http://informahealthcare.com/phb ISSN 1388-0209 print/ISSN 1744-5116 online Editor-in-Chief: John M. Pezzuto Pharm Biol, Early Online: 1–6 ! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2014.942868
ORIGINAL ARTICLE
Green biosynthesis of silver nanoparticles using Quercus brantii (oak) leaves hydroalcoholic extract Hassan Korbekandi1, Mohammad Reza Chitsazi2, Gholamreza Asghari3, Rahim Bahri Najafi2, Akbar Badii4, and Siavash Iravani3
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Department of Genetics & Molecular Biology, Faculty of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran, 2Department of Pharmaceutics, 3Department of Pharmacognosy, and 4Department of Biochemistry, Faculty of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran Abstract
Keywords
Context: There is an ever-growing need to develop green, non-toxic, and eco-friendly procedures for synthesis and assembly of nanoparticles (NPs) with the desired morphologies and sizes. The hydroalcoholic extract of Persian oak leaves [Quercus brantii Lindl. (Fagaceae)] contains high content of phenolic and flavonoid compounds with strong antioxidant activities, and it seems that this plant can be considered a good candidate for metal nanoparticle synthesis. Objective: The potential of Q. brantii leaves in the production of silver NPs and the effect of the extract ethanol concentration on the produced NPs were studied. Materials and methods: Quercus brantii leaves were freshly collected, air-dried at room temperature, powdered, and sieved. Hydroalcoholic extracts (70% and 96%) were prepared by percolation of the plant powder. The reaction mixtures contained (final concentrations): AgNO3 (1 mM) as the substrate, plant extract as the biocatalyst, and phosphate buffer (pH ¼ 7, 100 mM) as the reaction medium. Silver ions were determined using atomic absorption analysis. Particle size distribution of NPs was analyzed using Nano-Zeta Sizer (Malvern Instruments Ltd, Malvern, UK). Samples for TEM were prepared by drop-coating the silver nanoparticle suspensions onto carbon-coated copper grids. Results: Hydroalcoholic extract (96%) of Q. brantii successfully produced quite small (as small as 0.83 nm and the mean size of 6 nm), spherical, and poly-dispersed NPs with low aggregates. The conversion was fast and completed in 5 h. Discussion and conclusion: This plant and the extraction method seem to be quiet attractive for industrial scale production of NPs.
Bioreduction, flavonoids, green synthesis, nanoparticle synthesis, phenolic compounds, tannins
Introduction Silver nanoparticles (NPs) are the most commercialized nanomaterials, and have the potential for large-scale applications in the formulation of dental resin composites, bone cement, water and air filters, clothing and textiles, medical devices and implants, cosmetics, and packaging. In addition to their antimicrobial and antiviral properties, silver NPs and silver nanocomposites or nanohybrids have other interesting characteristics which will enable them to be used in catalysts, biosensors, drug delivery, tissue regeneration, conductive inks, electronic devices, and solar cells (Iravani et al., 2014b; Korbekandi & Iravani, 2012). Silver NPs are prepared by a variety of methods, but majority of them are either expensive or environmentally hazardous. Moreover, the use of toxic chemical materials, high temperature, and production of
Correspondence: Dr. Hassan Korbekandi, Department of Genetics & Molecular Biology, Faculty of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran. E-mail: [email protected]
History Received 4 June 2014 Revised 21 June 2014 Accepted 5 July 2014 Published online 20 February 2015
hazardous by-products make it necessary to develop safe, non-toxic, and eco-friendly (green chemistry) methods for synthesis and assembly of silver NPs. The use of microorganisms and biological systems in this area is rapidly developing due to their growing success and ease of formation of NPs (Iravani et al., 2014a,b; Mariselvam et al., 2014; Nalawade et al., 2014; Sathiya & Akilandeswari, 2014; Sun et al., 2014; Suresh et al., 2014; Umoren et al., 2014; Velmurugan et al., 2014). Recent studies tend to provide a controlled and up-scalable process for synthesis of monodispersed and highly stable NPs. Plants were found to be good candidates for synthesizing silver NPs (Iravani, 2011; Iravani & Zolfaghari, 2013; Iravani et al., 2014a,b; Jain et al., 2009; Jha & Prasad, 2010; Jha et al., 2009a,b; Mariselvam et al., 2014; Nalawade et al., 2014; Sathiya & Akilandeswari, 2014; Sun et al., 2014; Suresh et al., 2014; Umoren et al., 2014; Velmurugan et al., 2014). Persian oak, Quercus brantii Lindl. (Fagaceae), is one of the most important and abundant plant species in Iran, specifically in Zagros zone (South Zagros)
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(Chahardooli et al., 2014). Extract of Q. brantii is useful for wound healing. Furthermore, Quercus species can be used for gastrointestinal (GI) diseases, cancer, diarrhea, inflammation, and burns (Mirzaei & Mirzaei, 2013; Safary et al., 2009; Seddik et al., 2010). The hydroalcoholic extract of Q. brantii leaves contains high content of phenolic and flavonoid compounds with strong antioxidant activities, and it seems that this plant can be considered as a good candidate for metal nanoparticle synthesis. Thus, the objectives were to study the potential of Q. brantii leaves in production of NPs, and the effect of the extract ethanol concentration on the produced NPs.
