Reactivity of Crystalline ZnO Superstructures against Fungi and

Article pubs.acs.org/crystal

Reactivity of Crystalline ZnO Superstructures against Fungi and Bacterial Pathogens: Synthesized Using Nerium oleander Leaf Extract T. R. Lakshmeesha,† M. K. Sateesh,† B. Daruka Prasad,‡ S. C. Sharma,§ D. Kavyashree,∥ M. Chandrasekhar,⊥ and H. Nagabhushana*,# †

Molecular Diagnostic Laboratory, Department of Microbiology & Biotechnology, Jnana Bharathi Campus, Bangalore University, Bangalore 560056, India ‡ Department of Physics, B.M.S. Institute of Technology, Bangalore 560 064, India § Chhattisgarh Swamy Vivekananda Technical University, North Park Avenue, Sector 8, Bhilai, Chhattisgarh 490 009, India ∥ Department of Physics, Channabasaveshwara Institute of Technology, Gubbi 572 216, India ⊥ Department of Physics, Acharya Institute of Technology, Bangalore 560 107, India # Prof. C.N.R. Rao Centre for Advanced Materials, Tumkur University, Tumkur 572 103, India ABSTRACT: For the first time, different morphologies of zinc oxide (ZnO) superstructures are synthesized by a simple and environmental friendly route using Nerium oleander leaf extract as fuel. Powder X-ray diffraction, scanning electron microscopy, UV−visible spectroscopy, and photoluminescence studies are performed to ascertain the formation and characterization of ZnO. X-ray diffraction confirmed the crystalline nature of the compound with hexagonal Wurtzite structure. When the concentration of the leaf extract is varied, different morphologies are formed. ZnO are tested for antifungal using soybean seed-borne fungi by food-poison method and antibacterial activity against bacterial human pathogens by a broth microplate dilution method using 96-well plates. Among the screened soybean seed-borne fungi, Fusarium equisiti was found to be more susceptible, which was followed by Macrophomina phaseolina for ZnO nanoparticles (NPs) prepared using 0.2188 mol/dm3 N. oleander leaf extract. It was observed that NPs exhibited pronounced antifungal activity in a dose-dependent manner with a relatively high percentage of mycelial inhibition. ZnO obtained with the concentration of 0.2188 mol/dm3 leaf extract showed both minimum inhibitory concentration and minimum bactericidal concentration effectiveness compared to other synthesized compounds. It is observed that the samples with small crystallite size show greater antibacterial activity than those of larger crystallite size. Further, we found that crystallite size and morphology significantly affects the antibacterial activity of ZnO. Prepared compounds showed significant inhibition against Escherichia coli, Staphylococcus aureus, Bacillus subtilis, and Pseudomonas aeurginosa. Among the tested bacteria, P. aeurginosa is more susceptible and E. coli is the least effective against bacterial pathogens. The antibacterial activities of the as-formed ZnO are preliminarily studied against Gram-positive (B. subtilis and S. aureus) and Gram-negative (E. coli and P. aeruginosa) bacteria and are found to be dependent on the shape of the nanostructures.

1. INTRODUCTION Microbial contamination is a serious matter in the healthcare and food industry, so that growth of antimicrobial agents and surface coatings has been gaining attention in recent years.1−3 It has been well documented that toxicity is more common in the case of microsized particles. Therefore, antimicrobial properties with nanosized particles are of considerable interest.4−6 The morphology-controlled synthesis of inorganic oxides has recently been extensively investigated due to their catalytic, sensors, and solar cells applications.7−9 In recent studies, zinc oxide nanoparticles with superstructures have demonstrated antimicrobial efficacy against microbes, which significantly have low toxicity and low cost and are ecofriendly compared to conventional synthesis methods.10−13 Because of the larger surface to volume ratio in nanosized particles, the antibacterial activity is much stronger than that of micrometer-sized ZnO particles. © 2014 American Chemical Society

Further, generation of hydrogen peroxide (H2O2) from the surface of ZnO is an effective means for the inhibition of bacterial growth.14 The antibacterial properties mainly depend on the morphologies, i.e., on their surface atomic arrangements. The specific atomic arrangement on the surface can be selected by finetuning the morphology of the inorganic oxide. Morphologycontrolled synthesis of inorganic oxides can thus be developed by simply adjusting the synthesis conditions and examining the resulting morphologies.15 Biological entities in synthesis of nanoparticles may vary from simple prokaryotic bacteria to eukaryotes such as fungi and plants. Compared to microorganisms, plants Received: May 12, 2014 Revised: June 20, 2014 Published: June 26, 2014 4068

dx.doi.org/10.1021/cg500699z | Cryst. Growth Des. 2014, 14, 4068−4079

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have better advantages wherein plant-mediated synthesis is a one-step protocol towards synthesis, whereas microorganisms

