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ScienceDirect Journal of Taibah University for Science 9 (2015) 7–12
Green synthesis of CuO nanoparticles using Gloriosa superba L. extract and their antibacterial activity H. Raja Naika a,∗ , K. Lingaraju a , K. Manjunath b , Danith Kumar c , G. Nagaraju c , D. Suresh d,e , H. Nagabhushana e a
Department of Studies and Research in Environmental Science, Tumkur University, Tumkur 572103, Karnataka, India b Centre for Nano and Material Sciences, Jain University, Bangalore 562112, Karnataka, India c Department of Chemsitry, BMS Institute of Technology, Avalahalli, Bangalore 562164, Karnataka, India d Department of Studies and Research in Chemistry, Tumkur University, Tumkur 572103, Karnataka, India e Prof. CNR Rao Center for Advanced Materials, Tumkur University, Tumkur 572103, Karnataka, India Available online 11 July 2014
Abstract The investigation aims at the synthesis of copper oxide nanoparticles (CuO Nps) using Gloriosa superba L. plant extract as fuel by solution combustion synthesis, their characterization and studies on antibacterial activities against selected pathogenic bacteria. Xray diffraction studies showed that the particles are monoclinic in nature. The UV–visible absorption spectrum of CuO Nps indicates the blue shift with increase of concentration of plant extract. SEM images reveal that the particles are spherical in nature. TEM image indicates that as-formed CuO Nps are spherical in shape, and the size is found to be in the range 5–10 nm. Further, as-formed CuO Nps exhibit significant antibacterial activity against pathogenic bacterial strains namely Gram −ve Klebsiella aerogenes, Pseudomonas desmolyticum, and Escherichia coli, Gram +ve bacteria Staphylococcus aureus. The current study demonstrates convenient utilization of Gloriosa superba L. extract as a fuel for the efficient synthesis of CuO nanoparticles through a green synthesis method to obtain significantly active antibacterial material. © 2014 Taibah University. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/). Keywords: Metallic nanoparticles; Anti-bacterial activity; Solution combustion; Fuel
1. Introduction
∗
Corresponding author. Tel.: +91 0816 2254546; fax: +91 0816 2270719; mobile: +91 8496866876. E-mail addresses:
[email protected],
[email protected],
[email protected] (H.R. Naika). Peer review under responsibility of Taibah University
Copper oxide nanostructures have attracted significant attention because of their wide range of applications such as high-Tc superconductors [1], sensors [2,3], catalytic [4], optical [5], electrical [6], giant magnet resistance materials [7,8] gas sensors [9–11], solar energy transformation and preparation of organic–inorganic nanostructure composites [12]. CuO is a p-type semiconductor with the band gap of ∼1.7 eV [13]. Further it can be used as an antimicrobial, anti-biotic and anti-fungal agent when incorporated in coatings, plastics textiles, etc. [14]. Copper and
http://dx.doi.org/10.1016/j.jtusci.2014.04.006 1658-3655 © 2014 Taibah University. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).
H.R. Naika et al. / Journal of Taibah University for Science 9 (2015) 7–12
2. Materials and methods 2.1. Plant material and extraction Leaves of G. superba were collected from Devarayana durga Forest of Tumkur district region, Karnataka, India. Fresh Leaves of G. superba were collected and then washed in running tap water, and shade dried at room temperature. Dried leaves were powdered using mixer grinder, mechanically, sieved (10/44) and subjected to Soxhlet extraction using deionized water for 72 h. The aqueous solution obtained after extraction
(d)
(c) Intensity (a.u.)
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30
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(311)
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(-311) (113)
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(a) (020)
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copper-based compounds are efficient biocidal properties, which are generally used in pesticidal formulations [15] and several health related applications. Different methods available to prepare CuO Nps namely sol–gel technique [16], Sonochemical [17], alkoxide based route [18], electrochemical methods [19], precipitationpyrolysis [20], microwave irradiations [21], solid-state reaction method [22], thermal decomposition of precursor [23], etc. Chemical synthesis methods lead to presence of some toxic chemicals absorbed on the surface that may cause adverse effects in medical applications. Recently green synthesis of different nanoparticles by plants such as neem [24], alfalfa [25,26], Cinnamomum camphora [27], Emblica officinalis [28], lemon grass [29], tamarind [30], and Euphorbia tirucalli [31] have been reported. Gloriosa superba L. is a species of flowering plant in the family Colchicaceae. It is native of Africa and Asia, but it is known worldwide as an ornamental plant, a medicinal, poisonous plant as a noxious weed. G. superba is one of the alkaloid-rich plant has long been used as a traditional medicine in many cultures. It has been used in the treatment of gout, infertility, open wounds, snakebite, ulcers, arthritis, cholera, colic, kidney problems, typhus, itching, leprosy, bruises, sprains, hemorrhoids, cancer, impotence, nocturnal emission, smallpox, sexually transmitted diseases, and many types of internal parasites. Solution combustion synthesis (SCS) is one of the simple and easiest methods for synthesis of metal oxide nanoparticles [32]. In this paper, CuO Nps were prepared by solution combustion method using G. superba extract as a fuel. The obtained product was characterized with the aid of PXRD, UV–visible, SEM and TEM. Further, the synthesized nanoparticles were evaluated for antibacterial activities by employing Gram −ve Klebsiella aerogenes, Pseudomonas desmolyticum, and Escherichia coli, Gram +ve bacteria Staphylococcus aureus using agar well diffusion method.
