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Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
Green synthesis of silver nanoparticles using Eucalyptus leucoxylon leaves extract and evaluating the antioxidant activities of extract a
b
Mehdi Rahimi-Nasrabadi , Seied Mahdi Pourmortazavi , Seyed c
de
Ataollah Sadat Shandiz , Farhad Ahmadi a
f
& Hossein Batooli
Nano-Science Center, Imam Hossein University, Tehran, Iran
b
Faculty of Material and Manufacturing Technologies, Malek Ashtar University of Technology, Tehran, Iran c
Department of Biology, Tehran Science and Research Branch, Islamic Azad University, Tehran, Iran d
Faculty of Pharmacy, Nano Drug Delivery Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran e
Department of Medicinal Chemistry, Faculty of Pharmacy, Kermanshah University of Medical Sciences, Kermanshah, Iran f
Isfahan Research Center of Natural Sources and Agriculture, Kashan Station, Kashan, Iran Published online: 27 May 2014.
To cite this article: Mehdi Rahimi-Nasrabadi, Seied Mahdi Pourmortazavi, Seyed Ataollah Sadat Shandiz, Farhad Ahmadi & Hossein Batooli (2014) Green synthesis of silver nanoparticles using Eucalyptus leucoxylon leaves extract and evaluating the antioxidant activities of extract, Natural Product Research: Formerly Natural Product Letters, 28:22, 1964-1969, DOI: 10.1080/14786419.2014.918124 To link to this article: http://dx.doi.org/10.1080/14786419.2014.918124
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors,
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This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Natural Product Research, 2014 Vol. 28, No. 22, 1964–1969, http://dx.doi.org/10.1080/14786419.2014.918124
Green synthesis of silver nanoparticles using Eucalyptus leucoxylon leaves extract and evaluating the antioxidant activities of extract Mehdi Rahimi-Nasrabadia*, Seied Mahdi Pourmortazavib, Seyed Ataollah Sadat Shandizc, Farhad Ahmadid,e and Hossein Batoolif
Downloaded by [University of Maastricht] at 01:14 28 October 2014
a
Nano-Science Center, Imam Hossein University, Tehran, Iran; bFaculty of Material and Manufacturing Technologies, Malek Ashtar University of Technology, Tehran, Iran; cDepartment of Biology, Tehran Science and Research Branch, Islamic Azad University, Tehran, Iran; dFaculty of Pharmacy, Nano Drug Delivery Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran; eDepartment of Medicinal Chemistry, Faculty of Pharmacy, Kermanshah University of Medical Sciences, Kermanshah, Iran; fIsfahan Research Center of Natural Sources and Agriculture, Kashan Station, Kashan, Iran (Received 5 March 2014; final version received 22 April 2014) This study was designed to examine the in vitro antioxidant activity of essential oil and methanol extracts of Eucalyptus leucoxylon. Furthermore, the polar fraction of the extract was used as a reducing agent for the green synthesis of silver nanoparticles (Ag NPs). Antioxidant activities of the samples were determined by using three different test systems, namely DPPH and b-carotene/linoleic acid and reducing power. The structure and composition of the prepared Ag NPs were characterised by X-ray diffraction, scanning electron microscopy, transmission electron microscopy and UV – vis spectroscopy. Synthesised Ag NPs were almost spherical in shape with an average diameter of about , 50 nm and synthesised within 120 min reaction time at room temperature. Keywords: Eucalyptus leucoxylon; antioxidant activity; silver nanoparticles; green synthesis
1. Introduction Reactive oxygen species (ROS), including singlet oxygen, superoxide anion, hydroxyl radical and so on, are highly toxic and are generated in cells under normal metabolic activities (Bahramikia et al. 2009). The presence of ROS is necessary for some living process such as signal transduction and production of energy to fuel biological process; but at high concentrations, ROS can damage macromolecules such as DNA, proteins and lipids (Kowaltowski et al. 2009). Researchers showed that oxidative damage is linked to the pathogenesis of oxidative diseases such as cancer, vascular problems and diabetes. It has been proved that antioxidants can play an important role in protection against these disorders. Antioxidants can delay or retard the initiation or propagation steps in oxidative chain reaction and thus prevent the damage mediated by ROS (Steinbrenner & Sies 2009). Furthermore, lipid oxidation, which is caused by free radicals, is an important factor in food deterioration during its processing and storage. It decreases the quality of food products (Gachkar et al. 2007; Ebrahimzadeh et al. 2009). Therefore, synthetic antioxidants such as butylated hydroxyl toluene (BHT), butylated hydroxyl anisole (BHA), gallic acid (GA) and some other synthetic antioxidants are widely used for food
