molecules Article
Green Microwave-Assisted Combustion Synthesis of Zinc Oxide Nanoparticles with Citrullus colocynthis (L.) Schrad: Characterization and Biomedical Applications Susan Azizi 1, *, Rosfarizan Mohamad 1,2, * and Mahnaz Mahdavi Shahri 3 1 2 3
*
Department of Bioprocess Technology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, UPM Serdang, Selangor 43400, Malaysia Laboratory of Biopolymer and Derivatives, Institute of Tropical Forestry and Forest Products, Universiti Putra Malaysia, UPM Serdang, Selangor 43400, Malaysia Department of Chemistry, Shiraz Branch, Islamic Azad University, Shiraz 74731-71987, Iran;
[email protected] Correspondence:
[email protected] (S.A.);
[email protected] (R.M.); Tel.: +60-1-7622-8029 (S.A.); +60-1-3263-6029 (R.M.)
Academic Editor: Derek J. McPhee Received: 18 January 2017; Accepted: 13 February 2017; Published: 16 February 2017
Abstract: In this paper, a green microwave-assisted combustion approach to synthesize ZnO-NPs using zinc nitrate and Citrullus colocynthis (L.) Schrad (fruit, seed and pulp) extracts as bio-fuels is reported. The structure, optical, and colloidal properties of the synthesized ZnO-NP samples were studied. Results illustrate that the morphology and particle size of the ZnO samples are different and depend on the bio-fuel. The XRD results revealed that hexagonal wurtzite ZnO-NPs with mean particle size of 27–85 nm were produced by different bio-fuels. The optical band gap was increased from 3.25 to 3.40 eV with the decreasing of particle size. FTIR results showed some differences in the surface structures of the as-synthesized ZnO-NP samples. This led to differences in the zeta potential, hydrodynamic size, and more significantly, antioxidant activity through scavenging of 1, 1-Diphenyl-2-picrylhydrazyl (DPPH) free radicals. In in vitro cytotoxicity studies on 3T3 cells, a dose dependent toxicity with non-toxic effect of concentration below 0.26 mg/mL was shown for ZnO-NP samples. Furthermore, the as-synthesized ZnO-NPs inhibited the growth of medically significant pathogenic gram-positive (Bacillus subtilis and Methicillin-resistant Staphylococcus aurous) and gram-negative (Peseudomonas aeruginosa and Escherichia coli) bacteria. This study provides a simple, green and efficient approach to produce ZnO nanoparticles for various applications. Keywords: ZnO nanoparticles; Combustion method; green synthesis; Citrullus colocynthis; antimicrobial; antioxidant
1. Introduction Zinc oxide nanoparticle as a non-toxic, low-cost, and non-hygroscopic metal oxide is very economical and safe polar inorganic crystalline material which has extensive applications in different areas. Nano-sized ZnO particles are an important metal oxide material which has a wide direct band gap (3.37 eV) and large exciton binding energy (60 meV) at room temperature [1,2]. The ZnO-NPs has been broadly explored for applications in catalysis [3], gas sensing [4], cosmetics [5], drug delivery [6], and solar cells [7]. There are numerous researches available on the synthesis of ZnO-NPs using various chemical and physical methods such as precipitation [8], solvothermal and hydrothermal synthesis [9,10], laser ablation [11], sonochemical [12], sol-gel [13] and flame spray synthesis [14]
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approaches. However, most of these synthetic methods require costly equipment and need to be operated under very severe conditions.with In comparison withtechniques, the mentioned techniques, combustion very severe conditions. In comparison the mentioned combustion route has more route has more advantages, such as fast, simple fast, low cost, high purity and homogeneous advantages, such as simple process, lowprocess, cost, high purity and homogeneous products [15,16]. products [15,16]. The method is interesting for the synthesis of multi-component oxide materials The method is interesting for the synthesis of multi-component oxide materials in a short time with in a short time high surface areapure products and highly crystalline particle [17,18]. size at low high surface areawith products and highly crystalline particlepure size at low temperature The temperature [17,18].combustion The microwave-assisted combustion approach has been extensively used microwave-assisted approach has been extensively used to produce various metal oxide to produce various materials suchand as ZnO Y2 O3[22]. [19],However TiO2 [20], CeO2researches, [21] and ZnO [22]. materials such as Y2Ometal 3 [19], oxide TiO2 [20], CeO2 [21] in most chemical However in most used as fuel which to synthesize metal oxide compounds have researches, been used chemical as fuel tocompounds synthesize have metalbeen oxide materials may restricts their materials which may restricts their biomedical applications. To overcome this limitation, we applied a biomedical applications. To overcome this limitation, we applied a green microwave-assisted green microwave-assisted combustion approach to synthesize ZnO-NPs using fruit, seed and pulp combustion approach to synthesize ZnO-NPs using fruit, seed and pulp extracts obtained from extracts from colocynthis colocynthis) plant as bio-fuels. Green synthesiswith is more Citrullusobtained colocynthis (C. Citrullus colocynthis) plant as(C. bio-fuels. Green synthesis is more eco-friendly less eco-friendly with less toxic effects in comparison with chemical processes [23]. The C. colocynthis toxic effects in comparison with chemical processes [23]. The C. colocynthis a valuable cucurbit plant, abroadly valuable cucurbit plant, broadly tropical areas of and the nutraceutical world, has values many distributed in the tropical areas distributed of the world,in hasthe many pharmaceutical pharmaceutical and nutraceutical [24]. The fruitsispossess a soft, white spongy pulp which is [24]. The fruits possess a soft, whitevalues spongy pulp which filled with many smooth, compressed and filled with many smooth, compressed and oval-shaped seeds (Figure 1). Based on the previous studies, oval-shaped seeds (Figure 1). Based on the previous studies, C. colocynthis has shown very effective C. colocynthis anti-inflammatory, has shown very effective antibacterial, anti-inflammatory, antioxidant and anticancer antibacterial, antioxidant and anticancer activities [25]. The therapeutic effects of activities [25]. The therapeutic effects of C. colocynthis are usually attributed to its polyphenolic C. colocynthis are usually attributed to its polyphenolic compounds such as isosaponarin, isovitexin compounds such as isosaponarin, isovitexin and isoorientin with 3-O-methyl ethereffects [26,27]. These and isoorientin 3-O-methyl ether [26,27]. These compounds medicinal have thecompounds potential to with medicinal have the potential to beduring absorbed the surface of nanoparticles duringZnO the be absorbed oneffects the surface of nanoparticles theon synthesis process. Therefore, stable synthesis process. Therefore, stable ZnO nanoparticles synthesized with C. colocynthis could be nanoparticles synthesized with C. colocynthis could be extremely suitable for drug delivery, gene extremely suitable for drug delivery, gene delivery applications, where there is a direct delivery and biosensor applications, where thereand is abiosensor direct contact of these nanoparticles with contact of these with blood. This study attempts to exploit C.synthesis colocynthis as blood. This studynanoparticles attempts to exploit C. colocynthis extract as a bio-fuel for the of extract ZnO-NPs afor bio-fuel for the synthesis of ZnO-NPs for biomedical applications. biomedical applications.