during the course of time may lose their ability to synthesize nanoparticles due to mutations. In addition, preservation of microorganisms and maintenance of cultures in active form are very laborious and time-consuming.16−18 Therefore, the search for cleaner methods of synthesis has ushered in the development of bioinspired approaches. Bioinspired methods are advantageous compared to other synthetic methods as they are economical and restrict the use of toxic chemicals as well as high pressure, energy, and temperatures.19−21 ZnO is one of the most important semiconducting materials with unique properties and versatile applications. ZnO has a stable wurtzite structure consisting of a number of planes composed of tetrahedrally coordinated O2− and Zn2+ ions, which are stacked alternately along the c-axis. Because of its noncentral symmetry, ZnO is piezoelectric, which is a key property in building electromechanical coupled sensors and transducers.22 ZnO is an environmental friendly material and biocompatible which is desirable especially for biomedical applications.23 Further, it has wide direct band gap (3.37 eV) and large exciton binding energy (60 meV) at room temperature as a result ZnO is a promising material for short wavelength optoelectronic devices, self-powered nanosystems, sensors, ultraviolet lasers, and field-emission devices.24−26

Figure 1. Flowchart for the preparation of ZnO SS.

Figure 2. Mechanism of ZnO SS stabilization from (a) ascorbic and (b) gallic acids of N. oleander leaf extract. 4069



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Figure 3. PXRD patterns of ZnO SS prepared using various concentrations of N. oleander leaf extract.

Table 1. Various Structural Parameters of ZnO SS Compound strain (10−3)

D (nm) concentration of N. oleander leaf to obtain ZnO SS (mol/dm )

Scherrer’s

W−H plots

W−H plots

ε

Dx (gm cm−3)

a(311) (Å)

δ (1014 lin m−2)

0.0438 0.0875 0.1313 0.1750 0.2188 0.3282 0.4376 0.5470

29 27 21 14 11 15 18 17

36 31 27 19 13 17 24 21

0.86 0.97 0.91 1.78 1.57 3.02 3.10 0.89

0.86 0.91 0.95 1.79 1.59 3.23 2.48 0.97

5.602 5.597 5.596 5.594 5.593 5.591 5.607 5.590

3.247 3.249 3.242 3.245 3.241 3.243 3.241 3.239

6.92 11.89 17.36 34.60 51.02 138.4 67.47 27.13

3

The medicinal plant Nerium oleander (N. oleander) is an evergreen shrub that belongs to family Apocynaceae and is native to Mauritania, Morocco, and Portugal. It is widely used in traditional medicine for its cardiotonic, emetic, anti-inflammatory, antibacterial, antifungal, etc. properties. The latex of the leaves and stem is toxic to livestock due to the presence of cardenolides, triterpenoids, and arabinogalactan.27 N. oleander leaf mainly contains phenolic contents with gallic acid, ascorbic acid (vitamin C), cardiac glycoside, minerals, α-tocopherol, glucose, paraffin, ursolic acid, and essential oil.28 Among them, the role of ascorbic, cardiac glycoside, and gallic acid counterbalance the forces to obtain the superstructures of ZnO compound as shown in Figure 2. A similar kind of stabilization mechanism can be explained with other contents of N. oleander leaf extract. According to the egg-box model, the ZnO that gets trapped and stabilized in the polymerization network of organic chains of N. oleander leaf extract leads to the superstructures of ZnO. To the best of our knowledge, for the first time we report the synthesis of ZnO superstructures (SS) from N. oleander leaves extract. Further, antifungal properties using soybean seed-borne fungi by the food-poison method and antibacterial activity against bacterial human pathogens by the broth

microplate dilution method using 96-well plates are studied and discussed in detail.

2. MATERIALS AND METHODS 2.1. Collection of Plant Materials. Fresh leaves of N. oleander leaves, free from disease, were collected early in the morning during the month of August/September 2013 from Dhanvantrivana (12056139.2811N; 77030143.9111E), Bangalore University, Bangalore. The voucher specimen number (MBMDL84) was assigned and housed in the Molecular Diagnostic Laboratory, Department of Microbiology and Biotechnology, Bangalore, Karnataka, India. The collected leaves were washed thoroughly 2−3 times with running tap water and sterilized with double-distilled water. Leaf material is then air-dried on sterile blotter paper under shade for 7 days and pulverized using a sterile electric blender to obtain a fine powdered form and stored in sterile polyethylene bags before its use.29 2.2. Synthesis of Zinc Oxide. ZnO SSs were prepared by ecofriendly green combustion route using N. oleander leaf extract as a fuel. The zinc nitrate hexahydrate (Zn(NO3)2·6H2O) used in this experiment was procured from Sigma-Aldrich analytical grade and used without further purification. Stoichiometric amounts of Zn (NO3)2· 6H2O were dissolved in 10 mL of double distilled water. To this reaction mixture, volumes ranging from 2, 4, 6, 8, 10, 15, 20, and 25 mL of plant extract are added and mixed well using a magnetic stirrer for 4070



