- Email: [email protected]
Biosynthesis and characterization of Acalypha indica mediated copper oxide nanoparticles and evaluation of its antimicrobial and anticancer activity
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 129 (2014) 255–258
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Biosynthesis and characterization of Acalypha indica mediated copper oxide nanoparticles and evaluation of its antimicrobial and anticancer activity Rajeshwari Sivaraj a,b,⇑,1, Pattanathu K.S.M. Rahman a,1, P. Rajiv b,2, S. Narendhran b,2, R. Venckatesh c,3 a b c
School of Science and Engineering, Teesside University, Middlesbrough – TS1 3BA, Tees Valley, UK Department of Biotechnology, School of Life Sciences, Karpagam University, Eachanari Post, Coimbatore 641 021, Tamil Nadu, India Faculty of Chemistry, Government Arts College, Udumalpet 642 126, Tamil Nadu, India
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Highly stable copper oxide
nanoparticles are successfully synthesized. A. indica mediated copper oxide nanoparticles showed antimicrobial activity. A. indica mediated copper oxide nanoparticles have anticancer activity.
a r t i c l e
i n f o
Article history: Received 14 December 2013 Received in revised form 3 March 2014 Accepted 18 March 2014 Available online 28 March 2014 Keywords: Acalypha indica Antimicrobial Biological method Copper oxide nanoparticles Cytotoxicity
a b s t r a c t Copper oxide nanoparticles were synthesized by biological method using aqueous extract of Acalypha indica leaf and characterized by UV–visible spectroscopy, XRD, FT-IR, SEM TEM and EDX analysis. The synthesised particles were highly stable, spherical and particle size was in the range of 26–30 nm. The antimicrobial activity of A. indica mediated copper oxide nanoparticles was tested against selected pathogens. Copper oxide nanoparticles showed efficient antibacterial and antifungal effect against Escherichia coli, Pseudomonas fluorescens and Candida albicans. The cytotoxicity activity of A. indica mediated copper nanoparticles was evaluated by MTT assay against MCF-7 breast cancer cell lines and confirmed that copper oxide nanoparticles have cytotoxicity activity. Ó 2014 Elsevier B.V. All rights reserved.
Introduction ⇑ Corresponding author at: Department of Biotechnology, School of Life Sciences, Karpagam University, Eachanari Post, Coimbatore 641 021, Tamil Nadu, India. Tel./ fax: +91 422 647 1113. E-mail address: [email protected] (R. Sivaraj). 1 Tel.: +44 7440902654. 2 Tel./fax: +91 422 647 1113. 3 Tel.: +91 4252 223062. http://dx.doi.org/10.1016/j.saa.2014.03.027 1386-1425/Ó 2014 Elsevier B.V. All rights reserved.
Green synthesis of nanoparticles is budding into an important approach in nanotechnology [1,2]. Green chemistry method highlights the usage of natural organisms as reliable, simple, nontoxic and eco-friendly [3,4]. Hence, investigators have focused the synthesis of nanoparticles using biological systems in the last years
256
R. Sivaraj et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 129 (2014) 255–258
[5]. Synthesis of nanoparticles by using microorganisms, enzyme and plant or plant extract, have been proposed by several authors [6,7]. In the biological methods, it is found that the extracts of living organisms act both as reducing and capping agents in the synthesizing process of the nanoparticles [8]. Copper oxide nanoparticles are used as gas sensors, catalysis, batteries, high temperature superconductors, solar energy conversion tools, etc. [9–11]. It is very stable, strong and has a longer shelf life compared to organic antimicrobial agents [9,12]. Cioffi et al. [13] has reported the antifungal and bacteriostatic properties of copper nanoparticles/polymer composites. Berntsen et al. [14] studied the impaired cell viability in human airway smooth muscle cells and observed that reduced in cell contractility occurred due to exposure of copper oxide nanoparticles. Acalypha indica L. (family: Euphorbiaceae) is commonly distributed throughout the plains of India. It has been reported to be beneficial in treating pneumoniae, asthma, rheumatism and several other ailments [15]. In this research paper we have reported biosynthesis of copper oxide nanoparticles using aqueous extract of A. indica leaf. The present study is the continuation to assess the antimicrobial and cytotoxicity activities of A. indica mediated copper oxide nanoparticles. Materials and methods Materials All the chemical reagents (analytical grade) were purchased from sigma–aldrich chemicals, India. The bacterial and fungal strains were obtained from Department of Microbiology, Karpagam University, Coimbatore, Tamil Nadu. Bacterial and fungal cultures namely Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Proteus vulgaris, Pseudomonas fluorescens and Candida albicans were maintained in the respective medium.