Figure 1. Photograph of fruits of C. colocynthis. Figure 1. Photograph of fruits of C. colocynthis.
2. Results and Discussion 2. Results and Discussion Zinc oxide nanoparticles using combustion process can be produced through the combination Zinc oxide nanoparticles using combustion process can be produced through the combination of zinc nitrates in an aqueous solution with a fuel. In the experiments, fruit, seed and pulp extracts of of zinc nitrates in an aqueous solution with a fuel. In the experiments, fruit, seed and pulp extracts C. colocynths were suitable fuels because they contain various compounds such as Cucurbitacins, of C. colocynths were suitable fuels because they contain various compounds such as Cucurbitacins, glycosides, flavonoids and phenolic acids [28,29] that can act as complexing agents of the zinc ion in glycosides, flavonoids and phenolic acids [28,29] that can act as complexing agents of the zinc ion in the solution, as well as contributing as fuel for the synthesis of ZnO nanoparticles. the solution, as well as contributing as fuel for the synthesis of ZnO nanoparticles. During the formation of ZnO nanoparticles an exothermic reaction takes place between oxidizing During the formation of ZnO nanoparticles an exothermic reaction takes place between oxidizing and reducing agent. Nitrate could act as an oxidizer agent and fuels could act as reducing agents for and reducing agent. Nitrate could act as an oxidizer agent and fuels could act as reducing agents for the formation of ZnO nanoparticles [30–32]. the formation of ZnO nanoparticles [30–32]. The possible mechanism for formation of ZnO-NPs is as below: The nitrate group is a The possible mechanism for formation of ZnO-NPs is as below: The nitrate group is stronger oxidizer and can oxidize hydroxyl groups present in biomolecules to carbonyl groups and a stronger oxidizer2+and can oxidize hydroxyl groups present in biomolecules to carbonyl groups and simultaneously Zn ions form a complex compound inside the nanoscopic templates of metabolites simultaneously Zn2+ ions form a complex compound inside the nanoscopic templates of metabolites through transfer of the π electrons from the carbonyl groups to the transition metal–dative coordinate through transfer of the π electrons from the carbonyl groups to the transition2+metal–dative coordinate bonding and finally, the ZnO-NPs formed by thermal decomposition of Zn complex (Scheme 1). bonding and finally, the ZnO-NPs formed by thermal decomposition of Zn2+ complex (Scheme 1).
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Scheme 1. 1. The The possible possible mechanism mechanism for for formation formation of of ZnO-NPs ZnO-NPs with with Isovitexin Isovitexin as as aa flavone. flavone. Scheme The possible mechanism
103 103
110 110
102 102
101 101
002 002
cc
200 200 112 112 201 201
Absorbance Absorbance(a.u) (a.u)
100 100
2.1. Structural and and Morphology Characterization Characterization of ZnO-NPs ZnO-NPs 2.1. 2.1. Structural Structural and Morphology Morphology Characterization of of ZnO-NPs Figure 2 shows the the XRD patterns patterns of synthesized synthesized ZnO-NPs. All All the recorded recorded peaks of of the (100), (100), Figure Figure 22 shows shows the XRD XRD patterns of of synthesized ZnO-NPs. ZnO-NPs. All the the recorded peaks peaks of the the (100), (002), (101), (101), (102), (110), (110), (103), (200), (200), (112) and and (201) can can be indexed indexed to reflection reflection lines lines of of hexagonal hexagonal (002), (002), (101), (102), (102), (110), (103), (103), (200), (112) (112) and (201) (201) can be be indexed to to reflection lines of hexagonal wurtzite ZnO (JCPDS 36-1451). The widening of the diffraction peaks indicates that the crystalline wurtzite ZnO ZnO(JCPDS (JCPDS36-1451). 36-1451).The Thewidening widening diffraction peaks indicates the crystalline wurtzite ofof thethe diffraction peaks indicates thatthat the crystalline size size of of obtained obtained particles particles is is in in nanoscale nanoscale range. range. size of obtained particles is in nanoscale range.
bb
aa 25 25
35 35
45 45
55 55 2θ (degree) 2θ (degree)
65 65
75 75
Figure 2.The The XRD XRD patterns patterns of of (a) (a) ZnO ZnO fruit fruit (ZnO-F), (ZnO-F), (b) (b) ZnO ZnO seed seed (ZnO-S) and and (c) (c) ZnO ZnO pulp pulp (ZnO-P) (ZnO-P) Figure Figure 2. 2. The XRD patterns of (a) ZnO fruit (ZnO-F), (b) ZnO(ZnO-S) seed (ZnO-S) and (c) ZnO pulp nanoparticles. nanoparticles. (ZnO-P) nanoparticles.
The wurtzite wurtzite lattice lattice parameters, parameters, such such as as the the values values of of d, d, the the distance distance between between adjacent adjacent planes planes in in The The wurtzite lattice parameters, such as the values of d, theangle distance between adjacentthe planes in the Miller indices (h k l); lattice constants a, b, and c, interplanar (the angle φ between planes the Miller indices (h k l); lattice constants a, b, and c, interplanar angle (the angle φ between the planes the (h d k l);and lattice constants and c, interplanar (the angle φ between the planes (h11 kkMiller ), of ofindices spacing the plane plane (h (h22 kka,22 llb, of spacing spacing andangle unit cell cell volumes volumes (V), are are calculated calculated (h 11 ll11), spacing d11 and the 22)) of dd22),), and unit (V), (h k l ), of spacing d and the plane (h k l ) of spacing d ), and unit cell volumes (V), are calculated 1 1 1 1 2 2 2 2 from the the lattice lattice geometry geometry [33]. [33]. It It was was observed observed that that there there was was small small change change in in the the lattice lattice parameters parameters from from the lattice geometry [33]. It was observed thatextracts. there was small change in the lattice parameters of nanoparticles produced by fruit, seed and pulp The variation in lattice parameters can be be of nanoparticles produced by fruit, seed and pulp extracts. The variation in lattice parameters can of nanoparticles produced by fruit, seed and pulp extracts. The variation in lattice parameters can ascribed to to the the change change of of particle particle size size and and quantum quantum size size effects effects [34]. [34]. The The lattice lattice parameters parameters of of the the ascribed be ascribed toZnO-NPs the change of particle sizeinand quantum size effects [34]. The lattice parameters of the synthesized are summarized Table 1. synthesized ZnO-NPs are summarized in Table 1. synthesized ZnO-NPs are summarized in Table 1. Table 1. 1. The The lattice lattice parameters parameters of of the the synthesized synthesized ZnO-NPs. ZnO-NPs. Table Table 1. The lattice parameters of the synthesized ZnO-NPs.
