the X-ray wavelength of Cu Kα radiation (1.54 Ǻ), θ is the Bragg diffraction angle, and β is the full width at half maximum (FWHM) of the respective diffraction peak. Based on the scherrer equation, the crystallite size of the ZnONPs was estimated to be 13.3 and 11.3 nm for CH and MI methods, respectively. It appears that the influence of temperature gradient and reaction time through both methods has no noticeable effect on the size of the synthesized ZnONPs. 3.1.2. EDX pattern The presence of both zinc and oxygen elements of the ZnONPs can be confirmed by EDX pattern (Fig. 1b). The showing signal of gold element in EDX spectra is because of the gold-covering process for SEM analysis. Moreover EDX spectra show the signal of carbon element which is usually seen in the organic substances attached to the ZnONPs due to

During this study, the size, size distribution and surface charge of dispersed ZnONPs in a liquid was monitored by DLS. The value of size obtained by this technique can be larger than the value obtained by using SEM owing to the entity of DLS. However, DLS is a suitable and noninvasive technique. Fig. 3 shows that biosynthesized ZnONPs using CH and MI methods have sizes between 25 and 204 and 21–243 nm (where CH; 92% and MI; 98% smaller than 50 nm), respectively. In addition, the polydispersity indices (PDIs) for CH and MI methods are 0.884 and 1.021, respectively (Table 1). Based on our findings, the amounts of zeta potential for colloidal suspensions of ZnONPs were found at −30.5 and −18.2 mV for CH and MI methods, respectively (Table 1). Based on DLVO theory, the high absolute zeta potential value exerts a strong repulsive force among the particles and prevents aggregation. During measurement the time dependence of the zeta potential was not observed, which demonstrated that ZnONPs were stable. Based on Table 1, the data obviously highlight that the ZnONPs obtained from MI method did not appreciably differ from those obtained using CH method. Notably, these results prove that the colloidal properties of ZnONPs prepared by using both methods are approximately independent of thermal source. MI (reaction time, 8 min) leads to spherical aggregates with the more or less similar properties like those of CH method (reaction time, 5 h). This rapid reaction rate of MI could be justified owing to: (i) efficient internal heating (in-core volumetric heating) by direct coupling of MI with reactant s in the reaction mixture; (ii) increased diffusion rate of reactants, and (iii) reduced activation energy of the reaction [21].

3.4. FTIR characterization In general, the stability of NPs is very important for their applications. Therefore the NPs are usually stabilized with capping agents such as surfactants and polymers. However, ZnONPs produced in this research are stable due to in situ bio-capping by the residues of the flesh of the fruit which could be observed in FTIR characterization (Fig. 4). This is due to the presence of metabolites in fruits such as phenolic acids, proanthocyanidins, tannins, flavonoids, fatty acids, pectin, carotenoids, sugars and fruit acids such as ascorbic acid, malic acid, and citric metabolites which possess antioxidant activity [19]. The biocapping phenomenon could be due to the presence of metabolites such as phenolic and carboxylic acids (like ascorbic acid) that adhered to the surface of ZnONPs. The spectrum obtained clearly shows Zn\\O absorption bands on 470 and 433 cm− 1 for CH and MI, respectively [22–24]. Moreover, the bands at 1107, 1049 and 1117 cm−1 correspond to C\\O stretching mode of esters. The band at 2855 cm− 1 and 2924 cm−1 can be attributed to C\\H stretching. The synthesized ZnONPs possess peaks at 1742 and 1741 cm−1 which indicates C_O of esters. In FT-IR spectrum of synthesized ZnONPs, the typical peak at 3400 cm−1 corresponding to the hydroxyl group has been eliminated. The elimination of typical corresponding peaks of hydroxyl groups and appearance of carbonyl peak imply the oxidation process of fruit metabolites [25–30] (Fig. 4b,c). Thus, the presence of fruit acids as capping agents in the matrix of ZnONPs was confirmed.

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Fig. 2. SEM figures of ZnONPs prepared by a) CH method and b) MI method; c) TEM figures of ZnONPs prepared by CH method as a typical image.