Materials and methods Pharmaceutical Biology Downloaded from informahealthcare.com by McMaster University on 02/24/15 For personal use only.
Preparation of Q. brantii extract Quercus brantii leaves were freshly collected between August and September 2013 from Isfahan Agricultural and Natural Resources Research Center (Herbarium number: 1743). Plant identities were confirmed by Dr. Rahimnejad (Biology Department, Isfahan University). After washing, air drying at room temperature, and powdering, the plant powder was screened using a 17 mesh sieve. Hydroalcoholic extract was prepared by percolation (about 48 h). The plant powder (200 g) was extracted with hydroalcoholic solution (70% and 96%, 500 mL) using a 2 L percolator. The extract was concentrated in rotary evaporator to 50 mL, and then freezedried (ChristÕ , Martin Christ, Osterode am Harz, Germany).
Pharm Biol, Early Online: 1–6
Atomic absorption (AA) spectrophotometer (Perkin-Elmer Zeeman 3030, E. Merck, Darmstadt, Germany) with pyrocoated tube with platform, pretreatment temperature of 650 C and atomize temperature of 1600 C were used. The samples (1 mL) were centrifuged (Sigma D-37520, Sigma Laborzentrifugen, Harz, Germany) at 2240 g for 45 min before the analysis.
Results and discussion Monitoring the synthesis of silver NPs Hydroalcoholic extract (70%) of Q. brantii leaves transformed 99.8% of AgNO3 to Ag NPs after 96 h (Figure 1). The reaction was almost complete (conversion ¼ 99.8%) after 5 h and the conversion rate was maximum (0.985 mmole h1) between hour 0 and 1. Hydroalcoholic extract (96%) of Q. brantii leaves transformed 100% of AgNO3 to Ag NPs after 70 h (Figure 2). The reaction was also complete (conversion% ¼ 100%) after 5 h and the conversion rate was maximum (0.965 mmole h1) between hour 0 and 1.
Preparation of the reaction mixtures The reaction mixtures contained (final concentrations): AgNO3 (1 mM) as the substrate, concentrated and freezedried plant extract (equivalent to 100 g plant powder) as the biocatalyst, and phosphate buffer (pH ¼ 7, 100 mM) as the medium in the reaction mixture (50 mL). The aforementioned ingredients were added in appropriate volumes into DuranÕ bottles (Martin Christ, Osterode am Harz, Germany) (100 ml) and were incubated (70 rpm) at room temperature. Characterization and analysis
Figure 1. Time course of silver nitrate conversion to silver NPs by 70% hydroalcoholic extract of Q. brantii leaves.
Transmission electron microscopy analysis Transmission electron microscopy (TEM) was performed on selected samples in order to investigate the process of the formation of silver NPs and study their sizes and shapes. Samples for TEM were prepared by drop-coating the silver nanoparticle suspensions onto carbon-coated copper grids. Micrographs were obtained using EM 900 ZEISS transmission electron microscope (Malvern Instruments Ltd, Malvern, UK). Dynamic light scattering analysis Particle size distribution of NPs was analyzed using Nano-Zeta Sizer (Nano ZS, ZEN 3600, Malvern NanoÕ , Malvern Instruments Ltd, Malvern, UK). Atomic absorption analysis In order to quantify the substrate (Ag+) and calculate the conversion of it to Ag0 NPs, Ag+ ions were determined using atomic absorption analysis by a Ag lamp at 328 nm.