Determination of antimicrobial activity of copper oxide nanoparticles The antimicrobial activity was determined by well diffusion method [16]. The Muller Hinton Agar plates were prepared and 100 ll of microbial culture was swabbed. After 5 min the well (5 mm size) was cut by gel puncher and a volume of 100 ll (25 lg/ml) of the copper oxide nanoparticles was added into the well. The effects were compared with that of the standard antibiotic tetracycline (positive control) at a concentration of 10 lg/ml. The plates were incubated at 37 °C for 24 h (bacteria) and room temperature for 48 h (fungi). The assessment of the antimicrobial activity was based on the measurement of the diameter of the inhibition zone formed around the well and the mean values are recorded. Determination of cytotoxicity studies of copper oxide nanoparticles Human breast MCF-7 cell lines (cell culture) were obtained from the National Centre for Cell Science (NCCS), Pune, India. The cells were cultured in Eagles Minimum Essential Medium (EMEM) added with FBS (10%, v/v) at 37 °C in a CO2 incubator (95% air, 5% CO2 and 100% relative humidity). In order to evaluate the cytotoxic effect of the green synthesised copper oxide nanoparticles against MCF-7 cells, the cells were collected in the exponential stage of growth, seeded into 96-well plates (15,000 per well) and permitted to adhere for 48 h. Then, Different concentrations (6.5, 12.5, 25, 50, 100 lg/ml) of A. indica mediated copper oxide nanoparticles were added to the desired wells and incubated for 48 h. A 20 ll of EMEM medium having MTT (5 mg/mL) was added to each well and incubated for 4 h at 37 °C. Later, the medium was altered with 100 lL of DMSO, and optical densities were measured at 570 nm. All studies were performed in triplicates and expressed as the mean ± standard error. Results and discussion
Synthesis of copper oxide nanoparticles Synthesis and characterization of copper oxide nanoparticles A. indica plants were collected from in and around Karpagam University, Coimbatore, Tamil Nadu, India. 100 g of leaves were washed with tap water, ground and boiled with 500 ml of de-ionized water for 10 min. Finally the product was filtered and stored in freezer for further investigations. A 50% of leaf extract was made up to 250 ml. The analytical grade copper sulphate solution was prepared using de-ionized water and was mixed in the 50% leaf extract under constant stirring using magnetic stirrer. The mixture of this solution was kept under vigorous stirring at 100 °C for 7–8 h. After this process, a brownish black colour product obtained. The solid product obtained was washed twice with de-ionized water and dried at 80 °C for 8 h. Finally the dried powder was stored in properly labelled containers and used for further studies.
Fig. 1 shows the UV–Visible absorption spectrum of green synthesized copper oxide nanoparticles. It has an optical absorbance range around 220 nm. The XRD analysis of green synthesized copper oxide nanoparticles is shown in Fig. 2. The XRD peaks were obtained at (1 1 0), (1 1 1), (2 0 0), (2 0 2), (0 2 0), (2 0 2), (1 1 3), (3 1 1), (2 2 0) and (4 0 0) Bragg’s reflection based on the crystal of copper
Characterization of copper oxide nanoparticles UV–vis spectrophotometer (UV-2450, Shimadzu) was used to determine the optical property of copper oxide nanoparticles in 200–800 nm wave length range. The X-ray diffractometry (XRD) study was done using Perkin–Elmer spectrum one instrument. The functional groups in the copper oxide nanoparticles were predicated by Fourier transform infrared (FT-IR) spectrometer (Perkin– Elmer 1725). Scanning electron microscopy (SEM) (Model JSM 6390LV, JOEL, USA) and Transmission electron microscope (TEM) (JEOL JEM-3100F) were used to study the morphology and size of the copper oxide nanoparticles. Energy dispersive X-ray spectrometer (RONTEC’s EDX system, Model QuanTax 200, Germany) was used for elemental analysis of copper oxide nanoparticles.