80
2θ(degrees)
Fig. 1. PXRD patterns of CuO nanoparticles prepared at different concentration of plant extract (a) 5 mL (b) 10 mL (c) 15 mL and (d) 20 mL.
is subjected to concentration under reduced pressure at 40 ± 5 ◦ C by rotary flash evaporator (Büchi, Flawil, Switzerland) after it is dried in hot air oven at 50–60 ◦ C to give crude extract (15.50 g). A small amount of the extract (0.1 g/mL) is used for the synthesis of CuO NPs. 2.2. Synthesis of CuO nanoparticles In solution combustion method [33] the reaction mixture was prepared by adding 1 mL of the plant extract (fuel) and cupric nitrate (2.32 g) as a source of copper. Small amount of double distilled water was added 20ml 15 ml 10 ml 05 ml
Absorbance, au.
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300
400
500
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Wavelength(nm)
Fig. 2. UV–visible spectra of CuO nanoparticles of given concentration of plant extract of (1) 5 mL (2) 10 mL (3) 15 mL (4) 20 mL.
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Fig. 3. SEM images of CuO nanoparticles show Spherical in shape with different concentration of plant extract (a) 5 mL (b) 10 mL (c) 15 mL and (d) 20 mL of plant extract.
to get homogenous mixture under constant stirring for ∼10 min. This reaction mixture was kept in a pre-heated muffle furnace maintained at 400 ± 10 ◦ C. CuO Nps are formed within 3–4 min. The obtained products of CuO Nps were stored in air tight container for further analysis. To illuminate the effects of the plant extract, different concentrations (5, 10, 15, and 20 mL) were used by keeping the source of copper at constant level (2.32 g). 2.3. Material characterization The phase identity and crystalline size of CuO Nps were characterized by Shimadzu X-ray diffractometer (PXRD-7000) using Cu-K␣ radiation of wavelength ˚ The absorption spectrum of the sample λ = 1.541 A. was measured on a Shimadzu UV-1800 UV–visspectrophotometer. Morphological features were studied by using Hitachi-7000 Scanning Electron Microscopy (SEM), Transmission electron microscopy (TEM, TECNAI F-30).
NCIM-2098, P. desmolyticum NCIM-2028, and E. coli NCIM-5051, Gram +ve bacteria S. aureus NCIM-5022 by agar well diffusion method [34] Nutrient Agar plates were prepared and swabbed using Sterile L-shaped glass rod with 100 l of 24 h mature broth culture of individual bacterial strains. The wells were made by using sterile cork borer (6 mm) wells was created into the each Petri-plates. Varied concentrations of CuO Nps (500 and 1000 g/well) were used to assess the activity of the nanoparticles. The compounds were dispersed in sterile water and it was used as a negative control and simultaneously the standard antibiotics Ciprofloxacin (5 g/50 l) (Hi Media, Mumbai, India) as positive control were tested against the bacterial pathogens. Then the plates were incubated at 37 ◦ C for 24–36 h, the zone inhibition measured in millimeter (mm) of the every well and also the values were noted. Triplicates were maintained in every concentration and also the average values were calculated for the ultimate antibacterial activity. 3. Results and discussions
2.4. Antibacterial activity Antibacterial activity was screened against four bacterial strains namely Gram −ve K. aerogenes
Fig. 1 shows typical XRD patterns of the formed CuO Nps which are identical to the single phase monoclinic (JCPDS: 80-1916) structure with a lattice
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˚ b = 3.4324 A ˚ and c = 5.1329 A, ˚ constant a = 4.6965 A, β = 99.5287◦ . No impurity peaks other than CuO were observed in the XRD pattern indicating the high phase purity. The broadening of the diffraction peaks indicates that the crystal size is small. The average crystallite size of the CuO Nps calculated by Debye–Scherrer’s formula as kλ D= β cos θ where D is the particle size (nm), k is a constant equal to ˚ 0.94, λ is the wave length of X-ray radiation (1.5406 A), β is the full-width at half maximum (FWHM) of the peak (in radians) and 2θ is the Bragg angle (degree). The average crystallite size was found to be in the range 8–17 nm. Fig. 2 shows the UV–vis spectra of the as-formed CuO Nps dispersed in water exhibiting the maximum absorption peaks at about 380 nm. In the spectrum, the peaks at 380 nm are due to surface plasmon absorption of metal oxide. The surface plasmon absorption in the metal oxide nanoparticles is due to the collective oscillation of the free conduction band electrons which is excited by the incident electromagnetic radiation. This type of resonance is seen when the wavelength of the incident light far exceeds the particle diameter. Surface plasmon absorption band with a maximum at 380 nm indicates the formation of CuO nanoparticles [35]. Fig. 3(a–d) shows the SEM images of CuO Nps. It clearly shows that the particles are almost spherical in nature which is free from agglomeration. Further it is observed that micro structure is independent concentration of plant extract. Fig. 4 shows the TEM image of CuO Nps. The TEM study is carried out to understand the crystalline characteristics of the nanoparticles. The particles are observed to be spherical in shape and the size is found to be in the range 5–10 nm. 4. Antibacterial activity The antibacterial properties of the CuO Nps was evaluated against Gram −ve K. aerogenes, E. coli, and P.