Sample 2θ 2θ ±± 0.1 0.1 hh kk ll Sample Sample 2θ ±67.96 0.1 hk1 l1 11 22 67.96 ZnO-F ZnO-F 69.10 1 1 222 00 11 67.9669.10 ZnO-F 69.1068.08 68.08 2 0 111 11 22 ZnO-S ZnO-S 69.18 1 1 222 00 11 68.0869.18 ZnO-S 69.1867.92 2 0 11 1 2 112 ZnO-P 67.92 ZnO-P 67.9269.15 1 1 22 0 1 69.15 201 ZnO-P 69.15
201
Structure Lattice Lattice Parameter Parameter (nm) (nm) V V (A˚) (A˚)33 cosφ cosφ Structure ◦ )3 Structure Lattice Parameter (nm) cosφ V (A wurtzite wurtzite 0.3222, c/a c/a == 1.599 1.599 48.17 0.8480 aa == 0.3222, 48.17 0.8480 wurtzite
wurtzite wurtzite wurtzite
wurtzite wurtzite wurtzite
a = 0.3222, c/a = 1.599
0.3248, c/a c/a == 1.592 1.592 aa == 0.3248, a = 0.3248, c/a = 1.592
0.3249, c/a c/a == 1.601 1.601 aa == 0.3249,
a = 0.3249, c/a = 1.601
48.17
47.88 47.88 47.88
47.62 47.62
47.62
0.8480
0.7193 0.7193 0.7193
0.7180 0.7180
0.7180
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The average crystallite size calculated using Scherrer equation D = 0.89λ βcosθ with the width of the . nm (002) andThe (101) peaks are found to be 27 ± 2 nm, 64 ± 2 nm, and 85 ± 2 for ZnO prepared average crystallite size calculated using Scherrer equation D = with the width of the with seed,(002) pulpand and(101) fruitpeaks extract, respectively. are found to be 27 ± 2 nm, 64 ± 2 nm, and 85 ± 2 nm for ZnO prepared with The morphology, size, and structure of the ZnO samples were investigated through FESEM, and seed, pulp and fruit extract, respectively. Molecules 2017, 22, 301 4 of 12 TEM. As The shown in Figure 3a the quantity flower shaped nanostructures, 85–100 nm in size morphology, size, andlarge structure of theofZnO samples were investigated through FESEM, . produced with fruit extract. The nanostructures resulted assemblage of numerous and TEM. shown in Figure 3a floral the large quantity of flower shapedfrom nanostructures, 85–100 nm TheAs average crystallite size calculated using Scherrer equation D = the with the width of in thesize produced with fruit extract. The floral nanostructures resulted from the assemblage of numerous of of ZnO nanoflakes, which were created from a single axis. In ZnO-S (Figure 3b), the hexagonal (002) and (101) peaks are found to be 27 ± 2 nm, 64 ± 2 nm, and 85 ± 2 nm for ZnO prepared with ZnO nanoflakes, which were created from a single axis. In ZnO-S (Figure 3b), the hexagonal nanostructures, nmextract, in sizerespectively. were generated. It is interesting to mention that the flower-like and seed, pulp20–35 and fruit nanostructures, 20–35 nm in size generated. is interesting to entirely mention that the flower-like and The morphology, size, andwere structure of the samples were investigated through FESEM, hexagonal nanostructure produced with fruit andItZnO seed changed into a blocky morphology hexagonal nanostructure produced with fruit and seed changed entirely into a blocky morphology and TEM. As shown in Figure 3a the large quantity of in flower shaped 85–100 in size in the ZnO sample prepared by pulp, as presented Figure 3c.nanostructures, The dimensions ofnm these irregular in the ZnO sample prepared presented inresulted Figure 3c. The these irregular produced with fruit extract. by Thepulp, floral as nanostructures from thedimensions assemblage of of numerous of block-shaped nanoparticles ranged from 30 to 80 nm. The results demonstrate that bio-fuel functions as block-shaped nanoparticles ranged from from 30 to 80 nm. The results demonstrate functions ZnO nanoflakes, which were created a single axis. In ZnO-S (Figure that 3b),bio-fuel the hexagonal a structure-directing agent, greatly affecting the anisotropic development of ZnO in differentand structures as nanostructures, a structure-directing agent, greatly affecting the of flower-like ZnO in different 20–35 nm in size were generated. It is anisotropic interesting todevelopment mention that the of flower-shaped, hexagonal and block-like morphology. hexagonal nanostructure produced with fruit and seedmorphology. changed entirely into a blocky morphology structures of flower-shaped, hexagonal and block-like in the ZnO sample prepared by pulp, as presented in Figure 3c. The dimensions of these irregular block-shaped nanoparticles ranged from 30 to 80 nm. The results demonstrate that bio-fuel functions as a structure-directing agent, greatly affecting the anisotropic development of ZnO in different structures of flower-shaped, hexagonal and block-like morphology.
Figure 3. FESEM images of: (a) ZnO-F; (b) ZnO-S and (c) ZnO-P nanoparticles.
Figure 3. FESEM images of: (a) ZnO-F; (b) ZnO-S and (c) ZnO-P nanoparticles.