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mined as the lowest concentration of the drug that prevented the color change from colorless to red. The colorless tetrazolium salt acts as an electron acceptor and is reduced to a red-colored formazan product by biologically active organisms. The lowest concentration at which no visible bacterial growth observed was considered as MIC. For the determination of MBC, 50 μL of cultured broth (without INT) was transferred onto the Mueller Hinton (MH) agar and incubated for 24 h at 37 °C. The lowest concentration which showed complete absence of the growth on the agar surface was defined as the MBC (Figure 9). 2.4. Antifungal Activity. The antifungal activities of the synthesized ZnO SS counterpoise by N. oleander leaf extract is determined using soybean seed-borne fungal pathogen Macrophomina phaseolina and Fusarium equisiti by the food-poison method with minor modifications.32 The autoclaved Sabouraud dextrose agar (SDA) media with ZnO SS at concentrations of 500, 400, 300, and 200 μg/ mL and a ZnO SS-free solution are poured into the Petri dishes (9 cm diameter). A disc of seven-day old culture, punched aseptically with a sterile cork borer of 5 mm diameter, was placed in the center of Petri dish. The plates were then incubated at 26 ± 1 °C. Antifungal index is calculated as mentioned below.33 percent inhibition =

dc − dt × 100 dc

(1)

where dc is the average increase in mycelia growth in control, and dt is the average increase in mycelia growth in treatment. 2.5. Statistical Analysis. The antifungal experimental data is analyzed by mean ± SE subjected to univariate analysis. Means are separated by Tukey’s multiple range test at 0.5 significant (P < 0.05) using SPSS software (version 19).

3. CHARACTERIZATION The phase purity and the crystallinity of the superstructures are examined by powder X-ray diffractometer (Shimadzu) using Cu Kα (1.5418 Å) radiation with a nickel filter. The morphology of the products is examined by SEM (Hitachi Table top TM-3000). The UV−visible absorption of the samples is recorded on SL 159 ELICO UV−visible spectrophotometer. The transmission electron microscopy (TEM), high resolution transmission microscopy (HRTEM), and selected area electron diffraction (SAED) studies were carried out using JEOL JEM 2100 HRTEM. The photoluminescence (PL) measurements were performed on a Horiba, fluorolog-3 spectrofluorometer.

Figure 4. Rietveld refinement for (a) (0.0438 mol/dm3) and (b) (0.547 mol/dm3) N. oleander leaf extract. ∼5−10 min. With the above volume of plant extract, the obtained concentration of active agents are 0.0438, 0.0875, 0.1313, 0.175, 0.2188, 0.3282, 0.4376, and 0.547 mol/dm3 for 2, 4, 8, 10, 15, 20, and 25 mL respectively. During this process, the resulting mixture is then placed in preheated muffle furnace maintained at 400 ± 10 °C, where the reaction mixture boils, froths, and thermally dehydrates forming foam in less than 3 min. The product is further calcined at 700 °C for 2 h and used for structural, antibacterial, and antifungal activities. The flowchart used for the preparation of ZnO nanostructures is shown in Figure 1. The particle size and structures are dependent on the kinetics of nucleation and growth from a supersaturated solution as well as processes such as coarsening, oriented attachment, and aggregation. 2.3. Antibacterial Activity. The test organisms Bacillus subtilis (MTCC 121), Staphylococcus aureus (MTCC 9760), Escherichia coli (MTCC 443), and Pseudomonas aeruginosa (MTCC 424) were procured from Institute of Microbial Technology (IMTECH), Chandigarh, India. The antibacterial activity, minimum inhibitory concentrations (MIC), and minimum bactericidal (MBC) of the synthesized ZnO nanoparticles (NPs) were determined by broth microdilution method30 with minor modifications. Briefly, the stock solution of ZnO NPs was serially 2-fold diluted in Mueller Hinton (MH) broth medium to obtain desired different concentrations (500 to 3.90 μg/mL) and was added separately to the wells of a sterile 96-well microtiter plate. Twenty microliters of the bacterial suspension (108 CFU/mL) was added to each well and incubated at 37 °C for 24 h. ZnO NPs-free solution was used as a blank control. Tetracycline at the concentration of 30 μg/mL was used as a positive control.31 All the tests were performed in triplicate. After a 24 h incubation period, the MIC values of the compounds were detected by the addition of 50 μL of iodonitrotetrazolium chloride (INT) (0.5 mg/mL) in each well; the samples were further incubated at 37 °C for 60 min. MICs were deter-