Figure 2. Time course of silver nitrate conversion to silver NPs by 96% hydroalcoholic extract of Q. brantii leaves.
DOI: 10.3109/13880209.2014.942868
DLS analysis Hydroalcoholic extract (70%) Three peaks were seen in DLS analysis of the reaction mixture after 5 h; therefore, the NPs were polydispersed (Figure 3). Single NPs with a diameter of 455 nm were
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Figure 3. Particle size analysis of NPs produced by 70% hydroalcoholic extract of Q. brantii leaves after 5 h of biotransformation.
Figure 4. Particle size analysis of NPs produced by 70% hydroalcoholic extract of Q. brantii leaves after 70 h of biotransformation.
Green biosynthesis of silver NPs using oak leave extract
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the most frequent ones (81.4%) and the mean size was 433 nm (Z-Average). The smallest NPs were 4.1 nm (7.6%). The aggregates with a diameter of 4786 nm were 11.1% of the NPs. The poly-dispersity index (PDI) was 0.349 (which was near the optimum value). By increasing the time of reaction (70 h), the NPs were aggregated further (Figure 4).
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Hydroalcoholic extract (96%) Three peaks were seen in DLS analysis of the reaction mixture after 4 h; therefore, the NPs were poly-dispersed (Figure 5). Single NPs with a diameter of 8.7 nm were the
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Figure 5. Particle size analysis of NPs produced by 96% hydroalcoholic extract of Q. brantii leaves after 4 h of biotransformation.
Figure 6. Particle size analysis of NPs produced by 96% hydroalcoholic extract of Q. brantii leaves after 48 h of biotransformation.
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most frequent ones (66.6%) and the mean size was 6 nm (Z-Average). The second most frequent (19.3%) particles were 0.82 nm (smallest NPs). The aggregates with a diameter of 2566 nm were 14.1% of the NPs. The poly-dispersity index (PDI) was 0.323 (near to optimal value). By increasing
DOI: 10.3109/13880209.2014.942868
the time of reaction (48 h, Figure 6), the peak related to 0.83 nm was disappeared and the sizes of NPs were increased. TEM analysis
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TEM micrographs of the silver NPs synthesized by using 70% hydroalcoholic extract of Q. brantii leaves after 24 h of biotransformation are shown in Figure 7. The produced silver NPs were spherical in shape. Figure 8 shows TEM micrographs of silver NPs synthesized using 96% hydroalcoholic extract of Q. brantii leaves, 24 h after the start of the reaction. The produced NPs were spherical and in some places aggregated and assembled in structures. Effect of ethanol concentration of hydroalcoholic extract on the produced NPs Biotransformation time-courses, bioconversion percents, and bioreduction rates of Ag+ to Ag0 NPs by both the extracts (Figures 1 and 2) were almost the same. By comparing the NP shapes and size distributions produced by 70% hydroalcoholic extract of Q. brantii (Figures 3, 4, and 7) and 96 hydroalcoholic extract (Figures 5, 6, and 8), it can be seen that 96 hydroalcoholic extract produced smaller and more separated silver NPs, which might be interpreted that 96% hydroalcoholic solution can extract the nanoparticle producing compounds and nanoparticle coating proteins better than 70% hydroalcoholic solution. Figure 7. TEM micrographs of the produced NPs by 70% hydroalcoholic extract of Q. brantii leaves after 24 h of biotransformation.
Figure 8. TEM micrographs of the produced NPs by 96% hydroalcoholic extract of Q. brantii leaves after 24 h of biotransformation.