Fig. 1. UV spectra of Acalypha indica mediated copper oxide nanoparticles.
R. Sivaraj et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 129 (2014) 255–258
257
obtained in gram negative bacteria (E. coli) with a zone diameter of 15 ± 1 mm at a concentration of 25 lg/ml, and lowest zone of inhibition was observed in P. vulgaris with a zone diameter of 9 ± 1 mm at same concentration. A. indica mediated copper oxide nanoparticles showed the best antifungal activity in C. albicans. Azam et al. reported the antibacterial activity of copper oxide nanoparticles against both gram-positive and negative bacterial strains [18]. Dasa et al. [19] reported that copper nanoparticles have efficient and bactericidal effect against E. coli and P. aeruginosa. The growth inhibition of cells may be due to distractions of cell membrane by Copper oxide nanoparticles which results in breakdown of cell enzyme [20]. The results reveal that A. indica mediated copper oxide nanoparticles show effective antimicrobial activity. Cytotoxicity studies Fig. 2. XRD pattern of Acalypha indica mediated copper oxide nanoparticles.
The cytotoxicity of the copper oxide nanoparticles was evaluated against MCF-7 breast cancer cell lines at a various concentrations (6.5–100 lg/ml). Supplementary information A5 shows the cytotoxic activity of copper oxide nanoparticles and IC50 value for copper oxide nanoparticles was found to be 56.16 lg/ml. Maximum concentration of copper oxide nanoparticles (100 lg/ml) effectively inhibits the growth of cell by more than 97%. Sankar et al. [21] reported the anticancer activity of Origanum vulgare mediated silver nanoparticles and cytotoxic effects of green synthesized O. vulgare mediated silver nanoparticles against human lung cancer A549 cells. Conclusion
Fig. 3. FT-IR spectrum of Acalypha indica mediated copper oxide nanoparticles.
oxide nanoparticles. These phases were indexed to spherical shape. The Scherrer formula was used to calculate the particles sizes and found to be in the range of 26 ± 4 nm. Sangeetha et al. [17] stated that Aloe barbadensis mediated copper oxide nanoparticles were spherical in nature and sizes ranging from 15 to 30 nm. FTIR spectra of green synthesised copper oxide nanoparticles are shown in Fig. 3. The spectrum showed bands at 446 and 558 cm 1 corresponding to metal–oxygen (M–O). The bands at 1384 and 1528 cm 1 refer to N–H bending mode. A band at 2488 cm 1 represents alkynes. The synthesized copper oxide nanoparticles show peak at 3612 cm 1 which corresponds to phosphorous compounds. The band at 1228 cm 1 corresponding to C–O–C stretch was also obtained. The size and shape of green synthesised copper oxide nanoparticles were analysed using SEM and TEM, which is shown in Supplementary information A1 and A2. Particles are spherical and particle size is in the range of 26–30 nm. Particles are well dispersed, which was confirmed by SEM and TEM studies. This is very similar to previous literature [17]. Supplementary information A3 refers to the energy dispersive X-ray analysis (EDAX) of green synthesized copper oxide nanoparticles, which denotes that the strong signal in the copper and confirmed the formation of copper oxide nanoparticles. Antimicrobial activity Supplementary information A4 shows the results of antimicrobial activity of A. indica mediated copper oxide nanoparticles against pathogenic organisms. Maximum zone of inhibition was