4. RESULTS AND DISCUSSION The pictorial form of mechanism of formation of superstructures is shown in Figure 2. PXRD patterns of ZnO SS synthesized for different leaf extract concentrations (0.0438− 0.547 mol/dm3) are shown in Figure 3. As indicated all of the diffraction patterns can be well indexed to pure hexagonal wurtzite structured ZnO (space group P63mc) with lattice constants close to the standard values, a = b = 3.25 Å and c = 5.25 Å, and it is well matched to JCPDS no. 89-7102. The diffraction peaks at 2θ = 31.82, 34.47, 36.30, 47.59, 56.63, 62.90, 66.41, 68.00, and 69.29 correspond to (100), (002), (101), (102), (110), (103), (200), (112), and (201) respectively. The results indicate that the product consists of pure phase, and no characteristic peaks appear either from other phases or impurities. The crystallite size and lattice strain can be extracted from X-ray peak width analysis. Crystallite size is a measure of the size of coherently diffracting domains. Lattice strain is a measure of the distribution of lattice constants arising from crystal imperfections such as lattice dislocations, contact or sinter stresses, stacking faults, and coherency stresses. Crystallite size and lattice strain affect the X-ray diffraction peak in different ways. 4071


0.33330 0.66666 0.38381 0.90614

x y z occupancy

5.718

5.716

4072

0.33330 0.66666 0.38399 0.89696

0.33330 0.66666 0.00000 0.88683

c = 5.1966 0.405 0.64 2.29 1.64 2.94 4.62 47.3764

c = 5.2002 0.492 0.70 3.06 4.00 5.80 5.74 47.4739

0.33330 0.66666 0.00000 0.88810

a = 3.2446

ZnO (N. oleander leaf extract of 0.875 mol/dm3)

a = 3.2468

x y z occupancy

χ2 GOF Rp RBragg RWP Rexp volume of unit cell/ formula unit (Å3) X-ray density (g/cm3)

crystal structure space group hall symbol lattice parameters (Å)


0.33330 0.66666 0.38790 0.72684

0.33330 0.66666 0.00000 0.75362

5.624

c = 5.2093 0.287 0.53 2.00 2.52 2.62 4.89 47.720

c = 5.2087 0.390 0.62 2.35 6.15 3.09 4.84 47.7122 5.701

a = 3.2523


a = 3.2523

Position Parameters and Occupancy State for Zn2+ Ions 0.33330 0.33330 0.33330 0.66666 0.66666 0.66666 0.00000 0.00000 0.00000 0.95939 0.89527 0.91125 Position Parameters and Occupancy State for O2− Ions 0.33330 0.33330 0.33330 0.66666 0.66666 0.66666 0.38397 0.38520 0.38616 0.93702 0.87176 0.94072

5.642

c = 5.2072 0.299 0.54 2.03 2.42 2.56 4.68 47.6578

c = 5.2097 0.398 0.63 1.35 1.82 1.74 2.76 47.7239 5.637

a = 3.2509


hexagonal P63mc (186) P6c2̅c


a = 3.2523


Table 2. Rietveld Refinement Data of ZnO Extracted from N. oleander Leaf Extracts: JCPDS No. 89-7102

0.33330 0.66666 0.38667 0.86241

0.33330 0.66666 0.00000 0.87249

0.33330 0.66666 0.38803 0.84984

0.33330 0.66666 0.00000 1.00204

5.503

c = 5.2070 0.303 0.55 2.51 2.78 3.25 5.90 47.6440

c = 5.2066 0.319 0.56 2.74 3.56 3.45 6.00 47.6189 5.663

a = 3.2504


a = 3.2497


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Figure 5. SEM images of the ZnO superstructures synthesized using N. oleander leaf extract with different concentrations (a) 0.0438 mol/dm3, (b) 0.0875 mol/dm3, (c) 0.1313 mol/dm3, (d) 0.175 mol/dm3, (e) 0.2188 mol/dm3, (f) 0.3282 mol/dm3, (g) 0.4376 mol/dm3, and (h) 0.547 mol/dm3 of N. oleander leaf extract.

performed by the Williamson-Hall (W−H) plot method.36 W−H plots may be expressed in the form

Both of these effects increase the peak width and intensity accordingly.34 On the full width at half-maximum (fwhm) of (100), (002), and (101) diffraction peaks, the crystallite sizes of ZnO SS are calculated from Debye−Scherrer’s equation. The lattice parameters and unit cell volumes for hexagonal ZnO compounds are calculated from the lattice geometry equations.35 1 4 ⎛ h2 + hk + k 2 ⎞ l2 = + ⎜ ⎟ 3⎝ ⎠ c2 d2 a2 V=

3 a 2c = 0.866a 2c 2

β cos θ =

kλ + 4ε sin θ D

where ε is the strain associated with the nanoparticles. The detailed description of W−H plot is discussed elsewhere37 Further, the X-ray density (Dx), dislocation density (δ), and microstrain (ε) are estimated by the relations

(2)

Dx = (3)