The difference in morphology and particle size using different fuels are directly associated to
The difference in morphology and using different directly the number of moles of3.combustion gases during process. Thefuels gases are disrupt bulkyassociated clusters to Figure FESEM images of:particle (a)escaped ZnO-F;size (b) ZnO-S and (c) ZnO-P nanoparticles. and make between particles. In fact, escaped the clusters are broken in conditions of disrupt more generating of the number ofholes moles of combustion gases during process. The gases bulky clusters The difference and particle size is using different directly associated to gaseous products andin inmorphology these conditions high heat released from the are system, hindering particle and make holes between particles. In fact, the clusters are broken infuels conditions of more generating of the number[35]. of moles of combustion gases escaped during process. The gases disrupt bulky clusters development gaseous products and in these conditions high heat is released from the system, hindering particle and make holes between particles. In fact, the ZnO clusters are broken inwas conditions of more generating of 4a Further structural characterization of the nanostructures performed by TEM. Figure development [35]. gaseous products and in these conditions high heat is released from the system, hindering particle displays the TEM image of the flower-shaped ZnO, which is accordance with the FESEM observations Further structural characterization of the ZnO nanostructures was performed by TEM. Figure 4a development (Figure 3a). ZnO[35]. with hexagonal and some irregular structures are clearly seen in the TEM image of Further structural characterization of theZnO, ZnO nanostructures was performed by TEM. Figure 4a displays the TEM image of the flower-shaped which is accordance with the FESEM observations ZnO-S, as shown in Figure 4b. The TEM image of ZnO-P shown in Figure 4c proves that the shape of displays the TEM image of the flower-shaped ZnO, which is accordance with the FESEM observations (Figure ZnO withishexagonal andofsome irregular structures are clearly seen inwith thethe TEM image of the3a). nanoparticles a combination irregular polygons. This outcome is consistent FESEM (Figure 3a). ZnO with hexagonal and some irregular structures are clearly seen in the TEM image of ZnO-S, as shown in Figure 4b. The TEM image of ZnO-P shown in Figure 4c proves that the shape of result (Figure 3c) ZnO-S, as shown in Figure 4b. The TEM image of ZnO-P shown in Figure 4c proves that the shape of the nanoparticles is a combination of irregular polygons. This outcome is consistent with the FESEM the nanoparticles is a combination of irregular polygons. This outcome is consistent with the FESEM result (Figure 3c) 3c) result (Figure
Figure 4. TEM images of: (a) ZnO-F; (b) ZnO-S and (c) ZnO-P nanoparticles. Figure 4. TEM images of: (a) ZnO-F; (b) ZnO-S and (c) ZnO-P nanoparticles.
Figure 4. TEM images of: (a) ZnO-F; (b) ZnO-S and (c) ZnO-P nanoparticles.
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FTIR ofof as-synthesized ZnO-NPs and extracts as shown in FTIRspectra spectraillustrate illustratethe thefunctional functionalgroups groups as-synthesized ZnO-NPs and extracts as shown −1−1can Figure 5a–f. TheThe broad absorption band is observed in in 3352 cmcm in Figure 5a–f. broad absorption band is observed 3352 canbe beascribed ascribedto toO-H O-Hstretching stretching −1−1 and andH-bonded H-bondedin inglycosides, glycosides,alcohol alcoholororphenol phenolgroups. groups.The Theabsorption absorptionpeak peakisisobserved observedatat1630 1630cm cm − 1 −1 corresponding to the theC=O C=Obands. bands. peak observed at 1420 cm attribute to C-C stretching in corresponding to TheThe peak observed at 1420 cm attribute to C-C stretching in aromatic −1 inof −1 in the cm aromatic groups. The absorption bands in of the390–350 range of the spectra of nanoparticles synthesized groups. The absorption bands in the range cm390–350 spectra synthesized nanoparticles to the Zn-O.clearly FT-IRshow spectra show the of absorption bandsand of O-H correspond to correspond the Zn-O. FT-IR spectra the clearly absorption bands O-H stretching C=O stretching and C=O after formation of ZnO-NPs reduced indicating the of these functional after formation of ZnO-NPs reduced indicating the participation ofparticipation these functional groups in the groups in the synthesis The FTIR results obviously demonstrate the surface structure the synthesis process. Theprocess. FTIR results obviously demonstrate that the that surface structure of theofZnO synthesized by fruit, seed seed and pulp extracts are different, and this the surface layer layer of Zn-O ZnO synthesized by fruit, and pulp extracts are different, andinfluences this influences the surface of bonds. This might affectaffect the properties of ZnO-NPs thatthat depend mainly on the surface structures. Zn-O bonds. This might the properties of ZnO-NPs depend mainly on the surface structures.
f
Absorbance (a.u)
e d c b a
Zn-O O-H
3700
3200
C=O C–C C-O 2700
2200
1700
1200
700
200
Wavenumber (cm-1) Figure5.5.FTIR FTIRspectra spectraof of(a) (a)fruit fruitextract; extract;(b) (b)ZnO-F; ZnO-F;(c) (c)pulp pulpextract; extract;(d) (d)ZnO-P; ZnO-P;(e) (e)seed seedextract extractand and Figure (f)ZnO-S. ZnO-S. (f)
2.2. Analysis of Optical Properties 2.2. Analysis of Optical Properties The UV-vis spectra of ZnO-NPs bio-synthesized by use of fruit, seed and pulp of C. colocynthis The UV-vis spectra of ZnO-NPs bio-synthesized by use of fruit, seed and pulp of C. colocynthis are shown in the inset of Figure 6. The relevant increase in the absorption at wavelengths more than are shown in the inset of Figure 6. The relevant increase in the absorption at wavelengths more than 350 nm can be assigned to the direct band-gap of ZnO due to the electron transitions from the 350 nm can be assigned to the direct band-gap of ZnO due to the electron transitions from the valence valence band to the conduction band (O2p-Zn3d) [36]. A redshift in the absorption edge was seen for band to the conduction band (O2p-Zn3d) [36]. A redshift in the absorption edge was seen for the ZnO the ZnO fruit (ZnO-F) and ZnO pulp (ZnO-P) compared to ZnO seed (ZnO-S). This might be owing fruit (ZnO-F) and ZnO pulp (ZnO-P) compared to ZnO seed (ZnO-S). This might be owing to changes to changes in its particle size, morphology and surface microstructure. Furthermore, the direct in its particle size, morphology and surface microstructure. Furthermore, the direct band-gap energies band-gap energies estimated from a plot of (αhν)2 versus the photo energy (hν) consistent with the estimated from a plot of (αhν)2 versus the photo energy (hν) consistent with the Kubelka-Munk model Kubelka-Munk model shown in Figure 6 were 3.40, 3.28 and 3.25 eV for the ZnO-NPs, synthesized shown in Figure 6 were 3.40, 3.28 and 3.25 eV for the ZnO-NPs, synthesized by seed, pulp, and by seed, pulp, and fruit extracts, respectively. Such a decrease in the ZnO band-gap energy is well fruit extracts, respectively. Such a decrease in the ZnO band-gap energy is well consistent with the consistent with the corresponding redshift seen in the absorption edge noted above. ZnO in the form corresponding redshift seen in the absorption edge noted above. ZnO in the form of bulk has a band of bulk has a band gap of 3.20 eV, which is in the close UV region. However, when ZnO is gap of ∼3.20 eV, which is in the close UV region. However, when ZnO is synthesized in nano-size synthesized in nano-size range, the band gap increases owing to the quantum confinement effect, range, the band gap increases owing to the quantum confinement effect, and this describes the larger and this describes the larger band gap of the ZnO-NP with smaller size. band gap of the ZnO-NP with smaller size.
