- Email: [email protected]
Characterization, antibacterial, antioxidant, and cytotoxic activities of ZnO nanoparticles using Coptidis Rhizoma
Bioorganic & Medicinal Chemistry Letters 24 (2014) 4298–4303
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Characterization, antibacterial, antioxidant, and cytotoxic activities of ZnO nanoparticles using Coptidis Rhizoma P. C. Nagajyothi a, , T. V. M. Sreekanth b, , Clement O. Tettey a, Yang In Jun a, Shin Heung Mook a,⇑ a b
Department of Physiology, College of Oriental Medicine, Dongguk University, Gyeongju, South Korea Department of Life Chemistry, Catholic University of Daegu, Gyeongsan 712-702, South Korea
a r t i c l e
i n f o
Article history: Received 29 April 2014 Revised 2 July 2014 Accepted 8 July 2014 Available online 15 July 2014 Keywords: Green synthesis Coptidis Rhizoma Antibacterial Antioxidant Cytotoxic activity
a b s t r a c t Here, we report a simple, eco-friendly and inexpensive approach for the synthesis of zinc oxide nanoparticles (ZnO NPs) using Coptidis Rhizoma. The ZnO NPs were characterized by UV–visible absorption spectroscopy, FTIR, SEM-EDX, TGA, TEM, SAED and XRD. TEM images confirmed the presence of spherical and rod shaped ZnO NPs in the range of 2.90–25.20 nm. Green synthesized ZnO NPS exhibited moderate antibacterial activity against Gram-positive and Gram-negative bacteria and excellent DPPH free radical scavenging activity. Synthesized ZnO NPs had no toxic effects on the RAW 264.7 cell line. Ó 2014 Published by Elsevier Ltd.
Nanoparticles with various sizes and shapes have garnered great interest in the past decade owing to their excellent optical, electronic and chemical properties, which are not displayed in the bulk state of metal. ZnO NPs are widely used in industrial applications such as pigments,1 dye-sensitized solar cells,2 photocatalysts,3 and sensors.4 ZnO is a wide band-gap semi-conductor (II–IV) with an energy gap of 3.37 eV at room temperature and ZnO NPs have a great advantage in their application as catalysts due to their large surface area and high catalytic activity.5 A wide variety of physical and chemical processes for the synthesis of ZnO nanostructures have been developed including laser ablation,6 hydrothermal,7 electrochemical depositions,8 sol–gel,9 chemical vapor deposition10 thermal decomposition,11 combustion,12,13 ultrasound,14 microwave-assisted combustion method,15 two-step mechano chemical–thermal synthesis,16 anodization,17 co-precipitation,18 and electrophoretic deposition19 methods. Even though, the exact mechanism of shape and size control of these structures have not been established and mentioned in the literature for the preparation of a number of self-assembled structures reported.20 Chemical synthesis methods lead to the adsorption of toxic chemical species on to the surface that may have adverse effects in medical applications. As a result, researchers in the field of synthesis of nanoparticles and assembly have turned to ⇑ Corresponding author. Tel.: +82 54 770 2372; fax: +82 54 742 5441.
E-mail address: [email protected] (S.H. Mook). Authors have made an equal contribution.
http://dx.doi.org/10.1016/j.bmcl.2014.07.023 0960-894X/Ó 2014 Published by Elsevier Ltd.
biosynthesis methods for nanoparticles production, which employ plants, fungi, bacteria and enzymes and represent possible ecofriendly alternatives to chemical and physical methods.21 To date, green synthesis of ZnO NPs by plants such as Calotropis procera,22 Aloe barbadensismiller,23 and Poncirus trifoliate24 have been reported. Coptidis Rhizoma is the dried rhizome of Coptis chinensis Franch (family; Ranunculaceae). C. Rhizoma, has common uses in traditional oriental medicine owing to its various pharmacological effects, which include anti-bacterial, antifungal, antiviral, antidiabetic, anticancer and anti-inflammatory activities,25 as well as the ability to relax blood vessels, reduce fever, stimulates circulation and prevent cardiovascular diseases.26 The present study was conducted to (i) characterize ZnO NPs prepared by the green synthesis method and (ii) investigate their antibacterial, antioxidant and cytotoxic activities. The UV–vis spectroscopy analysis was used for the preliminary characterization of ZnO NPs. The UV–vis absorption spectra for green synthesized ZnO NPs showed an adsorption maximum at 344 nm (Fig. 1). C. Rhizoma contains different classes of organic compounds (1° and 2° amines, aromatic, alkynes, nitro groups and alkyl halides) involved in the reduction and formation of ZnO NPs that were analyzed by FTIR. The bands of green synthesized ZnO NPs from C. Rhizoma were observed at 3410.28, 3062.89, 1603.74, 1506.42, 1479.71, 1384.47, 1275.27, 1232.66, 1102.80, 1004.44, 906.41, 825.50 and 630.93 cm1 (Fig. 2b). The bands of C. Rhizoma extract were observed at 3387, 2927, 1639,
P. C. Nagajyothi et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4298–4303
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Figure 1. UV–vis spectrum of ZnO NPs green synthesized by treating zinc nitrate with using Coptidis Rhizoma.