β cos θ 16M 1 , δ = 2, ε = 2 4 Na c D

where M is molecular mass, N is Avogadro’s number (6.0223 × 1023 particles mol−1), a and c are the lattice constants, (h k l) is Miller indices, and θ is Bragg’s angle. The estimated values of crystallite size, Dx, δ, ε are also tabulated in Table 1. It is evident from the table that a small variation in the crystallite size is observed when compared to Scherrer’s equation. Figure 5a−j shows the SEM micrographs of the prepared ZnO powder with different concentrations (0.0438−0.547 mol/dm3) of N. oleander leaf extract. From SEM micrographs, it is observed that the morphology dramatically depends on the concentration of the leaf extract. When the concentration of the extract is 0.0438 mol/dm3, the as-synthesized ZnO powders consisted of microparticles with a diameter of ∼12 μm. Many bell-shaped with approximately hexagonal-shaped particles are observed, and further the shape and patterns can be compared with sedimentary rock (Figure 5a). When the concentration of the extract is ∼0.0875 mol/dm3 and 0.1313 mol/dm3, each particle is analogues to a split twinned hemisphere. The patterns on the

where a and c are the lattice parameters, (hkl) is the Miller indices, dhkl is the interplanar spacing, which can be calculated from Bragg’s law. The estimated average crystallite size of ZnO for each N. oleander leaf extract concentration is given in Table 1. The PXRD patterns of all the compositions were subjected to the statistical validate by Rietveld refinement technique using Fullprof suite 2.05. The background was successfully fitted with a Chebyshev function (χ2) with a variable number of coefficients depending on its complexity and found good match. The refined fitment and the corresponding detailed outputs of the refinement are shown in Figure 4 and tabulated in Table 2 respectively. It was found that the occupancy of ions, cell volume, and X-ray densities were better optimized nearly to the standard values for ZnO stabilized with 10 mL of N. oleander leaf extract. The analysis of the random microstrain and crystallite size broadening effect on the diffraction maxima of the ZnO SS is 4073



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concentration, particles are almost converted to flaky flower bud morphology. With the concentration of the gel of 0.175 mol/dm3, 0.2188 mol/dm3, and 0.3282 mol/dm3, orchid, rose, and peony flower bud-like microstructures are obtained (Figure 5d−f). With a further increase in the concentration of the gel, many core−shell-like particles aggregated together, and an urchin morphology was obtained with each part resembling the part of babaco (Figure 5g). With a further increase in gel concentration to 0.547 mol/dm3 hard agglomerations with orange brown hexagonal diamond structures were obtained (Figure 5h). From these results, it is apparent that adjusting the concentration of the gel, significantly affected the formation of ZnO superstructures. Further, the uniformity of the morphology has been observed at higher volumes of plant leaf extract. The obtained ZnO morphology through solution combustion is due to the presence of soluble organic compounds particularly the vitamin C and gallic acid present in the leaf extract. Nature prefers the least utilization of energy in any structures. A similar type of mechanism is initiated in obtaining these ZnO superstructures. TEM, HRTEM images, and SAED patterns as shown in Figure 6A−C correspond to three ZnO compounds obtained for different N. oleander concentrations. The TEM image shows the agglomerated small particles of ZnO prepared using 0.0438, 0.2188, 0.4376 mol/dm3 of N. oleander leaf extract. The high resolution TEM image shows the well-defined crystal planes with an average spacing of ∼0.35 nm, ∼0.25 nm, and ∼0.21 nm respectively for the above-mentioned three compositions. There is a decrease in the interplanar spacing with more ordered superstructures of ZnO. The SAED patterns are well matched with the (hkl) values corresponding to the prominent peaks of the PXRD profiles. Further, it explains that the crystallinity of the compounds is increased with an increase in the N. oleander content. The in-depth details of the porosity and surface activity of the compounds can be studied from the BET surface area measurement. PL study is an effective method to evaluate defect characteristics and optical properties. Figure 7a shows an excitation spectrum of ZnO prepared using N. oleander leaf extract. Figure 7b shows the PL spectra of the ZnO superstructures are measured with an excitation wavelength of 376 nm at room temperature. A broad green emission is observed at ∼501 nm along with weak emission peak at 547 nm for all the samples. The green luminescence from the ZnO is associated with the intrinsic defect centers such as oxygen vacancy (VO), zinc vacancy (VZn), zinc interstitial (Zni), oxygen interstitial (Oi), or antisite oxygen (OZn).38−40 Though the origin of the green emission generally refers to the deep level or trapped state emission, there is no universally accepted mechanism. There are many proposed models to explain the emission from defect structure in the ZnO crystals. The commonly cited reason is that the green band emission originates from the singly ionized oxygen vacancy in ZnO, and the emission results from the recombination of a photogenerated hole with the singly ionized charge state of this defect.41 Vanheusden et al.42 proposed that the singly ionized VO center is the main cause for green emission of ZnO crystal, whereas Lima43 suggested that it might be ascribed to the lowest lattice microstrain. Dijken et al.44 reported that the green luminescence might be due to the transition from the conduction band to the deeply trapped hole. The donor−acceptor transitions are also reported45 as the origin of green emission. UV−visible absorption spectra (Figure 8a) and their corresponding optical energy band gap (Figure 8b) of ZnO superstructures show a strong absorption peak at 353 nm. It is known that bulk

Figure 6. TEM, HRTEM images, and SAED patterns for ZnO superstructures prepared using (A) 0.0438 mol/dm3, (B) 0.2188 mol/dm3, and (C) 0.4376 mol/dm3 N. oleander leaf extract.

structures are similar to martian sedimentary rock or coral reef structures (Figure 5b,c). Further, with the increase of leaf extract 4074



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Figure 7. (a) PL excitation spectrum with 501 nm emission and (b) PL emission spectra of ZnO obtained for various N. oleander leaf extract concentration (excited at 376 nm).