The present study reported that copper oxide nanoparticles can be synthesized in a simple method using A. indica leaf extract. The TEM analysis showed that the sizes of the synthesized copper oxide nanoparticles ranged from 26 to 30 nm. Plant mediated copper oxide nanoparticles showed best antimicrobial and anticancer activity. Acknowledgements We thank the Association of Commonwealth Universities and Commonwealth Commission, United Kingdom for the Commonwealth Academic Fellowship. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.saa.2014.03.027. References [1] P. Raveendran, J. Fu, S.L. Wallen, Green Chem. 8 (2006) 34–38. [2] P. Magudapathy, P. Gangopadhyay, B.K. Panigrahi, K.G.M. Nair, S. Dhara, Physica B 299 (2001) 142–146. [3] K.B. Narayanan, N. Sakthivel, Adv. Colloid Interface Sci. 156 (2010) 1–13. [4] M. Sathishkumar, K. Sneha, Y.S. Yun, Bioresource Technol. 101 (2010) 7958– 7965. [5] N. Tsibakhashvil, T. Kalabegishvili, V. Gabunia, E. Gintury, N. Kuchava, N. Bagdavadze, D. Pataraya, M. Gurielidzse, D. Gvarjaladze, L. Lomidze, Nano. Stud. 2 (2010) 179–182. [6] S. Schultz, D.R. Smith, J.J. Mock, D.A. Schultz, Proc. Natl. Acad. Sci. 97 (2000) 996–1001. [7] B. Nair, T. Pradeep, Growth Des. 2 (2002) 293–298. [8] S. Rashmi, V. Preeti, Bioresource Technol. 100 (2009) 501–504. [9] G. Ren, D. Hu, E.W.C. Cheng, M.A.V. Reus, P. Reip, R.P. Allaker, Int. J. Antimicrob. Agents 33 (2009) 587. [10] C.T. Hsieh, J.M. Chen, H.H. Lin, H.C. Shih, Appl. Phys. Lett. 82 (2003) 3316. [11] X. Zhang, G. Wang, X. Liu, J. Wu, M. Li, J. Gu, H. Liu, B. Fang, J. Phys. Chem. 112 (2008) 16845. [12] M. Raffi, S. Mehrwan, T.M. Bhatti, J.I. Akhter, A. Hameed, W. Yawar, M.M.U. Hasan, Ann. Microbiol. 60 (2010) 75.
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[13] N. Cioffi, L. Torsi, N. Ditaranto, G. Tantillo, L. Ghibelli, L. Sabbatini, T. BleveZacheo, M.D. Alessio, P.G. Zambonin, E. Traversa, Chem. Mater. 17 (2005) 5255. [14] P. Berntsen, C.Y. Park, B.R. Rutishauser, A. Tsuda, T.M. Sager, R.M. Molina, T.C. Donaghey, A.M. Alencar, D.I. Kasahara, T. Ericsson, E.J. Millet, J. Swenson, D.J. Tschumperlin, J.P. Butler, J.D. Brain, J.J. Fredberg, P. Gehr, E.H. Zhou, J.R. Soc, Interface 7 (2010) 331–340. [15] R.N. Chopra, S.L. Nayar, I.C. Chopra, Glossary of Indian Medical Plants, CSIR, New Delhi, 1956. [16] A.W. Bauer, W.M. Kirby, J.C. Sherris, M. Turck, Am. J. Clin. Pathol. 45 (1966) 493–496.
[17] G. Sangeetha, S. Rajeshwari, R. Venckatesh, Mater. Res. Bull. 12 (2011) 2560– 2566. [18] S. Ohara, T. Mousavand, T. Sasaki, M. Umetsu, T. Naka, T. Adschiri, J. Mater. Sci. 43 (2008) 2393–2396. [19] Dhaneswar Dasa, Bikash Chandra Nathb, Pinkee Phukonc, Swapan Kumar Doluia, Colloids Surf. B 101 (2013) 430–433. [20] G. Ren, D. Hu, E.W.C. Cheng, M.A.V. Reus, P. Reip, R.P. Allaker, Int. J. Antimicrob. Agents. 33 (2009) 587. [21] R. Sankar, A. Karthik, A. Prabu, S. Karthik, K.S. Shivashangari, V. Ravikumar, Colloids Surf. B 108 (2013) 80–84.