Figure 2. FTIR spectrum of (a) Coptidis Rhizoma (b) green synthesized ZnO NPs.
1509, 1459, 1363, 1273, 1235, 1150, 1029, 863 and 614 cm1 (Fig. 2a). C. Rhizoma extract bands shifted to 3410.28, 3062.89, 1603.74, 1506.42, 1479.71, 1384.47, 1275.27, 1232.66, 1102.80, 1004.44, 906.41, 825.50 and 630.93 cm1. The absorbance bands
were observed in the region of 500–3500 cm1and consisted of 3410.28 cm1 (secondary amines), 3062.89 cm1 (aromatic), 1603.74 (1° amines), 1506.42 and 1479.71 (aromatic), 1384.47 (nitro groups), 1275.27 (aromatic amines), 1232.66 and 1102.80
4300
P. C. Nagajyothi et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4298–4303
Figure 3. TGA analysis of ZnO NPs synthesized by Coptidis Rhizoma.
(aliphatic amines), 1004.44 (alcohols, carboxylic acids, esters and ethers), 906.41 (1° and 2° amines), 825.50 (alkyl halides) and 630.93 (alkynes). Figure 3 shows the TGA and DTA curves of ZnO NPs measured in static air at 0–1000 °C. There were clearly two primarily weight
loss processes in the TGA curve at 235 °C and 871 °C. The DTA curve shows an endothermic peak at about 235 °C, which is related to the first decomposition of organic compound from ZnO NPs. The second endothermic peak at about 871 °C reflected decomposition of ZnO to metal Zn and O2. At temperatures above 1000 °C there was no weight loss in the TGA curve, indicating that the metal Zn was stable within this temperature range. Figure 4 shows the green synthesized ZnO NPs, which was confirmed by the characteristic peaks observed in the XRD image (JCPDS CARD NO: 36-1451, ICDD (The International Centre for Diffraction Data ver. 2002)). Diffraction patterns were observed at the 2h angles of 31.71, 34.41, 36.23, 47.67 and 57.61°, 62.98, 66.51, 68.05 and 69.21°, which were equivalent to (1 0 0), (0 0 2), (1 0 1), (1 0 2), and (1 1 0), (1 0 3), (2 0 0), (1 1 2) and (2 0 1), respectively. No other characteristic peaks corresponding to the impurities of the zinc nitrate. Figure 5 shows the SEM image of the green synthesized ZnO NPs. This image indicated that ZnO NPs were predominantly spherical in shape. The EDX results revealed that the particles consisted of 18.33% Zn and 32.41% O2 (Fig. 6). Taken together, the SEM and EDX results confirm the presence of ZnO NPs.
Figure 4. XRD-ray diffraction patterns of ZnO NPs green synthesized by Coptidis Rhizoma.
Figure 5. SEM image of ZnO NPs.
P. C. Nagajyothi et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4298–4303
Figure 6. EDX spectrum of green synthesized ZnO NPs.
Figure 7. (a) TEM images (b) high resolution TEM image (c and d) selected area electron diffraction (SAED).