Figure 8. (a) UV−vis spectra and (b) energy band gap of ZnO superstructure of all the samples.

where α is the absorbance, h is the Planck constant, ν is the frequency, Eg is the optical band gap, and k is a constant associated with the different types of electronic transitions (k = 1/2, 2, 3/2, or 3 for direct allowed, indirect allowed, direct forbidden and indirect forbidden transitions, respectively). According to the literature,48 oxides are characterized by a direct allowed electronic transition, and hence the k = 2 value is adopted. Thus,

ZnO has absorption peak at 369 nm. The observed blue shift in ZnO superstructures may be due to a quantum size effect.46 The optical band gap energy (Eg) is estimated by the method proposed by Wood and Tauc.47 The optical band gap is associated with absorbance and photon energy by the following equation: (hvα) α(hν − Egopt)k

(4) 4075



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Figure 9. Photograph showing MIC and MBC values of different concentration of ZnO SS treated to (A) E. coli, (B) S. aureus, (C) B. subtilis, and (D) P. aeurginosa.

the Eg values are evaluated extrapolating the linear portion of the curve or tail [(hνα)1/k = 0)] (Figure 8B). The direct band gap is found to vary from 3.1 to 3.41 eV, which are well matched to reported literature.49 With the increase in the concentration of the N. oleander leaf extract, there is a slight red shift is observed and a similar kind of slight shift is observed in the PL intensity curves also (Figure 7B) . The mechanism of formation of the ZnO superstructures involves the cationic interactions with polymer chains of N. oleander extract leading to the cross-link between two adjacent polymer chains. In this case the Zn2+ cations can associate to form aggregates of the “egg-box” model of ion binding.50,51 In this model, arrangements of cations into electronegative cavities were considered as similar to the eggs in an egg-box. Within the domains, the Zn2+ cations form intermolecular bonds via two hydroxyl groups of one chain to two deprotonated carboxylate

groups of another chain. The formation of superstructures can be related to the interactions between reducing phenolic acids such as ascorbic, cardiac glycoside, gallic acid, and zinc ions. The probable reason for the change in the morphology of ZnO with the increase in concentration of N. oleander leaf extract is mainly due to increased active components such as phenolic acids. By acting like surfactants, these contents control the nucleation mechanism of ZnO leading to the controlled growth of the ZnO superstructures. The exact mechanism between the contents of any plant extract with metal ions leading to the superstructures needs more intensive research activities.52,53 Figure 9 shows the antibacterial effects of ZnO SS against human pathogens, Gram-positive, and Gram-negative bacteria using a broth microplate dilution assay (96-well plates). Exponentially grown bacteria are incubated with different concentrations of ZnO SS for 24 h. Bacteria grown without ZnO SS 4076



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factors as the lattice constant. That is, the generation of active oxygen species generated from crystalline materials may show a correlation with crystallographic factors.55 Crystallite size is one of the crystallographic factors associated with the formation of dislocations and point defects in the crystalline structure, which influences the material properties of crystalline ceramics. Among the different ZnO SSs obtained for N. oleander leaf extract concentrations, ZnO SS obtained with 10 mL concentration has shown better results of both MIC and MBC effective against all tested bacteria (Table 3). The reason for this may be the production of highly reactive oxygen species, namely, OH−, H2O2, and O22−. In ZnO SS, most of the defects can be activated by both UV and visible light; as a result electron−hole pairs (e−h+) can be created. The holes split H2O molecules from ZnO into OH− and H+. Dissolved oxygen molecules are transformed to superoxide radical anions (●O2−), which in turn react with H+ to generate (H2O2●) radicals, which upon successive collision with electrons create hydrogen peroxide anions (H2O2−). Then they react with hydrogen ions to generate molecules H2O2.56 The generated H2O2 molecules can penetrate the cell membrane and kill the bacteria. Because, the hydroxyl radicals and superoxides are negatively charged particles, they cannot penetrate into the cell membrane and must stay in direct contact with the outer surface of the bacteria; however, H2O2 can penetrate into the cell. Further, the reason for increasing the antibacterial activity with increasing concentration of ZnO SS is assumed due to the increase of H2O2 concentration from the surface of ZnO. Generally a smaller particle size of metal oxide nanoparticles is correlated with a larger band gap and consequently unfavorable conditions for recombination of excitons. Consequently more available excitons would lead to the generation of a higher concentration of reactive oxygen species (ROS) and subsequently to enhanced antibacterial activity. In addition, smaller sized nanoparticles are thought to be more efficient in causing abrasive action on the cell wall and might cause membrane damage.57 In this regard, synthesis of stable ZnO SS with small-sized particles would be appropriate to study the antibacterial activity. The antifungal effect of ZnO SS at different concentrations (200, 300, 400, and 500 μg/mL) is tested against soybean seedborne M. phaseolina and F. equisiti based on the food-poison method. The mycelial inhibition is shown in Figure 10, and