Green biosynthesis of silver NPs using oak leave extract
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Mechanistic aspects Antioxidant and antilipid peroxidation activities of the leaves and fruit components of Quercus species have been reported in the literature. Based on the previous studies, tannin is the most abundant compound in the plant fruits whose major effect is antidiarrhea due to its water absorption and protein precipitation effects. Phytochemicals such as tannins, flavonoids, alkaloids, and several other aromatic compounds are secondary metabolites of the plant (Mirzaei & Mirzaei, 2013; Safary et al., 2009; Seddik et al., 2010). Phenolic and flavonoid compounds are the largest group of antioxidant compounds, chelate metals and scavenge peroxide, lipid peroxyl, and hydroperoxide (Sakai et al., 1996). Antioxidant action of phenolic compounds is due to their high tendency to chelate metals. Phenolic compounds possess hydroxyl and carboxyl groups, which may inactivate iron ions by chelating and additionally suppressing the superoxide-driven Fenton reaction, which is believed to be the most important source of reactive oxygen species (Iravani & Zolfaghari, 2011, 2013, 2014). The hydroalcoholic extract of Q. brantii leaves contains high phenolic and flavonoid compounds with strong antioxidant activities. It seems that these phytochemicals and phenolic compounds were involved in phyto-reduction of silver ions, and phytosynthesis of silver NPs. However, more efforts need to be done to understand the effect of parameters regarding the effects of various biomolecules and
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phytochemicals available in the extract of Q. brantii leaves for synthesizing NPs.
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Conclusion Since the eco-friendly synthesis of NPs of different chemical compositions, sizes, shapes, and controlled dispersity is an important aspect of nanobiotechnology and green nanotechnology, this green method was developed. Hydroalcoholic extract (96%) of Q. brantii successfully produced quite small (as small as 0.83 nm and the mean size of 6 nm), spherical, and poly-dispersed NPs with low aggregates. Results from TEM and DLS analysis demonstrated that by increasing the time of reaction, the NPs were aggregated further. The conversion was fast and completed in 5 h. Therefore, this plant and the extraction method seems to be quiet attractive for industrial scale production of NPs.
Declaration of interest The authors report no conflicts of interest. This study was a part of the research project No. 392034 and was financially supported by Faculty of Pharmacy, Isfahan University of Medical Sciences.
References Chahardooli M, Khodadadi E, Khodadadi E. (2014). Green synthesis of silver nanoparticles using oak leaf and fruit extracts (Quercus) and its antibacterial activity against plant pathogenic bacteria. Int J Biosci 4:97–103. Iravani S. (2011). Green synthesis of metal nanoparticles using plants. Green Chem 13:2638–50. Iravani S, Korbekandi H, Mirmohammadi SV, Mekanik H. (2014a). Plants in nanoparticle synthesis. Rev Adv Sci Eng 3:261–74. Iravani S, Korbekandi H, Mirmohammadi SV, Zolfaghari B. (2014b). Synthesis of silver nanoparticles: Chemical, physical, and biological methods. Res Pharm Sci 9:385–406. Iravani S, Zolfaghari B. (2011). Pharmaceutical and nutraceutical effects of Pinus pinaster bark extract. Res Pharm Sci 6:1–11. Iravani S, Zolfaghari B. (2013). Green synthesis of silver nanoparticles using Pinus eldarica bark extract. Biomed Res Int 2013:639725. Iravani S, Zolfaghari B. (2014). Phytochemical analysis of Pinus eldarica bark. Res Pharm Sci 9:243–50. Jain D, Daima HK, Kachhwaha S, Kothari SL. (2009). Synthesis of plant-mediated silver nanoparticles using papaya fruit extract and evaluation of their antimicrobial activities. Dig J Nanomater Bios 4: 557–63.