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
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preservation and to prolong the storage period. Some disadvantages of the compounds such as BHT and BHA have been proved. Thus, there is an increasing interest in the extraction of antioxidants from natural sources by using various techniques (Pourmortazavi & Hajimirsadeghi 2007; Gholivand et al. 2009; Rahimi-Nasrabadi et al. 2009), and efforts have been made into identifying compounds that can act as appropriate antioxidants to replace synthetic food additives (Rahimi-Nasrabadi et al. 2012). In addition to the above-mentioned benefits of natural antioxidants, the extract of plants with antioxidant activity can be used as a reducing agent in the production of metal nanoparticles. Metal nanoparticles have been shown to possess enormous application potential in areas such as photography, catalysis, biological labelling, photonics, optoelectronics and surface-enhanced Raman scattering (Huang et al. 2010; Zhao et al. 2014). Various physical or chemical methods (Kim et al. 2012; Bera & Raj 2013; Zhang et al. 2013) have been suggested for the synthesis of silver nanoparticles (Ag NPs). However, all these methods suffer from some disadvantages such as need to a strictly controlled synthesis media, use of toxic reagent, high synthesis costs and complexities in synthesis reaction. More investigations are hence directed towards finding other easy-to-handle and economical methods for the synthesis of Ag NPs. Methods that are environmentally friendly, possess potentials for scaling-up and do not require the use of high pressure, energy, temperature and toxic chemicals are also preferred. From this point of view, the synthesis of Ag NPs using non-toxic and environmentally friendly green synthesis methods is attractive especially if it is intended for biomedical applications. Lately, methods using biological micro-organisms such as bacteria (Joerger et al. 2000) or fungus (Ahmad et al. 2003), yeast (Kowshik et al. 2003), honey (Philip 2010) and plant extracts (Dhar Dwivedi & Gopal 2010; Lukman et al. 2011; Kaviya et al. 2012) have appeared as a simple and practical option to more complex chemical synthetic procedures to produce nanomaterials; thus, much research and progress have been made in this field in the last few years. Utilisation of plant extracts for the synthesis of nanoparticles can be beneficial over other biological methods, due to its elimination of developing process of preserving cell cultures. Plant extracts can act both as reducing and stabilising agents in the synthesis of nanoparticles (VilchisNestor et al. 2008). Different extracts contain different concentrations and combinations of organic reducing agents and thus, the type of the plant extract is known to influence the characteristics of the produced nanoparticles (Kumar & Yadav 2009). Usually, a plant extractmediated reduction will be accomplished by mixing the aqueous extract with the solution of the relevant metal salt. This reaction will be carried out at room temperature within a few minutes. To the best of our knowledge, there is no information on the antioxidant properties of E. leucoxylon in the literature. The aim of this work is to evaluate in vitro antioxidant properties of the essential oil and methanol extracts of E. leucoxylon by using DPPH, b-carotene/linoleic acid and reducing power assays. Furthermore, the easy synthesis of Ag NPs by a simple and environmentally friendly procedure involving the reduction of aqueous Ag (I) by E. leucoxylon extract is reported. 2. Results and discussion 2.1. Antioxidant properties DPPH-scavenging activity is usually presented by IC50 value, defined as the concentration of the antioxidant needed to scavenge 50% of DPPH present in the test solution (Table 1). The presence of antioxidant in the sample leads to the disappearance of DPPH radical chromogens which can be detected by spectrophotometry at 517 nm. Lower IC50 value indicates higher antioxidant activity. The extract and the essential oil of E. leucoxylon exhibited remarkable
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Table 1. Antioxidant activities of E. leucoxylon essential oil and methanol extract. Sample Essential oil Polar subfraction Non-polar subfraction Ascorbic acid BHT
DPPH IC50 (mg/mL)
b-Carotene bleaching (RAA) (%)
Total phenol contents GAEs (mg/mg)
– 21.0 ^ 1.4 82.4 ^ 2.1 5.8 ^ 0.3 25.4 ^ 1.5
41.5 ^ 0.9 49.2 ^ 1.0 85.6 ^ 2.1 n.d. 100.0
n.d. 256.6 ^ 3.9 128.3 ^ 1.2 n.d. n.d.