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The TEM images of ZnO NPs are shown in Figure 7. The images revealed that green synthesized ZnO NPs were nearly spherical or rod shaped with sizes of 13.96, 25.20, 17.03, 6.55, 4.81, 11.61, 5.53, 6.32, 2.90, 4.08, 7.53, 9.28, 5.73, 4.20, 5.38 and 5.93 nm (mean size8.502). The selective area electron diffraction (SAED) patterns exhibited a set of rings containing bright spots, suggesting that ZnO NPs are crystalline in nature (Fig. 7c and d). TEM images clearly shows that the size of green synthesized ZnO NPs is below 26 nm compared to previous studies.23,24 The antibacterial activity of ZnO NPs was studied against Grampositive (Bacillus megaterium, Bacillus pumilus and Bacillus cereus) and Gram-negative (Escherichia coli) bacteria using the disc diffusion method (Fig. 8). The maximum antibacterial activity was observed against E. coli (11 mm) and the minimum against B. megaterium (10 mm), B. pumilus (9 mm) and B. cereus (9 mm). The size of the inhibition zone was different according to the type of pathogens, synthesis method, shape, size and the concentrations of ZnO NPs.27 Roselli et al.28 reported that ZnO NPs at concentrations of 104 to 103 M did not affect cell permeability. Sawai29 explained the viability of using ZnO as a biocidal agent and determined that the antibacterial activity was dependent on agent concentration. There are several mechanisms which have been proposed to explain the antibacterial activity of ZnO NPs. The production of hydrogen peroxide from the surface of ZnO is suggested as an efficient mean for the inhibition of bacterial growth.30 Another possible mechanism for ZnO antibacterial activity is the
release of Zn2+ ions which damage cell membrane and interact with intracellular contents.31 There is convincing evidence from the result that ZnO NPs may be useful in NPs-coated medical devices against microbes. Antioxidants are micro constituents that can scavenge reactive oxygen species (ROS) by terminating the oxidizing chain reaction.32,33 ROS play a vital role in the pathogenesis of a variety of degenerative conditions including cardiovascular diseases and carcinogenesis.34,35 DPPH has been widely applied to evaluate the radical scavenging ability of green synthesized nanoparticles.36 In the present study, the DPPH radical scavenging activity of the four concentrations were in the following order 1 mg/ml (52.34%) >0.5 mg/ ml (51.57%); >0.25 mg/ml (51.19%) >0.125 mg/ml (38.12%) in Figure 9, respectively. The cell viability of the green synthesized ZnO NPs was evaluated in vitro against the RAW 264.7 cell line at different concentrations. The ZnO NPs did not reduce RAW 264.7 cell viability. ZnO NPs have no cytotoxic effect at concentration of 1 mg/ml. These findings indicate that green synthesized ZnO NPs had no toxic effect on the RAW 264.7 cell line. In order to eliminate the probability of possible interference of ZnO NPs with XTT cell viability assay, a vehicular control consisting of ZnO NPs media and XTT reagent in the absence of cells was used. In conclusion, ZnO NPs with an average size of 8.50 nm and spherical and rod shapes were synthesized using C. Rhizoma extract. The ZnO NPs were characterized by UV–vis spectroscopy,
Figure 8. Antibacterial activity of ZnO NPs synthesized from Coptidis Rhizoma against Gram-positive and Gram-negative bacteria.
Figure 9. Antioxidant activity of the green synthesized ZnO NPs by DPPH assay using ascorbic acid as a standard.
P. C. Nagajyothi et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4298–4303
FTIR, TGA, XRD, TEM, SAED and SEM-EDX. Green synthesized ZnO NPs exhibited moderate antibacterial and significant antioxidant activity and demonstrated no cytotoxicity against RAW 264.7 cells, indicating that they have the potential for use in medical applications. Acknowledgment This work was supported by the Dongguk University Research Fund of 2013. Supplementary data Supplementary data (the details of the experiment and aqueous extract of C. Rhizoma, synthesis and characterization of ZnO NPs) associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.07.023.
11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.