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Figure6.6.Absorption Absorption edge edge (inset) ZnO-NPs. Figure (inset) and andband bandgap gapofofthe theprepared prepared ZnO-NPs.
2.3. Analysis on Colloidal Properties 2.3. Analysis on Colloidal Properties In this study, zeta potential and hydrodynamic size were measured by DLS. The zeta potential In this study, zeta potential and hydrodynamic size were measured by DLS. The zeta potential for colloidal suspensions of ZnO-NPs samples were found at −25.60, −19.5 and −15.3 mV for ZnO-F, for colloidal suspensions of ZnO-NPs samples were found at −25.60, −19.5 and −15.3 mV for ZnO-F, ZnO-P and ZnO-S, respectively. From the zeta potential values, it is suggested that the NPs were stable ZnO-P and ZnO-S, respectively. From theand zetaresponsible potential values, it is suggested that the NPs were stable and warped with anionic organic phases for electrostatic stabilization [37]. FTIR result and anionic organic phasesstructure and responsible for electrostatic stabilization [37].seed FTIRand result haswarped alreadywith revealed that the surface of the synthesized ZnO samples by fruit, has already revealed that the surface structure of the synthesized ZnO samples by fruit, seed and pulp pulp are somewhat different. This difference is expected to change the net surface charge density areand somewhat different. This difference is expected to change the net surface charge density and their their inter particle interactions. inter particle interactions. Nanoparticles generally suffer from agglomeration when dispersed in solutions, and this Nanoparticles generally from agglomeration dispersed in solutions, this behavior behavior has a major effect onsuffer the reactivity and responsewhen of nanomaterials upon contact and to various cells has major effect the reactivity and response ofthe nanomaterials upon contact to various cells or oraorganisms [38].on Hence, the hydrodynamic sizes of ZnO-NPs dispersed in nanopure water were recorded at physiological pH. The hydrodynamic of the ZnO-NPs producedin bynanopure seed, pulpwater and fruit organisms [38]. Hence, the hydrodynamic sizessizes of the ZnO-NPs dispersed were were 1050 ± 80 nm, 860 ±pH. 80 The nm, hydrodynamic and 750 ± 80 nm, respectively. Based produced on the Derjaguin-Landaurecorded at physiological sizes of the ZnO-NPs by seed, pulp and Verwey-Overbeek aggregation uncovered nanoparticles on the repulsive fruit were 1050 ± 80 (DLVO) nm, 860 model, ± 80 nm, and 750 ±of80 nm, respectively. Baseddepends on the Derjaguin-Landauforces arising from electrostatic and the van der Waals forces of attraction. Because the Verwey-Overbeek (DLVO) model, aggregation of uncovered nanoparticles dependssurface on thecharges repulsive of nanoparticles the electrostatic interaction, higher potential forces arising fromaffect electrostatic and the repulsive van der Waals forcesnanoparticles of attraction.with Because thezeta surface charges value will usually decrease hydrodynamic size. In addition, the polydispersity indices (PDIs) of of nanoparticles affect the electrostatic repulsive interaction, nanoparticles with higher zeta potential ZnO-NPs obtained from seed, pulp and fruit are 0.369, 0.385 and 0.407 (μ 2/Г2), respectively (Table 2). value will usually decrease hydrodynamic size. In addition, the polydispersity indices (PDIs) of These results show that the nanoparticles have an intermediate, moderately polydispersed distribution ZnO-NPs obtained from seed, pulp and fruit are 0.369, 0.385 and 0.407 (µ2 /Г2 ), respectively (Table 2). type, where the distribution is neither very polydispersed, or broad, nor in any sense narrow. These results show that the nanoparticles have an intermediate, moderately polydispersed distribution type, where the distribution is neither very polydispersed, or broad, nor in any sense narrow. Table 2. Colloidal properties of ZnO-S, ZnO-P and ZnO-F. Sample Zeta Potential Hydrodynamic Size (nm)ZnO-PPolydispersity Indices (μ2/Г2) Table(mV) 2. Colloidal properties of ZnO-S, and ZnO-F. ZnO-S −15.3 ± 2 1050 ± 80 0.369 ZnO-P −19.5Potential ±2 860 ± 80 0.385 Indices (µ2 /Г2 ) Sample Zeta (mV) Hydrodynamic Size (nm) Polydispersity ZnO-F −25.60 ±2 ±2 750 ±1050 80 ± 80 0.4070.369 ZnO-S −15.3 ZnO-P −19.5 ± 2 860 ± 80 0.385 ZnO-F −25.60 750 ± 80 0.407 2.4. In Vitro Cytotoxicity Study± 2 Cytotoxicity is a new property exposed by various materials, including ZnO, when produced 2.4. In Vitro Cytotoxicity Study with the nano-size dimensions. According to this, we attempted to investigate and compare the cytotoxicity of ZnO-NPs synthesized by seed, andmaterials, fruit extracts on 3T3ZnO, cells to determine if the Cytotoxicity is a new property exposed by pulp various including when produced with differences in the size, shape and surface structures of these three ZnO-NP samples have an the nano-size dimensions. According to this, we attempted to investigate and compare the cytotoxicity important effect on their interaction with these cells. To evaluate the cell viability of 3T3 cells treated of ZnO-NPs synthesized by seed, pulp and fruit extracts on 3T3 cells to determine if the differences in with each of the ZnO-NP samples, different ZnO-NP concentrations of 0, 0.05, 0.1, 0.15, 0.2, 0.3 and the size, shape and surface structures of these three ZnO-NP samples have an important effect on their 0.4 mg/mL were employed, and the cell viability was determined after 72 h of treatment. interaction with these cells. To evaluate the cell viability of 3T3 cells treated with each of the ZnO-NP