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Biosynthesis and characterization of Acalypha indica mediated copper oxide nanoparticles and evaluation of its antimicrobial and anticancer activity Rajeshwari Sivaraj a,b,⇑,1, Pattanathu K.S.M. Rahman a,1, P. Rajiv b,2, S. Narendhran b,2, R. Venckatesh c,3 a b c
School of Science and Engineering, Teesside University, Middlesbrough – TS1 3BA, Tees Valley, UK Department of Biotechnology, School of Life Sciences, Karpagam University, Eachanari Post, Coimbatore 641 021, Tamil Nadu, India Faculty of Chemistry, Government Arts College, Udumalpet 642 126, Tamil Nadu, India
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Highly stable copper oxide
nanoparticles are successfully synthesized. A. indica mediated copper oxide nanoparticles showed antimicrobial activity. A. indica mediated copper oxide nanoparticles have anticancer activity.
a r t i c l e
i n f o
Article history: Received 14 December 2013 Received in revised form 3 March 2014 Accepted 18 March 2014 Available online 28 March 2014 Keywords: Acalypha indica Antimicrobial Biological method Copper oxide nanoparticles Cytotoxicity
a b s t r a c t Copper oxide nanoparticles were synthesized by biological method using aqueous extract of Acalypha indica leaf and characterized by UV–visible spectroscopy, XRD, FT-IR, SEM TEM and EDX analysis. The synthesised particles were highly stable, spherical and particle size was in the range of 26–30 nm. The antimicrobial activity of A. indica mediated copper oxide nanoparticles was tested against selected pathogens. Copper oxide nanoparticles showed efficient antibacterial and antifungal effect against Escherichia coli, Pseudomonas fluorescens and Candida albicans. The cytotoxicity activity of A. indica mediated copper nanoparticles was evaluated by MTT assay against MCF-7 breast cancer cell lines and confirmed that copper oxide nanoparticles have cytotoxicity activity. Ó 2014 Elsevier B.V. All rights reserved.
Introduction ⇑ Corresponding author at: Department of Biotechnology, School of Life Sciences, Karpagam University, Eachanari Post, Coimbatore 641 021, Tamil Nadu, India. Tel./ fax: +91 422 647 1113. E-mail address: [email protected] (R. Sivaraj). 1 Tel.: +44 7440902654. 2 Tel./fax: +91 422 647 1113. 3 Tel.: +91 4252 223062. http://dx.doi.org/10.1016/j.saa.2014.03.027 1386-1425/Ó 2014 Elsevier B.V. All rights reserved.
Green synthesis of nanoparticles is budding into an important approach in nanotechnology [1,2]. Green chemistry method highlights the usage of natural organisms as reliable, simple, nontoxic and eco-friendly [3,4]. Hence, investigators have focused the synthesis of nanoparticles using biological systems in the last years
256
R. Sivaraj et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 129 (2014) 255–258
[5]. Synthesis of nanoparticles by using microorganisms, enzyme and plant or plant extract, have been proposed by several authors [6,7]. In the biological methods, it is found that the extracts of living organisms act both as reducing and capping agents in the synthesizing process of the nanoparticles [8]. Copper oxide nanoparticles are used as gas sensors, catalysis, batteries, high temperature superconductors, solar energy conversion tools, etc. [9–11]. It is very stable, strong and has a longer shelf life compared to organic antimicrobial agents [9,12]. Cioffi et al. [13] has reported the antifungal and bacteriostatic properties of copper nanoparticles/polymer composites. Berntsen et al. [14] studied the impaired cell viability in human airway smooth muscle cells and observed that reduced in cell contractility occurred due to exposure of copper oxide nanoparticles. Acalypha indica L. (family: Euphorbiaceae) is commonly distributed throughout the plains of India. It has been reported to be beneficial in treating pneumoniae, asthma, rheumatism and several other ailments [15]. In this research paper we have reported biosynthesis of copper oxide nanoparticles using aqueous extract of A. indica leaf. The present study is the continuation to assess the antimicrobial and cytotoxicity activities of A. indica mediated copper oxide nanoparticles. Materials and methods Materials All the chemical reagents (analytical grade) were purchased from sigma–aldrich chemicals, India. The bacterial and fungal strains were obtained from Department of Microbiology, Karpagam University, Coimbatore, Tamil Nadu. Bacterial and fungal cultures namely Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Proteus vulgaris, Pseudomonas fluorescens and Candida albicans were maintained in the respective medium.