References and notes 28. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
ˇ echalova, V.; Kalendova, A. J. Phys. Chem. Solids 2007, 68, 1096. C Suliman, A. E.; Tang, Y.; Xu, L. Sol. Energy Mater. Sol. Cells 2007, 91, 1658. Parida, K. M.; Dash, S. S.; Das, D. P. J. Colloid Interface Sci. 2006, 298, 787. Gao, T.; Wang, T. H. Appl. Phys. A 2005, 80, 1451. Chen, J. C.; Tang, C. T. J. Hazard. Mater. 2007, 142, 88. Scarisoreanu, N.; Metai, D. G.; Dinescu, G.; Epurescu, G.; Ghica, C.; Nistor, L. C.; Dinescu, M. Appl. Surf. Sci. 2005, 247, 518. Ni, Y. H.; Wei, X. W.; Hong, J. M.; Ye, Y. Mater. Sci. Eng., B 2005, 121, 42. Chang, S.; Yoon, S. O.; Park, H. J.; Sakai, A. Mater. Lett. 2002, 53, 432. Ristiac, M.; Musiac, S.; Ivanda, M.; Popoviac, S. J. Alloys Compd. 2005, 397, L1. Wu, J. J.; Liu, S. C. Adv. Mater. 2002, 14, 215.
29. 30. 31. 32. 33. 34. 35. 36.
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Wang, R. C.; Tsai, C. C. Appl. Phys. A 2009, 94, 241. Lamas, D. G.; Lascalea, G. E.; Walsoc, N. E. J. Eur. Ceram. Soc. 1998, 18, 1217. Badhuri, S.; Badhuri, S. B. Nanostruct. Mater. 1997, 8, 755. Khorsand, Z.; Abid, A.; Majid, W. H.; Wang, H. Z.; Yousefi, R.; Golsheikh, M.; Ren, Z. F. Ultrason. Sonochem. 2013, 20, 395. Kooti, M.; NagdhiSedish, A. J. Chem. 2013, 2013. Article ID 562028. Rajesh, D.; Vara Lakshmi, B.; Sunandana, C. S. Physica B 2012, 407, 4537. Shetty, A.; Nanda, K. Appl. Phys. A 2012, 109, 151. Singh, O.; Kohli, N.; Singh, R. C. Sens. Actuators, B 2013, 178, 149. Vazquez, A.; Lopez, I. A.; Gomez, I. J. Mater. Sci. 2013, 48, 2701. Shervani, Z.; Ikushima, Y.; Sato, M.; Kawanami, H.; Hakuta, Y.; Yokoyama, T.; Nagase, T.; Kuneida, H.; Aramaki, K. Colloid Polym. Sci. 2008, 286, 403. Nestor, A. R. V.; Mendieta, V. S.; Lopez, M. A. C.; Espinosa, R. M. G.; Alatorre, J. A. A. Mater. Lett. 2008, 62, 3103. Ravindra, P. S.; Vineet, K. S.; Raghvendra, S. Y.; Prashant, K. S.; Avinash, C. P. Adv. Mater. Lett. 2011, 2, 313. Sangeetha, G.; Rajeshwari, S.; Rajendran, V. Mater. Res. Bull. 2011, 46, 2560. Nagajyothi, P. C.; Minh An, T. N.; Sreekanth, T. V. M.; Jae-il, L.; Dong Joo, L.; Lee, K. D. Mater. Lett. 2013, 108, 160. Huang, K. C. The Pharmacology of Chinese Herbs; CRC Press: Boca Raton, FL, 1999. pp 381–382. Yu, Y. Y.; Wang, B. C.; Peng, L.; Wang, J. B.; Zeng, C.; Chongqing, C. J. Univ. (Nat. Sci. Ed.) 2006, 29, 107. Zhang, L.; Jiang, Y.; Ding, Y.; Daskalakis, N.; Jeuken, L.; Povey, M.; O0 Neill, A. J.; York, D. W. Prog. Nat. Sci. 2008, 18, 939. Roselli, M.; Finamore, A.; Garaguso, I.; Britti, M. S.; Mengheri, E. J. Nutr. 2003, 1334077. Sawai, J. J. Microbiol. Methods 2003, 54, 177. Yamamoto, O. Int. J. Inorg. Mater. 2001, 3, 643. Brayner, R.; Ferrari-Iliou, R.; Brivois, N.; Djediat, S.; Benedetti, M. F.; Fievet, F. Nano Lett. 2006, 6, 866. Tyug, T. S.; Johar, M. H.; Ismail, A. Int. J. Food Prop. 2010, 13, 682. Sowndhararajan, K.; Joseph, J. M.; Manian, S. Int. J. Food Prop. 2013, 16, 1717. Taniyama, Y.; Griendling, K. K. Hypertension 2003, 42, 1075. Waris, G.; Ahsan, H. J. Carcinog. 2006, 5, 14. Phang, C.-Weng; Abd Malek, S. N.; Ibrahim, H. BMC Complement. Alternat. Med. 2013, 243, 13.