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Jha AK, Prasad K. (2010). Green synthesis of silver nanoparticles using Cycas leaf. Int J Green Nanotech: Phys Chem 1:110–17. Jha AK, Prasad K, Kumar V, Prasad K. (2009a). Biosynthesis of silver nanoparticles using Eclipta leaf. Biotechnol Prog 25:1476–9. Jha AK, Prasad K, Prasad K, Kulkarni A. (2009b). Plant system: Nature’s nanofactory. Colloids Surf B: Biointerfaces 73:219–23. Korbekandi H, Iravani S. (2012). In: Abbass A. Hashim, ed. Silver Nanoparticles, The Delivery of Nanoparticles. Rijeka, Croatia: InTech. Available from: http://www.intechopen.com/books/ the-delivery-of-nanoparticles/silver-nanoparticles Mariselvam R, Ranjitsingh AJA, Usha Raja Nanthini A, et al. (2014). Green synthesis of silver nanoparticles from the extract of the inflorescence of Cocos nucifera (Family: Arecaceae) for enhanced antibacterial activity. Spectrochim Acta Part A: Mol Biomol Spectrosc 129:537–41. Mirzaei N, Mirzaei A. (2013). Antioxidant and antimutagenic property of Iranian oak and walnut plants. Int J Biol, Pharm, Allied Sci 2: 620–9. Nalawade P, Mukherjee P, Kapoor S. (2014). Biosynthesis, characterization and antibacterial studies of silver nanoparticles using pods extract of Acacia auriculiformis. Spectrochim Acta Part A: Mol Biomol Spectrosc 129:121–4. Safary A, Motamedi H, Maleki S, Seyyednejad SM. (2009). A preliminary study on the antibacterial activity of Quercus brantii against bacterial pathogens, particularly enteric pathogens. Int J Bot 5: 176–80. Sakai N, Inada K, Okamoto M, et al. (1996). Portuloside A, a monoterpene glucoside, from Portulaca oleracea. Phytochemistry 42:1625–8. Sathiya CK, Akilandeswari S. (2014). Fabrication and characterization of silver nanoparticles using Delonix elata leaf broth. Spectrochim Acta Part A: Mol Biomol Spectrosc 128:337–41. Seddik K, Smain A, Arrar L, Abderahmane B. (2010). Effect of some phenolic compounds and quercus tannin on lipid peroxidation. World Appl Sci J 8:1144–9. Sun Q, Caia X, Li J, et al. (2014). Green synthesis of silver nanoparticles using tea leaf extract and evaluation of their stability and antibacterial activity. Colloids Surf A: Physicochem Eng Asp 444:226–31. Suresh G, Gunasekar PH, Kokila D, et al. (2014). Green synthesis of silver nanoparticles using Delphinium denudatum root extract exhibits antibacterial and mosquito larvicidal activities. Spectrochim Acta Part A: Mol Biomol Spectrosc 127:61–6. Umoren SA, Obot IB, Gasem ZM. (2014). Green synthesis and characterization of silver nanoparticles using red apple (Malus domestica) fruit extract at room temperature. J Mater Environ Sci 5: 907–14. Velmurugan P, Anbalagan K, Manosathyadevan M, et al. (2014). Green synthesis of silver and gold nanoparticles using Zingiber officinale root extract and antibacterial activity of silver nanoparticles against food pathogens. Bioprocess Biosyst Eng 37:1935–43.
ORIGINAL ARTICLE
Green biosynthesis of silver nanoparticles using Quercus brantii (oak) leaves hydroalcoholic extract Hassan Korbekandi1, Mohammad Reza Chitsazi2, Gholamreza Asghari3, Rahim Bahri Najafi2, Akbar Badii4, and Siavash Iravani3
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Department of Genetics & Molecular Biology, Faculty of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran, 2Department of Pharmaceutics, 3Department of Pharmacognosy, and 4Department of Biochemistry, Faculty of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran Abstract
Keywords
Context: There is an ever-growing need to develop green, non-toxic, and eco-friendly procedures for synthesis and assembly of nanoparticles (NPs) with the desired morphologies and sizes. The hydroalcoholic extract of Persian oak leaves [Quercus brantii Lindl. (Fagaceae)] contains high content of phenolic and flavonoid compounds with strong antioxidant activities, and it seems that this plant can be considered a good candidate for metal nanoparticle synthesis. Objective: The potential of Q. brantii leaves in the production of silver NPs and the effect of the extract ethanol concentration on the produced NPs were studied. Materials and methods: Quercus brantii leaves were freshly collected, air-dried at room temperature, powdered, and sieved. Hydroalcoholic extracts (70% and 96%) were prepared by percolation of the plant powder. The reaction mixtures contained (final concentrations): AgNO3 (1 mM) as the substrate, plant extract as the biocatalyst, and phosphate buffer (pH ¼ 7, 100 mM) as the reaction medium. Silver ions were determined using atomic absorption analysis. Particle size distribution of NPs was analyzed using Nano-Zeta Sizer (Malvern Instruments Ltd, Malvern, UK). Samples for TEM were prepared by drop-coating the silver nanoparticle suspensions onto carbon-coated copper grids. Results: Hydroalcoholic extract (96%) of Q. brantii successfully produced quite small (as small as 0.83 nm and the mean size of 6 nm), spherical, and poly-dispersed NPs with low aggregates. The conversion was fast and completed in 5 h. Discussion and conclusion: This plant and the extraction method seem to be quiet attractive for industrial scale production of NPs.