Determination of antimicrobial activity of copper oxide nanoparticles The antimicrobial activity was determined by well diffusion method [16]. The Muller Hinton Agar plates were prepared and 100 ll of microbial culture was swabbed. After 5 min the well (5 mm size) was cut by gel puncher and a volume of 100 ll (25 lg/ml) of the copper oxide nanoparticles was added into the well. The effects were compared with that of the standard antibiotic tetracycline (positive control) at a concentration of 10 lg/ml. The plates were incubated at 37 °C for 24 h (bacteria) and room temperature for 48 h (fungi). The assessment of the antimicrobial activity was based on the measurement of the diameter of the inhibition zone formed around the well and the mean values are recorded. Determination of cytotoxicity studies of copper oxide nanoparticles Human breast MCF-7 cell lines (cell culture) were obtained from the National Centre for Cell Science (NCCS), Pune, India. The cells were cultured in Eagles Minimum Essential Medium (EMEM) added with FBS (10%, v/v) at 37 °C in a CO2 incubator (95% air, 5% CO2 and 100% relative humidity). In order to evaluate the cytotoxic effect of the green synthesised copper oxide nanoparticles against MCF-7 cells, the cells were collected in the exponential stage of growth, seeded into 96-well plates (15,000 per well) and permitted to adhere for 48 h. Then, Different concentrations (6.5, 12.5, 25, 50, 100 lg/ml) of A. indica mediated copper oxide nanoparticles were added to the desired wells and incubated for 48 h. A 20 ll of EMEM medium having MTT (5 mg/mL) was added to each well and incubated for 4 h at 37 °C. Later, the medium was altered with 100 lL of DMSO, and optical densities were measured at 570 nm. All studies were performed in triplicates and expressed as the mean ± standard error. Results and discussion
Synthesis of copper oxide nanoparticles Synthesis and characterization of copper oxide nanoparticles A. indica plants were collected from in and around Karpagam University, Coimbatore, Tamil Nadu, India. 100 g of leaves were washed with tap water, ground and boiled with 500 ml of de-ionized water for 10 min. Finally the product was filtered and stored in freezer for further investigations. A 50% of leaf extract was made up to 250 ml. The analytical grade copper sulphate solution was prepared using de-ionized water and was mixed in the 50% leaf extract under constant stirring using magnetic stirrer. The mixture of this solution was kept under vigorous stirring at 100 °C for 7–8 h. After this process, a brownish black colour product obtained. The solid product obtained was washed twice with de-ionized water and dried at 80 °C for 8 h. Finally the dried powder was stored in properly labelled containers and used for further studies.
Fig. 1 shows the UV–Visible absorption spectrum of green synthesized copper oxide nanoparticles. It has an optical absorbance range around 220 nm. The XRD analysis of green synthesized copper oxide nanoparticles is shown in Fig. 2. The XRD peaks were obtained at (1 1 0), (1 1 1), (2 0 0), (2 0 2), (0 2 0), (2 0 2), (1 1 3), (3 1 1), (2 2 0) and (4 0 0) Bragg’s reflection based on the crystal of copper
Characterization of copper oxide nanoparticles UV–vis spectrophotometer (UV-2450, Shimadzu) was used to determine the optical property of copper oxide nanoparticles in 200–800 nm wave length range. The X-ray diffractometry (XRD) study was done using Perkin–Elmer spectrum one instrument. The functional groups in the copper oxide nanoparticles were predicated by Fourier transform infrared (FT-IR) spectrometer (Perkin– Elmer 1725). Scanning electron microscopy (SEM) (Model JSM 6390LV, JOEL, USA) and Transmission electron microscope (TEM) (JEOL JEM-3100F) were used to study the morphology and size of the copper oxide nanoparticles. Energy dispersive X-ray spectrometer (RONTEC’s EDX system, Model QuanTax 200, Germany) was used for elemental analysis of copper oxide nanoparticles.