3.5. Performance at the bio-nano interface As it was known, the extract of rosehip has a high content of ascorbic acid (vitamin C). The pair electrons in the polar groups of ascorbic acid can occupy two orbits of the zinc ion to form a complex compound. The ascorbic acid and other polyphenols are capped with zinc ions as nanoscopic templates. Then ZnONPs are synthesized via calcination of complex at 400 °C inside the nanoscopic templates. In the presence of nanoscopic templates, small ZnONPs are easily formed [31]. 3.5.1. Antioxidant property of the extract and ZnONPs DPPH is a stable nitrogen-containing free radical and shows a typical absorptional peak at 517 nm. The free radical scavenging attribute of the Table 1 Colloidal properties of ZnONPs for both CH and MI methods. Method

T (°C)

P (W)a

D(nm) XRD

CH MI a b c d e

Fig. 3. DLS patterns of ZnONPs prepared by a) CH method and b) MI method.

f

150 –

– 320

13.3 11.3

b

SEM

c

40–50 20–40

PDI (μ2/Ƭ2)e

Ζf (mV)

0.884 1.021

−30.5 −18.2

d

DLS

32.2 26.37

Power of microwave irradiation. Theoretical diameter calculated by Origin software based on Scherrer's equation. Particle mean diameter measured by SEM. Hydrodynamic diameter measured by DLS in water. Polydispersity index based on DLS. Zeta potential.

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Fig. 4. FT-IR spectrum of a) the extract of rosehip, b) CH method and c) MI method.

fruit extract is presented in Fig. 5a. As shown, the antioxidants present in the fruit extract react with DPPH and convert it to 1,1-diphenyl-2picryl hydrazine with decolorization. The percent scavenging of DPPH increases with the increase in the concentration of the fruit extract in the test samples. In addition, the change of color from dark purple to light yellow is proportional to absorption of hydrogen atom form extract using DPPH (Fig. 5b). Moreover, the DPPH assay method was used to investigate the antioxidant property of ZnONPs. Various kinds of antioxidants of the extract could perform synergistically. During the synthesis of the ZnONPs, these metabolites are pooled into the NPs. They adsorbed onto the surface of the ZnONPs. Considering the high surface area to volume ratio, it appears that these ZnONPs show a high tendency to interact with and reduce DPPH. Among these metabolites, ascorbic acid (AA) has been known as a result of its good antioxidant activities. Therefore, it is possible to propose on Eq. (3): H–O–ðof AAÞ þ DPPH→O_ CH–ðof AAÞ þ 1; 1  diphenyl  2 picrylhydrazine

ð3Þ

Here hydroxyl of AA quenched the activity of DPPH by donating its electron. 3.5.2. Possible mechanism The exact mechanism for the biosynthesis of metal oxide NPs using plant extracts has not been confirmed. However, it was recommended that polar groups are responsible for the synthesis of NPs [32–34]. Fig. 6a shows the adapted mechanism for the capping effect of extract. It appears that, at first the lone pair electrons in the polar groups of AA can occupy orbital of the Zn 2 +. Then, Zn2 + is capped with polar groups to form a complex compound inside the nanoscopic

templates of metabolites. Finally, the reaction could result in ZnONPs by calcination. As mentioned earlier, based on the FTIR spectra for ZnONPs in both methods, the elimination of typical corresponding peaks of hydroxyl groups and appearance of carbonyl peak imply an oxidation process of active components of the extract. The nitrate group of the zinc salt is a stronger oxidant at low pH and can oxidize the extract [35]. Our experiments depicted that the equilibrium potential of rosehip extract is 0.153 V vs. SCE which is effective enough to reduce NO− 3 to NO which has the reduction potential of 0.960 V vs. SCE, respectively (Eq. (4)). NO3 − þ 4H3 Oþ þ 3e− ⇌ NOðgÞ þ 6H2 O; E0 ¼ 0:960 V

ð4Þ

According to the half reaction of reduction potential, the overall reaction was determined to have a potential of 0.807 V. Thermodynamically, therefore, the redox reaction between NO− 3 and the extract occurs spontaneously. Since H+ was released in the oxidation half reaction (Fig. 6b) and consume in Eq. (4), so the redox process continues properly. Our findings were confirmed by measuring of pH during biosynthesis reaction because in both CH and MI methods, we have not observed any noticeable change of pH (pHreaction = 6). 3.5.3. Antibacterial activity of ZnONPs Antibacterial activity of the synthesized ZnONPs was measured using disk diffusion method. The activity of ZnONPs differs based on the volume used against tested bacteria. A typical photo of inhibition zone represents the inhibition zone for the synthesized ZnONPs in the case of E. coli (Fig. 7). In general, the zone of inhibition increases with increasing concentration of ZnONPs shows the trend of 1.0 N 0.5 N 0.25