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Characterization, antibacterial, antioxidant, and cytotoxic activities of ZnO nanoparticles using Coptidis Rhizoma P. C. Nagajyothi a, , T. V. M. Sreekanth b, , Clement O. Tettey a, Yang In Jun a, Shin Heung Mook a,⇑ a b
Department of Physiology, College of Oriental Medicine, Dongguk University, Gyeongju, South Korea Department of Life Chemistry, Catholic University of Daegu, Gyeongsan 712-702, South Korea
a r t i c l e
i n f o
Article history: Received 29 April 2014 Revised 2 July 2014 Accepted 8 July 2014 Available online 15 July 2014 Keywords: Green synthesis Coptidis Rhizoma Antibacterial Antioxidant Cytotoxic activity
a b s t r a c t Here, we report a simple, eco-friendly and inexpensive approach for the synthesis of zinc oxide nanoparticles (ZnO NPs) using Coptidis Rhizoma. The ZnO NPs were characterized by UV–visible absorption spectroscopy, FTIR, SEM-EDX, TGA, TEM, SAED and XRD. TEM images confirmed the presence of spherical and rod shaped ZnO NPs in the range of 2.90–25.20 nm. Green synthesized ZnO NPS exhibited moderate antibacterial activity against Gram-positive and Gram-negative bacteria and excellent DPPH free radical scavenging activity. Synthesized ZnO NPs had no toxic effects on the RAW 264.7 cell line. Ó 2014 Published by Elsevier Ltd.
Nanoparticles with various sizes and shapes have garnered great interest in the past decade owing to their excellent optical, electronic and chemical properties, which are not displayed in the bulk state of metal. ZnO NPs are widely used in industrial applications such as pigments,1 dye-sensitized solar cells,2 photocatalysts,3 and sensors.4 ZnO is a wide band-gap semi-conductor (II–IV) with an energy gap of 3.37 eV at room temperature and ZnO NPs have a great advantage in their application as catalysts due to their large surface area and high catalytic activity.5 A wide variety of physical and chemical processes for the synthesis of ZnO nanostructures have been developed including laser ablation,6 hydrothermal,7 electrochemical depositions,8 sol–gel,9 chemical vapor deposition10 thermal decomposition,11 combustion,12,13 ultrasound,14 microwave-assisted combustion method,15 two-step mechano chemical–thermal synthesis,16 anodization,17 co-precipitation,18 and electrophoretic deposition19 methods. Even though, the exact mechanism of shape and size control of these structures have not been established and mentioned in the literature for the preparation of a number of self-assembled structures reported.20 Chemical synthesis methods lead to the adsorption of toxic chemical species on to the surface that may have adverse effects in medical applications. As a result, researchers in the field of synthesis of nanoparticles and assembly have turned to ⇑ Corresponding author. Tel.: +82 54 770 2372; fax: +82 54 742 5441.
E-mail address: [email protected] (S.H. Mook). Authors have made an equal contribution.
http://dx.doi.org/10.1016/j.bmcl.2014.07.023 0960-894X/Ó 2014 Published by Elsevier Ltd.
biosynthesis methods for nanoparticles production, which employ plants, fungi, bacteria and enzymes and represent possible ecofriendly alternatives to chemical and physical methods.21 To date, green synthesis of ZnO NPs by plants such as Calotropis procera,22 Aloe barbadensismiller,23 and Poncirus trifoliate24 have been reported. Coptidis Rhizoma is the dried rhizome of Coptis chinensis Franch (family; Ranunculaceae). C. Rhizoma, has common uses in traditional oriental medicine owing to its various pharmacological effects, which include anti-bacterial, antifungal, antiviral, antidiabetic, anticancer and anti-inflammatory activities,25 as well as the ability to relax blood vessels, reduce fever, stimulates circulation and prevent cardiovascular diseases.26 The present study was conducted to (i) characterize ZnO NPs prepared by the green synthesis method and (ii) investigate their antibacterial, antioxidant and cytotoxic activities. The UV–vis spectroscopy analysis was used for the preliminary characterization of ZnO NPs. The UV–vis absorption spectra for green synthesized ZnO NPs showed an adsorption maximum at 344 nm (Fig. 1). C. Rhizoma contains different classes of organic compounds (1° and 2° amines, aromatic, alkynes, nitro groups and alkyl halides) involved in the reduction and formation of ZnO NPs that were analyzed by FTIR. The bands of green synthesized ZnO NPs from C. Rhizoma were observed at 3410.28, 3062.89, 1603.74, 1506.42, 1479.71, 1384.47, 1275.27, 1232.66, 1102.80, 1004.44, 906.41, 825.50 and 630.93 cm1 (Fig. 2b). The bands of C. Rhizoma extract were observed at 3387, 2927, 1639,
P. C. Nagajyothi et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4298–4303
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Figure 1. UV–vis spectrum of ZnO NPs green synthesized by treating zinc nitrate with using Coptidis Rhizoma.