Downloaded by [University of Maastricht] at 01:14 28 October 2014
Note: n.d., not determined.
antioxidant activities. In DPPH system, polar subfraction of methanol extract showed the highest radical-scavenging activity (21.0 ^ 1.4 mg/mL). The antioxidant activities of the plant extract and the oil were evaluated by using the spectrophotometric b-carotene bleaching test. The relative antioxidative activities (RAAs) of the extracts were calculated from the equation, RAA ¼ Asample/ABHT, where ABHT is the absorbance of the control (BHT) and Asample is the absorbance of the extract or oil. The calculated RAAs of the extract and the oil are given in Table 1. The inhibition values of linoleic acid oxidation were estimated as 49.2 ^ 1.0%, 85.6 ^ 2.1% and 41.5 ^ 0.9% in the presence of the polar subfraction (PS) of methanol extract, non-polar subfraction (NS) and the essential oil, respectively. Different studies have indicated the electron donation capacity, reflecting the reducing power of bioactive compounds in association with antioxidant activity. Figure S1 (Supplementary material) shows the reducing power of the E. leucoxylon essential oil and methanol extracts as a function of their concentrations. In this assay, the yellow colour of the test solution changes to various shades of green and blue, depending on the reducing power of each compound. The presence of reducers causes the conversion of Fe3þ/ferric cyanide complex, used in this method, to ferrous form. By measuring the formation of Perl’s Prussian blue (Fe4[Fe (CN)6]3) at 700 nm, it is possible to determine the Fe2þ concentration. The reducing power of the essential oil and the methanol extract increased with their concentrations. At 100, 250, 500 and 1000 mg/mL, reducing powers were around 0.081, 0.205, 0.399 and 0.711 for essential oil and 0.487, 0.876, 0.926 and 0.932 for PS, respectively, while a solution of 250 mg/mL of ascorbic acid, as positive control used in this test, had a reducing power value of 0.929. The reducing power of other concentrations of extracts and essential oil are presented in Figure S1 (Supplementary material). Moreover, as can be seen from Figure S1 (Supplementary material), at higher concentrations the reducing power of extracts and essential oil are closer to the reducing power of ascorbic acid. The total phenolic content of extracts from E. leucoxylon was determined by the Folin and Ciocalteu method and calculated as gallic acid equivalents (GAEs). The data presented in Table 1 indicate that the amount of total phenol in PS (256.6 ^ 3.9 mg/mg as GAEs) was higher than NS (128.3 ^ 1.2 mg/mg). The high antioxidant activity in PS in comparison to NS is due to its high phenolic content. As can be seen from the results, there is a good agreement between the amounts of phenolic compound with antioxidant activity. The results indicated that the methanolic extract has higher total phenolic compounds than the oil. 2.2. Synthesis of Ag NPs Being a material of great technological importance, especially in medical applications, nanostructured silver is a subject of widespread investigations. This is also reflected in the field of polymer nanocomposites, where Ag NPs have been fabricated in various synthetic (Wang &
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Jeong 2011) and biopolymer matrices (Bozanic et al. 2007). Besides the well-known antimicrobial activity, the nano-sized silver has led to significant advancement in areas such as photonics, microelectronics, lithography, optics and catalysis (Wang et al. 2013). Therefore, the use of green compound for synthesising Ag NPs is of interest. As can be seen in antioxidant assays especially in reducing power assay, the reducing power of polar subfraction of extract used in the synthesis of Ag NPs is very high and comparable with ascorbic acid, and thus can be used as an efficient reducer in the production of Ag NPs. It was observed that upon addition of the extract into the reactor containing the aqueous silver nitrate solution, the colour of the medium changed to yellowish brown within 10 min. This indicates the formation of Ag NPs (Dhar Dwivedi & Gopal 2010; Lukman et al. 2011). The spectra were recorded after 10, 20, 30 and 60 min. By utilising the UV –vis spectroscopy and acquiring the spectra, the influence of the reaction time on synthesis of Ag NPs was appraised, and it is observed that the peak becomes sharper with an increase in time. The surface plasmon resonance band of Ag NPs emerged at 440 nm and even after 1 h of incubation only small change can be observed (Figure S2 (Supplementary material)). The increase in intensity could be due to the increasing number of nanoparticles produced as a result of reduction of silver ions present in the aqueous solution (Lukman et al. 2011). It was announced earlier that the absorbance at around 440 nm for silver is a distinctive trait of these metal particles (Dhar Dwivedi & Gopal 2010).