Fig. 1. UV spectra of Acalypha indica mediated copper oxide nanoparticles.
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obtained in gram negative bacteria (E. coli) with a zone diameter of 15 ± 1 mm at a concentration of 25 lg/ml, and lowest zone of inhibition was observed in P. vulgaris with a zone diameter of 9 ± 1 mm at same concentration. A. indica mediated copper oxide nanoparticles showed the best antifungal activity in C. albicans. Azam et al. reported the antibacterial activity of copper oxide nanoparticles against both gram-positive and negative bacterial strains [18]. Dasa et al. [19] reported that copper nanoparticles have efficient and bactericidal effect against E. coli and P. aeruginosa. The growth inhibition of cells may be due to distractions of cell membrane by Copper oxide nanoparticles which results in breakdown of cell enzyme [20]. The results reveal that A. indica mediated copper oxide nanoparticles show effective antimicrobial activity. Cytotoxicity studies Fig. 2. XRD pattern of Acalypha indica mediated copper oxide nanoparticles.
The cytotoxicity of the copper oxide nanoparticles was evaluated against MCF-7 breast cancer cell lines at a various concentrations (6.5–100 lg/ml). Supplementary information A5 shows the cytotoxic activity of copper oxide nanoparticles and IC50 value for copper oxide nanoparticles was found to be 56.16 lg/ml. Maximum concentration of copper oxide nanoparticles (100 lg/ml) effectively inhibits the growth of cell by more than 97%. Sankar et al. [21] reported the anticancer activity of Origanum vulgare mediated silver nanoparticles and cytotoxic effects of green synthesized O. vulgare mediated silver nanoparticles against human lung cancer A549 cells. Conclusion
Fig. 3. FT-IR spectrum of Acalypha indica mediated copper oxide nanoparticles.
oxide nanoparticles. These phases were indexed to spherical shape. The Scherrer formula was used to calculate the particles sizes and found to be in the range of 26 ± 4 nm. Sangeetha et al. [17] stated that Aloe barbadensis mediated copper oxide nanoparticles were spherical in nature and sizes ranging from 15 to 30 nm. FTIR spectra of green synthesised copper oxide nanoparticles are shown in Fig. 3. The spectrum showed bands at 446 and 558 cm 1 corresponding to metal–oxygen (M–O). The bands at 1384 and 1528 cm 1 refer to N–H bending mode. A band at 2488 cm 1 represents alkynes. The synthesized copper oxide nanoparticles show peak at 3612 cm 1 which corresponds to phosphorous compounds. The band at 1228 cm 1 corresponding to C–O–C stretch was also obtained. The size and shape of green synthesised copper oxide nanoparticles were analysed using SEM and TEM, which is shown in Supplementary information A1 and A2. Particles are spherical and particle size is in the range of 26–30 nm. Particles are well dispersed, which was confirmed by SEM and TEM studies. This is very similar to previous literature [17]. Supplementary information A3 refers to the energy dispersive X-ray analysis (EDAX) of green synthesized copper oxide nanoparticles, which denotes that the strong signal in the copper and confirmed the formation of copper oxide nanoparticles. Antimicrobial activity Supplementary information A4 shows the results of antimicrobial activity of A. indica mediated copper oxide nanoparticles against pathogenic organisms. Maximum zone of inhibition was