Figure 2. FTIR spectrum of (a) Coptidis Rhizoma (b) green synthesized ZnO NPs.
1509, 1459, 1363, 1273, 1235, 1150, 1029, 863 and 614 cm1 (Fig. 2a). C. Rhizoma extract bands shifted to 3410.28, 3062.89, 1603.74, 1506.42, 1479.71, 1384.47, 1275.27, 1232.66, 1102.80, 1004.44, 906.41, 825.50 and 630.93 cm1. The absorbance bands
were observed in the region of 500–3500 cm1and consisted of 3410.28 cm1 (secondary amines), 3062.89 cm1 (aromatic), 1603.74 (1° amines), 1506.42 and 1479.71 (aromatic), 1384.47 (nitro groups), 1275.27 (aromatic amines), 1232.66 and 1102.80
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Figure 3. TGA analysis of ZnO NPs synthesized by Coptidis Rhizoma.
(aliphatic amines), 1004.44 (alcohols, carboxylic acids, esters and ethers), 906.41 (1° and 2° amines), 825.50 (alkyl halides) and 630.93 (alkynes). Figure 3 shows the TGA and DTA curves of ZnO NPs measured in static air at 0–1000 °C. There were clearly two primarily weight
loss processes in the TGA curve at 235 °C and 871 °C. The DTA curve shows an endothermic peak at about 235 °C, which is related to the first decomposition of organic compound from ZnO NPs. The second endothermic peak at about 871 °C reflected decomposition of ZnO to metal Zn and O2. At temperatures above 1000 °C there was no weight loss in the TGA curve, indicating that the metal Zn was stable within this temperature range. Figure 4 shows the green synthesized ZnO NPs, which was confirmed by the characteristic peaks observed in the XRD image (JCPDS CARD NO: 36-1451, ICDD (The International Centre for Diffraction Data ver. 2002)). Diffraction patterns were observed at the 2h angles of 31.71, 34.41, 36.23, 47.67 and 57.61°, 62.98, 66.51, 68.05 and 69.21°, which were equivalent to (1 0 0), (0 0 2), (1 0 1), (1 0 2), and (1 1 0), (1 0 3), (2 0 0), (1 1 2) and (2 0 1), respectively. No other characteristic peaks corresponding to the impurities of the zinc nitrate. Figure 5 shows the SEM image of the green synthesized ZnO NPs. This image indicated that ZnO NPs were predominantly spherical in shape. The EDX results revealed that the particles consisted of 18.33% Zn and 32.41% O2 (Fig. 6). Taken together, the SEM and EDX results confirm the presence of ZnO NPs.
Figure 4. XRD-ray diffraction patterns of ZnO NPs green synthesized by Coptidis Rhizoma.
Figure 5. SEM image of ZnO NPs.
P. C. Nagajyothi et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4298–4303
Figure 6. EDX spectrum of green synthesized ZnO NPs.
Figure 7. (a) TEM images (b) high resolution TEM image (c and d) selected area electron diffraction (SAED).