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samples, different Molecules 2017, 22, 301 ZnO-NP concentrations of 0, 0.05, 0.1, 0.15, 0.2, 0.3 and 0.4 mg/mL were employed, 7 of 12 and the cell viability was determined after 72 h of treatment. The cell viability data of ZnO-NP samples are shown in Figure 7. It displays a slow decrease in cell viability with increasing concentration of ZnO-NP for all the ZnO samples. However, the ZnO-S cell viability viability with with increasing increasing nanoparticles nanoparticles concentration concentration displayed a slightly faster reduction in the cell compared to ZnO-P and ZnO-F, ZnO-F, so so that that the the half maximal inhibitory inhibitory concentration concentration IC50 of ZnO-S was compared mg/mL, while and 0.258 0.258 mg/mL, mg/mL, respectively. The ZnO-S 0.160 mg/mL, while those those of of ZnO-P and ZnO-F were 0.210 and ~1.6 times ZnO-F sample showed about ~1.3 and ~1.6 times higher higher IC50 compared to those of the ZnO-P and ZnO-F samples respectively, suggesting that ZnO-S NP is more toxic than the ZnO-P and ZnO-F nanoparticles. nanoparticles. cytotoxicity of synthesized ZnO-NPs can be related to their size size and and morphology. morphology. The smaller The cytotoxicity size nanoparticles can penetrate easily into cell membrane and caused more cytotoxicity compared to larger size nanoparticles [39]. This result is in agreement with previous findings [40,41] which showed factors, such as aa nanoparticle's nanoparticle's shape, shape, size, surface charge effect on cytotoxicity cytotoxicity that a variety of factors, of nanoparticles. 125 Reialavtive Cell Viability (%)
ZnO-F
100
ZnO-P ZnO-S
75 50 25 0 0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
Concentration (mg/mL) Figure 7. 7. Cytotoxic effect of of ZnO-NPs ZnO-NPs samples samples on on the the growth growth of of 3T3 3T3 cells. cells. Figure Cytotoxic effect
2.5. 1,1-Diphenyl-2-picrylhydrazyl (DPPH) Radical Scavenging Activity 2.5. 1,1-Diphenyl-2-picrylhydrazyl (DPPH) Radical Scavenging Activity The free radical scavenging activity of ZnO-NPs were examined by DPPH scavenging and are The free radical scavenging activity of ZnO-NPs were examined by DPPH scavenging and are shown in Figure 8. DPPH is a stable free radical and shows a typical absorption peak at 517 nm. shown in Figure 8. DPPH is a stable free radical and shows a typical absorption peak at 517 nm. The decrease in absorption is taken as a quantity of the level of radical scavenging. The change of The decrease in absorption is taken as a quantity of the level of radical scavenging. The change color from dark purple to light yellow is proportional to reduction of DPPH and conversion to of color from dark purple to light yellow is proportional to reduction of DPPH and conversion to 1,1-diphenyl-2-picryl hydrazine with decolorization (see inset in Figure 8). The DPPH activity of the 1,1-diphenyl-2-picryl hydrazine with decolorization (see inset in Figure 8). The DPPH activity of the ZnO-NPs was found to increase in a dose-dependent manner. Scavenging of DPPH radicals was ZnO-NPs was found to increase in a dose-dependent manner. Scavenging of DPPH radicals was found found to be increasing as the concentration of the ZnO-NPs samples increased. Among, these to be increasing as the concentration of the ZnO-NPs samples increased. Among, these nanoparticles nanoparticles the ZnO-F sample displayed a higher scavenging of DPPH radicals with increasing the ZnO-F sample displayed a higher scavenging of DPPH radicals with increasing concentration concentration than that of the ZnO-S and ZnO-P. Considering the more negative surface charge of than that of the ZnO-S and ZnO-P. Considering the more negative surface charge of ZnO-F compared ZnO-F compared to ZnO-S and ZnO-P, it appears that these ZnO-F NPs display a high tendency to to ZnO-S and ZnO-P, it appears that these ZnO-F NPs display a high tendency to interact with and interact with and reduce DPPH. The all-ZnO nanoparticles were proved to be potent at inhibiting reduce DPPH. The all-ZnO nanoparticles were proved to be potent at inhibiting the DPPH free radical the DPPH free radical scavenging activity with IC50 value of 0.22, 0.26 and 0.29 mg/mL for ZnO-F, scavenging activity with IC50 value of 0.22, 0.26 and 0.29 mg/mL for ZnO-F, ZnO-P and ZnO-S ZnO-P and ZnO-S NPs, respectively. NPs, respectively.
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125 125
ZnO-F ZnO-F ZnO-P ZnO-P ZnO-S ZnO-S
Inhibition Inhibition(%) (%)
100 100
75 75
50 50
25 25
00
00
0.05 0.05
0.1 0.3 0.5 0.1 0.3 0.5 Concentration (mg/mL) (mg/mL) Concentration
0.7 0.7
11
Figure Scavenging capacity of the (DPPH) Figure 8. the prepared prepared ZnO ZnO samples samples on on1,1-Diphenyl-2-picrylhydrazyl 1,1-Diphenyl-2-picrylhydrazyl (DPPH) Figure 8. Scavenging capacity of the prepared ZnO samples on 1,1-Diphenyl-2-picrylhydrazyl (DPPH) free radicals and color changes of DPPH with different concentration of ZnO-NPs (inset). (DPPH) free radicals and color changes of DPPH with different concentration of ZnO-NPs (inset). (DPPH) free radicals and color changes of DPPH with different concentration of ZnO-NPs (inset).