The present study reported that copper oxide nanoparticles can be synthesized in a simple method using A. indica leaf extract. The TEM analysis showed that the sizes of the synthesized copper oxide nanoparticles ranged from 26 to 30 nm. Plant mediated copper oxide nanoparticles showed best antimicrobial and anticancer activity. Acknowledgements We thank the Association of Commonwealth Universities and Commonwealth Commission, United Kingdom for the Commonwealth Academic Fellowship. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.saa.2014.03.027. References [1] P. Raveendran, J. Fu, S.L. Wallen, Green Chem. 8 (2006) 34–38. [2] P. Magudapathy, P. Gangopadhyay, B.K. Panigrahi, K.G.M. Nair, S. Dhara, Physica B 299 (2001) 142–146. [3] K.B. Narayanan, N. Sakthivel, Adv. Colloid Interface Sci. 156 (2010) 1–13. [4] M. Sathishkumar, K. Sneha, Y.S. Yun, Bioresource Technol. 101 (2010) 7958– 7965. [5] N. Tsibakhashvil, T. Kalabegishvili, V. Gabunia, E. Gintury, N. Kuchava, N. Bagdavadze, D. Pataraya, M. Gurielidzse, D. Gvarjaladze, L. Lomidze, Nano. Stud. 2 (2010) 179–182. [6] S. Schultz, D.R. Smith, J.J. Mock, D.A. Schultz, Proc. Natl. Acad. Sci. 97 (2000) 996–1001. [7] B. Nair, T. Pradeep, Growth Des. 2 (2002) 293–298. [8] S. Rashmi, V. Preeti, Bioresource Technol. 100 (2009) 501–504. [9] G. Ren, D. Hu, E.W.C. Cheng, M.A.V. Reus, P. Reip, R.P. Allaker, Int. J. Antimicrob. Agents 33 (2009) 587. [10] C.T. Hsieh, J.M. Chen, H.H. Lin, H.C. Shih, Appl. Phys. Lett. 82 (2003) 3316. [11] X. Zhang, G. Wang, X. Liu, J. Wu, M. Li, J. Gu, H. Liu, B. Fang, J. Phys. Chem. 112 (2008) 16845. [12] M. Raffi, S. Mehrwan, T.M. Bhatti, J.I. Akhter, A. Hameed, W. Yawar, M.M.U. Hasan, Ann. Microbiol. 60 (2010) 75.
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[13] N. Cioffi, L. Torsi, N. Ditaranto, G. Tantillo, L. Ghibelli, L. Sabbatini, T. BleveZacheo, M.D. Alessio, P.G. Zambonin, E. Traversa, Chem. Mater. 17 (2005) 5255. [14] P. Berntsen, C.Y. Park, B.R. Rutishauser, A. Tsuda, T.M. Sager, R.M. Molina, T.C. Donaghey, A.M. Alencar, D.I. Kasahara, T. Ericsson, E.J. Millet, J. Swenson, D.J. Tschumperlin, J.P. Butler, J.D. Brain, J.J. Fredberg, P. Gehr, E.H. Zhou, J.R. Soc, Interface 7 (2010) 331–340. [15] R.N. Chopra, S.L. Nayar, I.C. Chopra, Glossary of Indian Medical Plants, CSIR, New Delhi, 1956. [16] A.W. Bauer, W.M. Kirby, J.C. Sherris, M. Turck, Am. J. Clin. Pathol. 45 (1966) 493–496.
[17] G. Sangeetha, S. Rajeshwari, R. Venckatesh, Mater. Res. Bull. 12 (2011) 2560– 2566. [18] S. Ohara, T. Mousavand, T. Sasaki, M. Umetsu, T. Naka, T. Adschiri, J. Mater. Sci. 43 (2008) 2393–2396. [19] Dhaneswar Dasa, Bikash Chandra Nathb, Pinkee Phukonc, Swapan Kumar Doluia, Colloids Surf. B 101 (2013) 430–433. [20] G. Ren, D. Hu, E.W.C. Cheng, M.A.V. Reus, P. Reip, R.P. Allaker, Int. J. Antimicrob. Agents. 33 (2009) 587. [21] R. Sankar, A. Karthik, A. Prabu, S. Karthik, K.S. Shivashangari, V. Ravikumar, Colloids Surf. B 108 (2013) 80–84.