Fig. 5. a) %I value in terms of concentration of methanol extract; MI: microwave irradiation, CH: conventional heating and EX: extract, b) DPPH analysis of the rosehip; the change of color from dark purple to light yellow is proportional to absorption of hydrogen atom form extract using DPPH.

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Fig. 7. A typical photo of inhibition zone of the synthesized ZnONPs in the case of E. coli; antibacterial measurements have been performed using disk diffusion method.

Table 2 Inhibition zone (mm) of different concentrations against four pathogenic bacteria using disk diffusion method. Bacterial strain

S. typhimurium L. monocytogenes S. aureus E. coli

Inhibition zone (mm) 1.00a

0.50a

0.25a

–b 10 11 8

–b 8 9 8

–b 7 –b 6

a Concentrations (mg/mL); are expressed in terms of mg of synthesized nanoparticle on mL H2O. b It does not show any considerable inhibition zone.

Fig. 6. The possible mechanism for a) capping stage and formation of ZnONPs, b) oxidation mechanism of ascorbic acid of the rosehip.

that displays influence of the as-synthesized NPs in inhibiting the growth of pathogenic bacteria. Table 2 summarizes the antibacterial activity of ZnONPs. The bio-capped ZnONPs depicted a higher antibacterial activity against L. monocytogenes, S. aureus and lesser effectivity against E. coli. However, it is not active against S. typhimurium. Based on the obtained results, the NPs are relatively good antibacterial activity against both Gram-positive bacteria such as S. aureus, L. monocytogenes and gram-negative bacteria like E. coli. However, it is not an antibacterial activity against S. typhimurium.

are 84, 45 & 27%, respectively after incubation for 24 h. The results indicate that no significant harmful effect is imposed to the cells up to 0.1 mg/mL of ZnONPs. These findings are very interesting because according to the previous reports, ZnO was toxic although in the low concentration. It has been reported that, for example, the MTT reduction observed after 24 h of exposure of ZnO nanorods in A549 cells at the concentrations of 0.01, 0.025, 0.050 and 0.1 mg/mL was 73%, 60%, 49%, and 41%, respectively [36]. However, the green synthesized ZnONPs produced in this study exhibit poor cytotoxicity because of their

3.6. Cytotoxicity of the ZnONPs The cytotoxicity of the ZnONPs were investigated in the A549 cell line. Fig. 8 shows the relative cell viability ([Cr/C0] 100%) vs. different concentration of ZnONPs, determined by the MTT assay. Here, C0 is the viable cell numbers of the control sample, and Cr is the viable cell numbers treated with the ZnONPs. The error bars are the calculated standard deviation. The relative viability of cells treated with 0.05 mg/mL of ZnONPs is about 95 ± 5%. The relative viabilities (%) of cells treated with higher concentrations of ZnONPs (0.1, 0.25 & 0.5 mg/mL)

Fig. 8. The relative cell viability ([Cr/C0] 100%) vs. different concentrations of ZnONPs, determined by the MTT assay.