2.6. Antimicrobial Antimicrobial Activity Activity 2.6. 2.6. Activity this study, study, the the antimicrobial antimicrobial activity using new new bio-fuels bio-fuels was was evaluated. evaluated. In of ZnO-NPs synthesized using In this this study, the antimicrobial activity of ZnO-NPs synthesized In this thisanalysis, analysis,the the ZnO-NPs showed antimicrobial activity against range of different different bacteria In ZnO-NPs showed antimicrobial activity against a range different bacteria bacteria (Table 3, In this analysis, the ZnO-NPs showed antimicrobial activity against aa of range of (Table 3, Figure 9). The ZnO-S nanoparticles showed the highest antibacterial activity compared to Figure 9). The ZnO-S nanoparticles showed the highest antibacterial activity compared to the two (Table 3, Figure 9). The ZnO-S nanoparticles showed the highest antibacterial activity compared to the two two others ZnO-NPs ZnO-NPs samples. The reason for enhancement enhancement is probably due size to the the small size size of others ZnO-NPs samples. The reasonThe for reason enhancement is probably is due to the small of ZnO-S which the others samples. for probably due to small of ZnO-S which ispenetrate more likely likely to penetrate into the the cellthe membrane to kill theofbacteria. bacteria. The zone of is more which likely tois intoto thepenetrate cell membrane to kill bacteria. to Thekill zone inhibition in the case ZnO-S more into cell membrane the The zone of inhibition in the case of ZnO-S nanoparticles for each bacterium were determined to be approximately of ZnO-S nanoparticles for each bacterium were determined to be approximately 14.3 ± 0.51, 6.8 ± 0.36, inhibition in the case of ZnO-S nanoparticles for each bacterium were determined to be approximately 14.3 ± 0.51, 6.8 ±±13.4 0.36, 12.2 0.27respectively, and 13.4 13.4 ±± 0.47mm, 0.47mm, respectively, for B. B. subtilis, subtilis,Staphylococcus Methicillin-resistant 12.2 0.27 and ± 12.2 0.47mm, for B. subtilis, Methicillin-resistant aurous 14.3 ±± 0.51, 6.8 0.36, ±± 0.27 and respectively, for Methicillin-resistant Staphylococcus aurous (MRSA), P. aeruginosa and E. coli. The highest antimicrobial activity was (MRSA), P. aeruginosa and E. coli. The highest antimicrobial activity was observed against B.subtilis, Staphylococcus aurous (MRSA), P. aeruginosa and E. coli. The highest antimicrobial activity was observed against B.subtilis, P. aeruginosa and E. coli, while a lower activity was found against MRSA. P. aeruginosa and E. coli, while a lower activity was found against MRSA. This difference can be related observed against B.subtilis, P. aeruginosa and E. coli, while a lower activity was found against MRSA. This difference can be relatedcomposition to the the structural structural and chemical composition of the the cell cellThese membrane of to thedifference structuralcan andbe chemical of theand cellchemical membrane of micro-organisms. findings This related to composition of membrane of micro-organisms. These findings are consistent with earlier studies that studied the antimicrobial are consistent with earlier studies that studied the antimicrobial activity of ZnO against B. subtilis, micro-organisms. These findings are consistent with earlier studies that studied the antimicrobial activity of ZnO ZnO against B. subtilis, P. aeruginosa aeruginosa [42] [42] and and E. E. coli coli [43,44]. [43,44]. P. aeruginosa [42]against and E. B. colisubtilis, [43,44].P. activity of
Figure 9. 9. Inhibition Inhibition zone zone of of synthesized synthesized ZnO-NPs ZnO-NPs against against (a) (a) B. B. subtilis; subtilis; (b) (b) MRSA; MRSA; (c) (c) P. P. aeruginosa aeruginosa Figure Figure 9.E.Inhibition zone of synthesized ZnO-NPs against (a) B. subtilis; (b) MRSA; (c) P. aeruginosa and and (d) coli pathogens. and (d) E. coli pathogens. (d) E. coli pathogens.
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Table 3. Mean inhibition zone (mm) of synthesized ZnO-NPs against different pathogens. Pathogens B. subtilis MRSA P. aeruginosa Molecules 2017, 22, 301 E. coli
ZnO-S
ZnO-P
ZnO-F
14.3 ± 0.51 6.8 ± 0.36 12.2 ± 0.27 13.4 ± 0.47
11.2 ± 0.25 6.9 ± 0.12 10.7 ± 0.49 11.8 ± 0.28
12.2 ± 0.24 6.8 ± 0.39 10.4 ± 0.49 10.8 ± 0.10
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3. Materials Materials and and Methods Methods 3. 3.1. Preparation of the C. colocynthis colocynthis Extracts Extracts 3.1. from Koohzar district, Khuzestan, IranIran in April. The Mature fruits fruits of of C. C.colocynthis colocynthiswere werecollected collected from Koohzar district, Khuzestan, in April. ◦ whole fruit were oven-dried at 45 °C for 48 h, and then fruit, seed and pulp separately milled to fine The whole fruit were oven-dried at 45 C for 48 h, and then fruit, seed and pulp separately milled powder. About 5About g from5each fine each powder dispersed 50 mL ofin distilled and magnetically to fine powder. g from finewas powder was in dispersed 50 mL water of distilled water and ◦ C. The stirred for 30stirred min atfor 10030°C. The extracts were filtered andfiltered centrifuged to eliminatetoany bulks any and magnetically min at 100 extracts were and centrifuged eliminate ◦ kept atand −20kept °C before bulks at −20use. C before use. 3.2. 3.2. Synthesis Synthesis of of ZnO-NPs ZnO-NPs In 6H22O O was was dissolved dissolved in in 22 mL mL distilled distilled water In aa typical typical synthesis, synthesis, 66 gg of of Zn(NO Zn(NO33))22 6H water in in aa porcelain porcelain crucible crucible and and then then thoroughly thoroughly mixed mixed with with 55 mL mL of of the the extract extract (fruit, (fruit, seed seed or or pulp) pulp) under under stirring stirring and and finally was placed under microwave irradiation at 340 W. After boiling and evaporating, the mixture finally was placed microwave irradiation W. After boiling and evaporating, quickly and released gases. The The whole process was completed in 8 mininand quickly foamed foamedup, up, and released gases. whole process was completed 8 the minZnO-NPs and the were left aswere residue (Figure 10). The ZnO-NPs samples synthesized by fruit, seed andseed pulpand extracts ZnO-NPs left as residue (Figure 10). The ZnO-NPs samples synthesized by fruit, pulp called ZnO-F, ZnO-S and ZnO-P, respectively. extracts called ZnO-F, ZnO-S and ZnO-P, respectively.
Figure 10. Photograph formation of (a) foam and prepared (b) ZnO-NPs. Figure 10. Photograph formation of (a) foam and prepared (b) ZnO-NPs.