Table 3. MIC and MBC of ZnO SS against Gram-Positive Bacillus subtilis and Staphylococcus aureus, Gram-negative Escherichia coli, and Pseudomonas aeruginosa expressed as μg/mL organisms μg/mL concentration of leaf extract (mol/dm3) 0.0438 0.0875 0.1313 0.1750 0.2188 0.3282 0.4376 0.5470

MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC

E. coli

S. aureus

B. subtilis

P. aeurginosa

250 500 62.5 250 31.3 250 125 500 15.6 62.5 62.5 125 62.5 250 125 500

125 500 31.3 125 15.6 125 31.3 125 31.3 125 31.3 125 62.5 125 31.3 125

62.5 125 31.3 250 15.6 125 31.3 62.5 313 125 62.5 250 31.3 125 62.5 125

125 250 31.3 125 15.6 31.3 15.6 62.5 15.6 31.3 62.5 125 62.5 250 31.3 62.5

served as a control. The MIC and MBC values of all prepared ZnO SS samples against bacteria are shown in Table 3. All ZnO SS showed significant inhibition against E. coli, S. aureus, B. subtilis, and P. aeurginosa, with distinct differences in the susceptibility to ZnO SS in a dose-dependent manner. Among bacteria, P. aeurginosa is found to be more susceptible to ZnO SS. In contrast, E. coli is found to be least effective. The mechanism of ZnO SS is not fully understood; the discrepancy in activity among the Gram-positive and Gram-negative could be due to different cell wall compositions and the membrane structure. Liu et al.54 reported that ZnO SS leads to damage of the cell wall due the generation of highly reactive species such as H2O2. Further, the antibacterial activity of ZnO SS toward E. coli and S. aureus depends on particle size, powder concentration, morphology, specific surface area, etc. Furthermore, extensive research on antibacterial activity of crystalline ZnO SS has shown that the activity is dependent on crystallographic

Figure 10. Inhibitory effect of ZnO superstructures (0.2188 mol/dm3) on mycelial growth against soybean seed-borne 1 (Macrophomina phaseolina) and 2 (Fusarium equisiti) [C = control, A = 200 μg/mL, B = 300 μg/mL, D = 400 μg/mL, E = 500 μg/mL]. 4077



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Table 4. Effect of Dose-Dependent ZnO Superstructures (0.2188 mol/dm3) on Mycelial Growth of Macrophomina phaseolina and Fusarium equisitia concentration μg/mL organisms

200

300

400

500

negative control

positive control

Macrophomina phaseolina Fusarium equisiti

30.66 ± 0.0.50a 38.05 ± 0.0.07a

44.83 ± 0.0.21b 42.56 ± 0.0.17b

57.88 ± 0.0.11c 64.51 ± 0.0.23c

62.61 ± 0.0.17d 76.47 ± 0.0.19d

0 0

100 100

The above-mentioned readings are exclusive of the disc diameter. Observations are expressed as mean ± standard error, (n = 3). The values followed by different alphabets differ significantly when subjected to Tukey HSD (row by row analysis) P value ≤0.05.

a

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS H.N. thanks DST Nano Mission (Project No. SR/NM/NS-48/ 2010) New Delhi for sanctioning of the project.