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The TEM images of ZnO NPs are shown in Figure 7. The images revealed that green synthesized ZnO NPs were nearly spherical or rod shaped with sizes of 13.96, 25.20, 17.03, 6.55, 4.81, 11.61, 5.53, 6.32, 2.90, 4.08, 7.53, 9.28, 5.73, 4.20, 5.38 and 5.93 nm (mean size8.502). The selective area electron diffraction (SAED) patterns exhibited a set of rings containing bright spots, suggesting that ZnO NPs are crystalline in nature (Fig. 7c and d). TEM images clearly shows that the size of green synthesized ZnO NPs is below 26 nm compared to previous studies.23,24 The antibacterial activity of ZnO NPs was studied against Grampositive (Bacillus megaterium, Bacillus pumilus and Bacillus cereus) and Gram-negative (Escherichia coli) bacteria using the disc diffusion method (Fig. 8). The maximum antibacterial activity was observed against E. coli (11 mm) and the minimum against B. megaterium (10 mm), B. pumilus (9 mm) and B. cereus (9 mm). The size of the inhibition zone was different according to the type of pathogens, synthesis method, shape, size and the concentrations of ZnO NPs.27 Roselli et al.28 reported that ZnO NPs at concentrations of 104 to 103 M did not affect cell permeability. Sawai29 explained the viability of using ZnO as a biocidal agent and determined that the antibacterial activity was dependent on agent concentration. There are several mechanisms which have been proposed to explain the antibacterial activity of ZnO NPs. The production of hydrogen peroxide from the surface of ZnO is suggested as an efficient mean for the inhibition of bacterial growth.30 Another possible mechanism for ZnO antibacterial activity is the
release of Zn2+ ions which damage cell membrane and interact with intracellular contents.31 There is convincing evidence from the result that ZnO NPs may be useful in NPs-coated medical devices against microbes. Antioxidants are micro constituents that can scavenge reactive oxygen species (ROS) by terminating the oxidizing chain reaction.32,33 ROS play a vital role in the pathogenesis of a variety of degenerative conditions including cardiovascular diseases and carcinogenesis.34,35 DPPH has been widely applied to evaluate the radical scavenging ability of green synthesized nanoparticles.36 In the present study, the DPPH radical scavenging activity of the four concentrations were in the following order 1 mg/ml (52.34%) >0.5 mg/ ml (51.57%); >0.25 mg/ml (51.19%) >0.125 mg/ml (38.12%) in Figure 9, respectively. The cell viability of the green synthesized ZnO NPs was evaluated in vitro against the RAW 264.7 cell line at different concentrations. The ZnO NPs did not reduce RAW 264.7 cell viability. ZnO NPs have no cytotoxic effect at concentration of 1 mg/ml. These findings indicate that green synthesized ZnO NPs had no toxic effect on the RAW 264.7 cell line. In order to eliminate the probability of possible interference of ZnO NPs with XTT cell viability assay, a vehicular control consisting of ZnO NPs media and XTT reagent in the absence of cells was used. In conclusion, ZnO NPs with an average size of 8.50 nm and spherical and rod shapes were synthesized using C. Rhizoma extract. The ZnO NPs were characterized by UV–vis spectroscopy,
Figure 8. Antibacterial activity of ZnO NPs synthesized from Coptidis Rhizoma against Gram-positive and Gram-negative bacteria.
Figure 9. Antioxidant activity of the green synthesized ZnO NPs by DPPH assay using ascorbic acid as a standard.
P. C. Nagajyothi et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4298–4303
FTIR, TGA, XRD, TEM, SAED and SEM-EDX. Green synthesized ZnO NPs exhibited moderate antibacterial and significant antioxidant activity and demonstrated no cytotoxicity against RAW 264.7 cells, indicating that they have the potential for use in medical applications. Acknowledgment This work was supported by the Dongguk University Research Fund of 2013. Supplementary data Supplementary data (the details of the experiment and aqueous extract of C. Rhizoma, synthesis and characterization of ZnO NPs) associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.07.023.
11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.
References and notes 28. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
ˇ echalova, V.; Kalendova, A. J. Phys. Chem. Solids 2007, 68, 1096. C Suliman, A. E.; Tang, Y.; Xu, L. Sol. Energy Mater. Sol. Cells 2007, 91, 1658. Parida, K. M.; Dash, S. S.; Das, D. P. J. Colloid Interface Sci. 2006, 298, 787. Gao, T.; Wang, T. H. Appl. Phys. A 2005, 80, 1451. Chen, J. C.; Tang, C. T. J. Hazard. Mater. 2007, 142, 88. Scarisoreanu, N.; Metai, D. G.; Dinescu, G.; Epurescu, G.; Ghica, C.; Nistor, L. C.; Dinescu, M. Appl. Surf. Sci. 2005, 247, 518. Ni, Y. H.; Wei, X. W.; Hong, J. M.; Ye, Y. Mater. Sci. Eng., B 2005, 121, 42. Chang, S.; Yoon, S. O.; Park, H. J.; Sakai, A. Mater. Lett. 2002, 53, 432. Ristiac, M.; Musiac, S.; Ivanda, M.; Popoviac, S. J. Alloys Compd. 2005, 397, L1. Wu, J. J.; Liu, S. C. Adv. Mater. 2002, 14, 215.