3.3. Characterization of ZnO-NPs 3.3. Characterization of ZnO-NPs The crystal structure of ZnO-NPs were analyzed by PXRD (Philips, X’pert, Cu Kα) in the 2θ The crystal structure of ZnO-NPs were analyzed by PXRD (Philips, X’pert, Cu Kα) in the 2θ range between (2–80°) at room temperature. FTIR spectra of the samples were recorded by FTIR range between (2–80◦ ) at room temperature. FTIR spectra of the samples were recorded by FTIR −1 at wavenumber spectrometer (Perkin-Elmer 1725X, Waltham, MA, USA), with a resolution of 4.0 cm spectrometer (Perkin-Elmer−1 1725X, Waltham, MA, USA), with a resolution of 4.0 cm−1 at wavenumber range from 400 to 4000 cm . The UV-Visible absorption of powder samples were examined by UV-Vis range from 400 to 4000 cm−1 . The UV-Visible absorption of powder samples were examined by UV-Vis spectrophotometer (a Lambda 25-Perkin Elmer, Waltham, MA, USA) in the range of 200–800 nm. spectrophotometer (a Lambda 25-Perkin Elmer, Waltham, MA, USA) in the range of 200–800 nm. Transmission electron microscope (HITACHI H-700, Tokyo, Japan) with an acceleration voltage of Transmission electron microscope (HITACHI H-700, Tokyo, Japan) with an acceleration voltage of 120 kV was applied to study size and morphology of the ZnO-NPs. TEM samples were prepared by 120 kV was applied to study size and morphology of the ZnO-NPs. TEM samples were prepared depositing a few drops of the sample suspension on a copper grid followed with drying at room by depositing a few drops of the sample suspension on a copper grid followed with drying at room temperature. The morphology of samples were further observed using FESEM (JSM-6360LA, temperature. The morphology of samples were further observed using FESEM (JSM-6360LA, Eindhoven, Eindhoven, The Netherlands). The small amounts of powder samples were mounted on a metal stub The Netherlands). The small amounts of powder samples were mounted on a metal stub using carbon using carbon tape and then gold-coated using a sputter coater. The particle sizes and the zeta tape and then gold-coated using a sputter coater. The particle sizes and the zeta potentials of ZnO-NPs potentials of ZnO-NPs were analyzed by photon correlation spectroscopy and laser Doppler anemometry, respectively, with a Zetasizer, Nano ZS (Malvern Instruments Ltd., Malvern, UK) at room temperature. 3.4. MTT Assay for Cell Viability The in vitro cytotoxicity of ZnO-NPs samples was assessed by the method using
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were analyzed by photon correlation spectroscopy and laser Doppler anemometry, respectively, with a Zetasizer, Nano ZS (Malvern Instruments Ltd., Malvern, UK) at room temperature. 3.4. MTT Assay for Cell Viability The in vitro cytotoxicity of ZnO-NPs samples was assessed by the method using 3-(4,5-dimethylthiazol-2-yl)-2,5-dephenyl-tetrazolium bromide (MTT) assay. Briefly, 3T3 cells were seeded at a density of 2 × 105 cells/mL in 96-well microplates and incubated for 24 h. Subsequently, the cells were treated with the various concentrations of samples in the presence of 10% FBS for 24 h. The samples were suspended separately in a stock solution at 5 µg/mL in a solution of dimethyl sulfoxide (DMSO)/double distilled water. After 24 h of incubation, 20 µL of 5 mg/mL MTT in the PBS buffer was added to each well, and the cells were incubated for another 4 h at 37 ◦ C. The 100 µL of DMSO was added to dissolve the formazan crystal formed by live cells. Optical absorbance was measured at 570 nm. Cell viability was calculated as the percentage of absorbent compared to control. The 50% inhibitory concentration (IC50 ) value, defined as the amount of sample that inhibits 50% of cell growth, was calculated from the concentration–response curves. 3.5. DPPH Radical Scavenging Assay Bio-synthesized ZnO-NPs samples were assessed for the scavenging effect on DPPH radical according to the method of Blois [45]. Different concentrations (0.05–1 mg/mL) of the samples were added, in equal volume (3 mL), to 0.1 mM methanolic DPPH solution. The reaction mixture was incubated for 30 min at room temperature under vigorous shaking and the absorbance was recorded at 517 nm. Glutation (GSH) was used as control. All determinations were performed in triplicate. The DPPH radical scavenging activity (RSA) was expressed in percentage of inhibition using the following formula. ASample ]×100 (1) %RSA = [1 − AControl 3.6. Antimicrobial Assessment In the present study, in vitro antimicrobial activities of prepared ZnO samples towards gram-positive (Bacillus subtilis (B. subtilis) B29 and Methicillin-resistant Staphylococcus aurous (MRSA)) and gram-negative (Peseudomonas aeruginosa (P. aeruginosa) ATCC 15442 and Escherichia coli (E. coli) E266) pathogens were performed through a disc-diffusion assay. Briefly, the bacteria were grown overnight in nutrient broth. The bacterial inoculum was standardized to 0.5 MF units, meaning that approximately 108 colony-forming units of each bacterium were inoculated onto a plate. Previously prepared samples impregnated discs (6 mm) at the various concentrations were placed aseptically on plates inoculated with bacteria and incubated at 37 ◦ C for 24 h. After incubation, the zone of whole inhibition was measured. All tests were replicated three times. 4. Conclusions We have developed a simple, rapid and eco-friendly process to prepare ZnO nanocrystals using bio-fuels of fruit, seed and pulp extracts of C. colocynthis through microwave-assisted combustion. The bio-fuels could effectively mediate the nucleation and growth of ZnO nanoparticles under microwave radiation. Results reveal that the bio-fuels significantly affect the morphology of ZnO. Flower-like, hexagonal, and block-shaped nanostructures were produced by fruit, seed and pulp, respectively. The particle size of nanoparticles was varied between ~27 to ~85 nm. The ZnO samples with different shapes, sizes and chemical surfaces revealed some differences in biological activities. The as-synthesized nanoparticles with low toxicity exhibited desirable antioxidant and antimicrobial activities. This study showed that different parts of C. colocynthis fruit are suitable fuels to prepare ZnO-NPs and could be extended to synthesize other related metal oxide materials by microwave-assisted combustion.
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Acknowledgments: The authors are grateful to the Department of Bioprocess Technology, Faculty of Biotechnology and Biomolecular Sciences for the laboratory facilities. Author Contributions: Susan Azizi has designed, analyzed data and written the manuscript. Rosfarizan Mohamad has supervised research, analyzed data and edited the manuscript. Mahnaz Mahdavi Shahri has performed experiments and contributed in writing of the manuscript. Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are not available from the authors. © 2017 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