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negative (−) charge as mentioned before in Section 3.3 (Investigation on colloidal properties). Therefore, the size of NPs, their surface charge and the type of cell line are important factors [37–40]. 4. Conclusions The present study has demonstrated that ZnONPs could be successfully biosynthesized by CH and MI methods using the aqueous fruit extract of Rosa canina. Analysis of the particle size and morphology of ZnONPs using DLS, SEM and XRD revealed only slight differences between the samples which were prepared by using both CH and MI methods. Therefore, the exploitation of MI method has the advantages such as fast reaction rates which make MI technique as a suitable method. This opens new windows to research challenges for designing the ZnONPs with defined sizes, morphologies and complex architectures in different experimental conditions. The cytotoxicity was studied by cell viability on the A549 cell line. Briefly, ZnONPs exhibit dosedependent toxicity to cells. In addition, very low toxicity was observed up to concentrations of 0.1 mg/mL. The findings provide preliminary information in the study of determining the potential use of ZnONPs in various medical and industrial applications. Acknowledgements The financial support by the Research Institute for Fundamental Sciences (RIFS), University of Tabriz is gratefully acknowledged. References [1] S. Jafarirad, Encyclopedia of Biomedical Polymers and Polymeric Biomaterials, in: M. Mishra (Ed.), Molecular Assemblies, 1st ed.Taylor & Francis Group, New York, 2015. [2] S. Jafarirad, Encyclopedia of Biomedical Polymers and Polymeric Biomaterials, in: M. Mishra (Ed.), Dendritic architectures, 1st ed.Taylor & Francis Group, New York, 2015. [3] S.S. Ashtaputre, A. Deshpande, S. Marathe, M.E. Wankhede, J. Chimanpure, R. Pasricha, J. Urban, S.K. Haram, S.W. Gosavi, S.K. Kulkarni, Synthesis and analysis of ZnO and CdSe nanoparticles, Pramana 65 (2005) 615–620. [4] Z. Deng, M. Chen, G. Gu, L. Wu, A facile method to fabricate ZnO hollow spheres and their photocatalytic property, J. Phys. Chem. B. 112 (2008) 16–22. [5] T. Krishnakumar, R. Jayaprakash, N. Pinna, V.N. Singh, B.R. Mehta, A.R. Phani, Microwave-assisted synthesis and characterization of flower shaped zinc oxide nanostructures, Mater. Lett. 63 (2009) 242–245. [6] B. Cao, W. Cai, From ZnO nanorods to nanoplates: chemical bath deposition growth and surface-related Emissions, J. Phys. Chem. C 112 (2008) 680–685. [7] M. Khatamian, B. Divband, A. Jodaei, Degradation of 4-nitrophenol (4-NP) using ZnO nanoparticles supported on zeolites and modeling of experimental results by artificial neural networks, Mater. Chem. Phys. 134 (2012) 31–37. [8] M. Khatamian, A.A. Khandar, B. Divband, M. Haghighi, S. Ebrahimiasl, Heterogeneous photocatalytic degradation of 4-nitrophenol in aqueous suspension by Ln (La3+, Nd3+ or Sm3+) doped ZnO nanoparticles, J. Mol. Catal. A Chem. 365 (2012) 120–127. [9] B. Divband, M. Khatamian, G.R. Kazemi Eslamian, M. Darbandi, Synthesis of Ag/ZnO nanostructures by different methods and investigation of their photocatalytic efficiency for 4-nitrophenol degradation, Appl. Surf. Sci. 284 (2013) 80–86. [10] K. Schilling, B. Bradford, D. Castelli, E. Dufour, J. Frank Nash, W. Pape, S. Schulte, I. Tooley, J. van den Bosch, F. Schellauf, Human safety review of “nano” titanium dioxide and zinc oxide, Photochem. Photobiol. Sci. 9 (2010) 495–509. [11] K. Gerloff, I. Fenoglio, E. Carella, J. Kolling, C. Albrecht, A.W. Boots, I. Förster, R.P.F. Schins, Distinctive toxicity of TiO2 rutile/anatase mixed phase nanoparticles on Caco-2 cells, Chem. Res. Toxicol. 25 (2012) 646–655. [12] Y.J. Zhu, F. Chen, Microwave-assisted preparation of inorganic nanostructures in liquid phase, Chem. Rev. 114 (12) (2014) 6462–6555. [13] A. Arumugam, C. Karthikeyan, A.S. Haja Hameed, K. Gopinath, S. Gowri, V. Karthika, Synthesis of cerium oxide nanoparticles using Gloriosa superba L. leaf extract and their structural, optical and antibacterial properties, Mater. Sci. Eng. C Mater. Biol. Appl. 49 (2015) 408–415. [14] J. Jacob, T. Mukherjee, S. Kapoor, A simple approach for facile synthesis of Ag, anisotropic Au and bimetallic (Ag/Au) nanoparticles using cruciferous vegetable extracts, Mater. Sci. Eng. C Mater. Biol. Appl. 32 (2012) 1827–1834.

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