Bioreduction, flavonoids, green synthesis, nanoparticle synthesis, phenolic compounds, tannins
Introduction Silver nanoparticles (NPs) are the most commercialized nanomaterials, and have the potential for large-scale applications in the formulation of dental resin composites, bone cement, water and air filters, clothing and textiles, medical devices and implants, cosmetics, and packaging. In addition to their antimicrobial and antiviral properties, silver NPs and silver nanocomposites or nanohybrids have other interesting characteristics which will enable them to be used in catalysts, biosensors, drug delivery, tissue regeneration, conductive inks, electronic devices, and solar cells (Iravani et al., 2014b; Korbekandi & Iravani, 2012). Silver NPs are prepared by a variety of methods, but majority of them are either expensive or environmentally hazardous. Moreover, the use of toxic chemical materials, high temperature, and production of
Correspondence: Dr. Hassan Korbekandi, Department of Genetics & Molecular Biology, Faculty of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran. E-mail: [email protected]
History Received 4 June 2014 Revised 21 June 2014 Accepted 5 July 2014 Published online 20 February 2015
hazardous by-products make it necessary to develop safe, non-toxic, and eco-friendly (green chemistry) methods for synthesis and assembly of silver NPs. The use of microorganisms and biological systems in this area is rapidly developing due to their growing success and ease of formation of NPs (Iravani et al., 2014a,b; Mariselvam et al., 2014; Nalawade et al., 2014; Sathiya & Akilandeswari, 2014; Sun et al., 2014; Suresh et al., 2014; Umoren et al., 2014; Velmurugan et al., 2014). Recent studies tend to provide a controlled and up-scalable process for synthesis of monodispersed and highly stable NPs. Plants were found to be good candidates for synthesizing silver NPs (Iravani, 2011; Iravani & Zolfaghari, 2013; Iravani et al., 2014a,b; Jain et al., 2009; Jha & Prasad, 2010; Jha et al., 2009a,b; Mariselvam et al., 2014; Nalawade et al., 2014; Sathiya & Akilandeswari, 2014; Sun et al., 2014; Suresh et al., 2014; Umoren et al., 2014; Velmurugan et al., 2014). Persian oak, Quercus brantii Lindl. (Fagaceae), is one of the most important and abundant plant species in Iran, specifically in Zagros zone (South Zagros)
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(Chahardooli et al., 2014). Extract of Q. brantii is useful for wound healing. Furthermore, Quercus species can be used for gastrointestinal (GI) diseases, cancer, diarrhea, inflammation, and burns (Mirzaei & Mirzaei, 2013; Safary et al., 2009; Seddik et al., 2010). The hydroalcoholic extract of Q. brantii leaves contains high content of phenolic and flavonoid compounds with strong antioxidant activities, and it seems that this plant can be considered as a good candidate for metal nanoparticle synthesis. Thus, the objectives were to study the potential of Q. brantii leaves in production of NPs, and the effect of the extract ethanol concentration on the produced NPs.
Materials and methods Pharmaceutical Biology Downloaded from informahealthcare.com by McMaster University on 02/24/15 For personal use only.
Preparation of Q. brantii extract Quercus brantii leaves were freshly collected between August and September 2013 from Isfahan Agricultural and Natural Resources Research Center (Herbarium number: 1743). Plant identities were confirmed by Dr. Rahimnejad (Biology Department, Isfahan University). After washing, air drying at room temperature, and powdering, the plant powder was screened using a 17 mesh sieve. Hydroalcoholic extract was prepared by percolation (about 48 h). The plant powder (200 g) was extracted with hydroalcoholic solution (70% and 96%, 500 mL) using a 2 L percolator. The extract was concentrated in rotary evaporator to 50 mL, and then freezedried (ChristÕ , Martin Christ, Osterode am Harz, Germany).