29. 30. 31. 32. 33. 34. 35. 36.
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Wang, R. C.; Tsai, C. C. Appl. Phys. A 2009, 94, 241. Lamas, D. G.; Lascalea, G. E.; Walsoc, N. E. J. Eur. Ceram. Soc. 1998, 18, 1217. Badhuri, S.; Badhuri, S. B. Nanostruct. Mater. 1997, 8, 755. Khorsand, Z.; Abid, A.; Majid, W. H.; Wang, H. Z.; Yousefi, R.; Golsheikh, M.; Ren, Z. F. Ultrason. Sonochem. 2013, 20, 395. Kooti, M.; NagdhiSedish, A. J. Chem. 2013, 2013. Article ID 562028. Rajesh, D.; Vara Lakshmi, B.; Sunandana, C. S. Physica B 2012, 407, 4537. Shetty, A.; Nanda, K. Appl. Phys. A 2012, 109, 151. Singh, O.; Kohli, N.; Singh, R. C. Sens. Actuators, B 2013, 178, 149. Vazquez, A.; Lopez, I. A.; Gomez, I. J. Mater. Sci. 2013, 48, 2701. Shervani, Z.; Ikushima, Y.; Sato, M.; Kawanami, H.; Hakuta, Y.; Yokoyama, T.; Nagase, T.; Kuneida, H.; Aramaki, K. Colloid Polym. Sci. 2008, 286, 403. Nestor, A. R. V.; Mendieta, V. S.; Lopez, M. A. C.; Espinosa, R. M. G.; Alatorre, J. A. A. Mater. Lett. 2008, 62, 3103. Ravindra, P. S.; Vineet, K. S.; Raghvendra, S. Y.; Prashant, K. S.; Avinash, C. P. Adv. Mater. Lett. 2011, 2, 313. Sangeetha, G.; Rajeshwari, S.; Rajendran, V. Mater. Res. Bull. 2011, 46, 2560. Nagajyothi, P. C.; Minh An, T. N.; Sreekanth, T. V. M.; Jae-il, L.; Dong Joo, L.; Lee, K. D. Mater. Lett. 2013, 108, 160. Huang, K. C. The Pharmacology of Chinese Herbs; CRC Press: Boca Raton, FL, 1999. pp 381–382. Yu, Y. Y.; Wang, B. C.; Peng, L.; Wang, J. B.; Zeng, C.; Chongqing, C. J. Univ. (Nat. Sci. Ed.) 2006, 29, 107. Zhang, L.; Jiang, Y.; Ding, Y.; Daskalakis, N.; Jeuken, L.; Povey, M.; O0 Neill, A. J.; York, D. W. Prog. Nat. Sci. 2008, 18, 939. Roselli, M.; Finamore, A.; Garaguso, I.; Britti, M. S.; Mengheri, E. J. Nutr. 2003, 1334077. Sawai, J. J. Microbiol. Methods 2003, 54, 177. Yamamoto, O. Int. J. Inorg. Mater. 2001, 3, 643. Brayner, R.; Ferrari-Iliou, R.; Brivois, N.; Djediat, S.; Benedetti, M. F.; Fievet, F. Nano Lett. 2006, 6, 866. Tyug, T. S.; Johar, M. H.; Ismail, A. Int. J. Food Prop. 2010, 13, 682. Sowndhararajan, K.; Joseph, J. M.; Manian, S. Int. J. Food Prop. 2013, 16, 1717. Taniyama, Y.; Griendling, K. K. Hypertension 2003, 42, 1075. Waris, G.; Ahsan, H. J. Carcinog. 2006, 5, 14. Phang, C.-Weng; Abd Malek, S. N.; Ibrahim, H. BMC Complement. Alternat. Med. 2013, 243, 13.