Fig. 4. TEM image of the as-formed CuO nanoparticles.
desmolyticum. Gram +ve bacteria S. aureus using agar well diffusion method. In agar well diffusion method the CuO Nps showed significant antibacterial activity on all the four bacterial strains. Some reports available for the mechanism behind the antimicrobial activities. In the present case, K. aerogenes and E. coli show significant zone of inhibition by CuO Nps. Naturally, Shigella flexneri and Bacillus subtilis have abundance of amine and carboxyl groups [36] on cell surface and high affinity of copper toward these groups [37]. Copper ions released subsequently may bind with DNA molecules and lead to disordering of the helical structure by cross-linking within and between the nucleic acid strands. Copper ions inside bacterial cells also disrupt the biochemical processes [38]. Cu2+ and Ag+ ions were also small studied well to disrupt the bacterial cell membranes and gain entry in order to disrupt enzyme function. Indirect effects through changes in the surrounding charge environment also have an impact on the effectiveness of nanoparticulate metals against microorganisms [39]. Another proposed mechanism is, there will be copper ions released from the nanoparticles that may attach to the negatively charged bacterial cell wall and rupture it, thereby leading to protein denaturation and cause cell death [40]. However, similar kind
Table 1 Antibacterial activity of CuO nanoparticles on pathogenic bacterial strains. Treatment
Klebsiella aerogenes (Mean ± SE)
Escherichia coli (Mean ± SE)
Staphyloccus aureus (Mean ± SE)
Pseudomonas desmolyticum (Mean ± SE)
Standard (5 g/50 L) CuO (500 g/50 L) CuO (1000 g/100 L)
21.67 ± 0.33** 12.00 ± 0.00 15.67 ± 0.33**
20.33 ± 0.33** 7.33 ± 0.33** 13.67 ± 0.33**
14.00 ± 0.00 3.33 ± 0.33** 6.00 ± 0.00
13.33 ± 0.33** 2.67 ± 0.33** 5.33 ± 0.33**
Values are the mean ± SE of inhibition zone in mm. Asterisks symbols represent statistical significance, *P < 0.05, **P < 0.01 as compared with the control.
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Fig. 5. zone of inhibition of (a) Klebsiella aerogenes (b) Escherichia coli (c) Staphylococcus aureus and (d) Pseudomonas desmolyticum.
of mechanism was proposed by Azam et al. [41], Line et al. [42] and Nawaz et al. [43] in CuO Samples. Fig. 5 shows the zone of inhibition of CuO Nps against the Bacterial strains namely Gram −ve, K. aerogenes, E. coli and P. desmolyticum. Gram +ve bacteria S. aureus with 500 and 1000 g concentration of CuO Nps. The details of zone of inhibition (mm) for the above bacterial strains are given in figure. It is evident from Table 1, P. desmolyticum and S. aureus is less susceptible to CuO Nps compare to K. aerogenes, E. coli.
significant antibacterial activity against all the four bacterial strains, i.e., Gram −ve K. aerogenes, E. coli, and P. desmolyticum, Gram +ve bacteria S. aureus. Among them, K. aerogenes and E. coli show significant zone of inhibition to CuO Nps compared to the positive control (Ciprofloxacin). S. aureus and P. desmolyticum strain shows the moderate zone of inhibition compared to positive control. The study successfully demonstrates the convenient utilization of G. superba extract as a fuel to get structurally and morphologically interesting and potentially antibacterial CuO nanoparticles.
5. Conclusion Acknowledgement This study reports that green synthesis of CuO Nps prepared by solution combustion method using Gloriosa superba extract as a fuel. The PXRD patterns showed monoclinic phase and the UV–visible absorption spectrum indicates blue shift with increasing concentration of the plant extract in the reaction mixture during the synthesis. SEM images reveal that the particles appear to be almost spherical in shape. TEM image of CuO Nps confirms the spherical shape and the size were found to be in the range 5–10 nm. CuO Nps exhibited
Raja Naika H wish to thank University Grant Commission (UGC), New Delhi for Major Research Project (UGC letter no. 42-179/2013(SR) for financial support. References [1] S.K. Yip, J.A. Sauls, Phys. Rev. Lett. 69 (1992) 2264–2267. [2] Y.S. Kim, I.S. Hwang, S.J. Kim, C.Y. Lee, J.H. Lee, Sens. Actuators B 135 (2008) 298–303.
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