Pharm Biol, Early Online: 1–6
Atomic absorption (AA) spectrophotometer (Perkin-Elmer Zeeman 3030, E. Merck, Darmstadt, Germany) with pyrocoated tube with platform, pretreatment temperature of 650 C and atomize temperature of 1600 C were used. The samples (1 mL) were centrifuged (Sigma D-37520, Sigma Laborzentrifugen, Harz, Germany) at 2240 g for 45 min before the analysis.
Results and discussion Monitoring the synthesis of silver NPs Hydroalcoholic extract (70%) of Q. brantii leaves transformed 99.8% of AgNO3 to Ag NPs after 96 h (Figure 1). The reaction was almost complete (conversion ¼ 99.8%) after 5 h and the conversion rate was maximum (0.985 mmole h1) between hour 0 and 1. Hydroalcoholic extract (96%) of Q. brantii leaves transformed 100% of AgNO3 to Ag NPs after 70 h (Figure 2). The reaction was also complete (conversion% ¼ 100%) after 5 h and the conversion rate was maximum (0.965 mmole h1) between hour 0 and 1.
Preparation of the reaction mixtures The reaction mixtures contained (final concentrations): AgNO3 (1 mM) as the substrate, concentrated and freezedried plant extract (equivalent to 100 g plant powder) as the biocatalyst, and phosphate buffer (pH ¼ 7, 100 mM) as the medium in the reaction mixture (50 mL). The aforementioned ingredients were added in appropriate volumes into DuranÕ bottles (Martin Christ, Osterode am Harz, Germany) (100 ml) and were incubated (70 rpm) at room temperature. Characterization and analysis
Figure 1. Time course of silver nitrate conversion to silver NPs by 70% hydroalcoholic extract of Q. brantii leaves.
Transmission electron microscopy analysis Transmission electron microscopy (TEM) was performed on selected samples in order to investigate the process of the formation of silver NPs and study their sizes and shapes. Samples for TEM were prepared by drop-coating the silver nanoparticle suspensions onto carbon-coated copper grids. Micrographs were obtained using EM 900 ZEISS transmission electron microscope (Malvern Instruments Ltd, Malvern, UK). Dynamic light scattering analysis Particle size distribution of NPs was analyzed using Nano-Zeta Sizer (Nano ZS, ZEN 3600, Malvern NanoÕ , Malvern Instruments Ltd, Malvern, UK). Atomic absorption analysis In order to quantify the substrate (Ag+) and calculate the conversion of it to Ag0 NPs, Ag+ ions were determined using atomic absorption analysis by a Ag lamp at 328 nm.
Figure 2. Time course of silver nitrate conversion to silver NPs by 96% hydroalcoholic extract of Q. brantii leaves.
DOI: 10.3109/13880209.2014.942868
DLS analysis Hydroalcoholic extract (70%) Three peaks were seen in DLS analysis of the reaction mixture after 5 h; therefore, the NPs were polydispersed (Figure 3). Single NPs with a diameter of 455 nm were
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Figure 3. Particle size analysis of NPs produced by 70% hydroalcoholic extract of Q. brantii leaves after 5 h of biotransformation.
Figure 4. Particle size analysis of NPs produced by 70% hydroalcoholic extract of Q. brantii leaves after 70 h of biotransformation.
Green biosynthesis of silver NPs using oak leave extract
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the most frequent ones (81.4%) and the mean size was 433 nm (Z-Average). The smallest NPs were 4.1 nm (7.6%). The aggregates with a diameter of 4786 nm were 11.1% of the NPs. The poly-dispersity index (PDI) was 0.349 (which was near the optimum value). By increasing the time of reaction (70 h), the NPs were aggregated further (Figure 4).
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Hydroalcoholic extract (96%) Three peaks were seen in DLS analysis of the reaction mixture after 4 h; therefore, the NPs were poly-dispersed (Figure 5). Single NPs with a diameter of 8.7 nm were the
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Figure 5. Particle size analysis of NPs produced by 96% hydroalcoholic extract of Q. brantii leaves after 4 h of biotransformation.
Figure 6. Particle size analysis of NPs produced by 96% hydroalcoholic extract of Q. brantii leaves after 48 h of biotransformation.