(1) Tam, K. H.; Djurisic, A. B.; Chan, C. M. N.; Xi, Y. Y.; Tse, C. W.; Leung, Y. H.; Chan, W. K.; Leung, F. C. C.; Au, D. W. T. Thin Solid Films 2008, 516, 6167. (2) Bintsis, E.; Litopoulou-Tzanetaki; Robinson, R. K. J. Sci. Food Agric. 2000, 90, 637. (3) Brayner, R.; Ferrari-Lliou, R.; Brivois, N.; Djediat, S.; Bebedetti, M. F.; Fievet, F. Nano Lett. 2006, 6, 866. (4) Liu, Y.; He, L.; Mustapha, A.; Li, H.; Hu, Z. Q.; Lin, M. J. Appl. Microbiol. 2009, 107, 1193. (5) Emami-Karvani, Z.; Chehrazi, P. Afr. J. Microbiol. Res. 2011, 5, 1368. (6) Xie, Y.; He, P. L.; Irwin, T.; Jin, X.; Shi. Appl. Environ. Microbiol. 2011, 77, 2325. (7) Pivin, J. C.; Socol, G.; Mihailescu, I.; Berthet, P.; Singh, F.; Patel, M. K. Thin Solid Films 2008, 517, 916. (8) Sato, K.; Katayama-Yoshida, H. Jpn. J. Appl. Phys. 2000, 39, 555. (9) Wei, A.; Pan, L. H.; Huang, W. Mater. Sci. Eng., B 2011, 176, 1409. (10) Jayaseelan, C.; Rahuman, A. A.; Kirthi, A. V.; Marimuthu, S.; Santhoshkumar, T.; Bagavan, A.; Gaurav, K.; Karthik, L.; Rao, K. V. B. Spectrochim. Acta Part A 2012, 90, 78. (11) Rajiv, P.; Sivaraj Rajeshwari, S.; Venckatesh, R. Spectrochim. Acta Part A 2013, 112, 384. (12) Jalal, R.; Goharshadi, E. K.; Abareshi, M.; Moosavi, M.; Yousefi, A.; Nancarrow, P. Mater. Chem. Phys. 2010, 121, 198. (13) Zhang, L.; Jiang, Y.; Ding, Y.; Daskalakis, N.; Jeuken, J.; Povey, M.; O’Neill, A. J.; York, D. W. J. Nanopart. Res. 2010, 12, 1625. (14) Premanathan, M.; Karthikeyan, K.; Jeyasubramanian, K.; Manivannan, G. Nanotech. Biol. Med. 2011, 7, 184. (15) Ramania, M.; Ponnusamy, S.; Muthamizhchelvan, C.; Cullen, J.; Krishnamurthy, S.; Marsili, E. Colloids Surf., B 2013, 105, 24. (16) Darroudi, B. N. M.; Sabouri, Z.; Kazemi Oskueeb, R.; Khorsand, Z. A.; Hamid, M. H. N. A. Ceram. Int. 2014, 40, 4827. (17) Samat, N. A.; Nor, R. M. Ceram. Int. 2013, 39, 545. (18) Sangeetha, G.; Rajeshwari, S.; Venkatesh, R. Mater. Res. Bull. 2011, 46, 2560. (19) Singh, R. P.; Shukla, V. K.; Yadav, R. S.; Sharma, P. K.; Singh, P. K.; Pandey, A. C. Adv. Mater. Lett. 2011, 2, 313. (20) Darroudi, M.; Sabouri, Z.; Kazemi Oskuee, R.; Khorsand Zak, A.; Kargar, H.; Hamid, M. H. N. A. Ceram. Int. 2013, 39, 9195. (21) Darroudi, M.; Ahmad, M. B.; Zamiri, R.; Zak, A. K.; Abdullah, A. H.; Ibrahim, N. A. Int. J. Nanomed. 2011, 6, 677. (22) Jin, D.-H.; Kim, D.; Seo, Y.; Park, H.; Huh, Y.-D. Mater. Lett. 2014, 115, 205. (23) Wang, Z. L. Mater. Sci. Eng. R: Rep. 2009, 64, 33. (24) An, K.; Hyeon, T. Nano Today 2009, 4, 359. (25) Zhang, J.; Wang, S.; Wang, Y.; Xu, M.; Xia, H.; Zhang, S.; Huang, W.; Guo, X.; Wu, S. Sens. Actuators, B 2009, 139, 411. (26) Lou, X. W.; Archer, L. A.; Yang, Z. Adv. Mater. 2008, 20, 3987.

Figure 11. Dose-dependent inhibition of fungi incubated with different concentrations of ZnO SS; the data are shown as mean ± standard error.

corresponding inhibition values are given in Table 4. The column graph (Figure 11) shows that all the concentrations show moderate mycelial inhibition in a dose-dependent manner against both fungi. M. phaseolina is less susceptible to ZnO SS compared to F. equisiti evaluated in this study. The results obtained indicate that ZnO SS are quite a useful and effective agent for the control of bacterial and fungal pathogens, which will be more specific and cost-effective. Moreover, it is worthwhile to conduct a detailed mechanism of inhibition studies of both bacteria and fungi.

5. CONCLUSIONS In conclusion, this paper reports leaves extract as a biotemplating/ capping agent for the green synthesis of ZnO superstructures. Further, this method offers several advantages, namely, a low cost, ecofriendly, nontoxic, simple and quick process, etc. It is evident from SEM studies that the plant extract is highly efficient in controlling the shape and size of ZnO. The photoluminescence band in the visible luminescence range resulting from the higher surface interstitial defects reduces the electrons or holes recombination and consequently increases the antibacterial activities. Antibacterial tests indicated that the P. aeurginosa showed more susceptibility and E. coli was the least effective against bacterial pathogens. Among the different ZnO obtained for leaf extract concentrations, ZnO obtained with 0.2188 mol/dm3 concentration showed better results of both minimum inhibitory concentration and minimum bactericidal concentrations effective against all tested bacteria. The antifungal effect was tested against soybean-seed borne M. phaseolina and F. equisiti. M. phaseolina is less succeptible to ZnO when compared to F. equisiti.

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REFERENCES

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*E-mail: [email protected]. 4078



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Reactivity of Crystalline ZnO Superstructures against Fungi and

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