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most frequent ones (66.6%) and the mean size was 6 nm (Z-Average). The second most frequent (19.3%) particles were 0.82 nm (smallest NPs). The aggregates with a diameter of 2566 nm were 14.1% of the NPs. The poly-dispersity index (PDI) was 0.323 (near to optimal value). By increasing
DOI: 10.3109/13880209.2014.942868
the time of reaction (48 h, Figure 6), the peak related to 0.83 nm was disappeared and the sizes of NPs were increased. TEM analysis
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TEM micrographs of the silver NPs synthesized by using 70% hydroalcoholic extract of Q. brantii leaves after 24 h of biotransformation are shown in Figure 7. The produced silver NPs were spherical in shape. Figure 8 shows TEM micrographs of silver NPs synthesized using 96% hydroalcoholic extract of Q. brantii leaves, 24 h after the start of the reaction. The produced NPs were spherical and in some places aggregated and assembled in structures. Effect of ethanol concentration of hydroalcoholic extract on the produced NPs Biotransformation time-courses, bioconversion percents, and bioreduction rates of Ag+ to Ag0 NPs by both the extracts (Figures 1 and 2) were almost the same. By comparing the NP shapes and size distributions produced by 70% hydroalcoholic extract of Q. brantii (Figures 3, 4, and 7) and 96 hydroalcoholic extract (Figures 5, 6, and 8), it can be seen that 96 hydroalcoholic extract produced smaller and more separated silver NPs, which might be interpreted that 96% hydroalcoholic solution can extract the nanoparticle producing compounds and nanoparticle coating proteins better than 70% hydroalcoholic solution. Figure 7. TEM micrographs of the produced NPs by 70% hydroalcoholic extract of Q. brantii leaves after 24 h of biotransformation.
Figure 8. TEM micrographs of the produced NPs by 96% hydroalcoholic extract of Q. brantii leaves after 24 h of biotransformation.
Green biosynthesis of silver NPs using oak leave extract
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Mechanistic aspects Antioxidant and antilipid peroxidation activities of the leaves and fruit components of Quercus species have been reported in the literature. Based on the previous studies, tannin is the most abundant compound in the plant fruits whose major effect is antidiarrhea due to its water absorption and protein precipitation effects. Phytochemicals such as tannins, flavonoids, alkaloids, and several other aromatic compounds are secondary metabolites of the plant (Mirzaei & Mirzaei, 2013; Safary et al., 2009; Seddik et al., 2010). Phenolic and flavonoid compounds are the largest group of antioxidant compounds, chelate metals and scavenge peroxide, lipid peroxyl, and hydroperoxide (Sakai et al., 1996). Antioxidant action of phenolic compounds is due to their high tendency to chelate metals. Phenolic compounds possess hydroxyl and carboxyl groups, which may inactivate iron ions by chelating and additionally suppressing the superoxide-driven Fenton reaction, which is believed to be the most important source of reactive oxygen species (Iravani & Zolfaghari, 2011, 2013, 2014). The hydroalcoholic extract of Q. brantii leaves contains high phenolic and flavonoid compounds with strong antioxidant activities. It seems that these phytochemicals and phenolic compounds were involved in phyto-reduction of silver ions, and phytosynthesis of silver NPs. However, more efforts need to be done to understand the effect of parameters regarding the effects of various biomolecules and
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phytochemicals available in the extract of Q. brantii leaves for synthesizing NPs.
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Conclusion Since the eco-friendly synthesis of NPs of different chemical compositions, sizes, shapes, and controlled dispersity is an important aspect of nanobiotechnology and green nanotechnology, this green method was developed. Hydroalcoholic extract (96%) of Q. brantii successfully produced quite small (as small as 0.83 nm and the mean size of 6 nm), spherical, and poly-dispersed NPs with low aggregates. Results from TEM and DLS analysis demonstrated that by increasing the time of reaction, the NPs were aggregated further. The conversion was fast and completed in 5 h. Therefore, this plant and the extraction method seems to be quiet attractive for industrial scale production of NPs.
Declaration of interest The authors report no conflicts of interest. This study was a part of the research project No. 392034 and was financially supported by Faculty of Pharmacy, Isfahan University of Medical Sciences.
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