2.3. Characterisation of Ag NPs The synthesised Ag NPs were characterised by X-ray powder diffraction for the determination of their composition and purity. Figure S3 (Supplementary material) shows the X-ray difrraction (XRD) pattern for the synthesised Ag NPs. Four major peaks with 2u values of 38.1758, 44.1758, 63.5258 and 78.3258 appeared in the XRD pattern of prepared Ag NPs. These peaks correspond to the (111), (200), (220) and (311) planes of face centred-cubic geometry of Ag NPs, which is in agreement with peaks from PC-APD Diffraction software No. 01-087-0719. The prepared Ag NPs were characterised by using scanning electron microscopy (SEM) for the determination of their morphology. As shown in Figure S4 (Supplementary material), the SEM result revealed that the prepared Ag NPs have about 50 nm average diameter under optimum conditions. Furthermore, the transmission electron microscopy (TEM) image of Ag NPs presents a cross-sectional view of the synthesised nanoparticles (Figure 1).
Figure 1. TEM image of synthesised Ag NPs via green synthesis method.
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3. Conclusions Methanolic extracts were found to be effective antioxidants in different in vitro assays including b-carotene bleaching and DPPH radical scavenging and can be suggested as a natural additive in food and pharmaceutical industries. E. leucoxylon extracts appear to contain compounds with antioxidant activities. However, the components responsible for the antioxidant activities of its extracts are currently unclear. Further works should be performed on the isolation and identification of the components in the extracts. In addition, the in vivo safety needs to be thoroughly investigated in experimental rodent models prior to its possible application. Furthermore, Ag NPs have been synthesised via simple and green procedure using the polar fraction of E. leucoxylon extract as a reducing agent. No toxic reagent has been used in this method. Thus, synthesised Ag NPs possess a great potential for biomedical application. The composition and structural characterisation of the produced Ag NPs were evaluated by using SEM, TEM, XRD and UV – vis proving the formation of Ag NPs with an average size of 50 nm.
Supplementary material Experimental details relating to this article are available online, alongside Figures S1– S4.
References Ahmad A, Mukherjee P, Senapati S, Mandal D, Khan MI, Kumar R, Sastry M. 2003. Extracellular biosynthesis of silver nanoparticles using the fungus Fusarium oxysporum. Colloid Surf B. 28:313–318. Bahramikia S, Ardestani A, Yazdanparast R. 2009. Protective effects of four Iranian medicinal plants against free radicalmediated protein oxidation. Food Chem. 115:37–42. Bera RK, Raj CR. 2013. A facile photochemical route for the synthesis of triangular Ag nanoplates and colorimetric sensing of H2O2. J Photochem Photobiol A Chem. 270:1–6. Bozanic DK, Djokovic V, Blanusa J, Nair PS, Georges MK, Radhakrishnan T. 2007. Preparation and properties of nanosized Ag and Ag2S particles in biopolymer matrix. Eur Phys J. 22:51–59. Dhar Dwivedi A, Gopal K. 2010. Biosynthesis of silver and gold nanoparticles using Chenopodium album leaf extract. Colloids Surf A Physicochem Eng Asp. 369:27–33. Ebrahimzadeh MA, Nabavi SM, Nabavi SF, Eslami B. 2009. Antioxidant activity of aqueous extract of Pyrus boissieriana fruit. Pharmacol Online. 1:1318–1323. Gachkar L, Yadegari D, Rezaei MB, Taghizadeh M, Alipoor Astaneh Sh, Rasooli I. 2007. Chemical and biological characteristics of Cuminum cyminum and Rosmarinus officinalis essential oils. Food Chem. 102:898–904. Gholivand MB, Rahimi-Nasrabadi M, Chalabi H. 2009. Determination of essential oil components of star anise (Illicium verum) using simultaneous hydrodistillation-static headspace liquid-phase microextraction-gas chromatography mass spectrometry. Anal Lett. 42:1382–1397. Huang NM, Lim HN, Radiman S, Khiew PS, Chiu WS, Hashim R, Chia CH. 2010. Sucrose ester micellar-mediated of Ag nanoparticles and the antibacterial properties. Colloids Surf A Physicochem Eng Asp. 353:69–76. Joerger R, Klaus T, Granqvist CG. 2000. Biologically produced silver–carbon composite, materials for optically functional thin-film coatings. Adv Mater. 12:407–409. Kaviya S, Santhanalakshmi J, Viswanathan B. 2012. Biosynthesis of silver nano-flakes by Crossandra infundibuliformis leaf extract. Mater Lett. 67:64–66. Kim SE, Park JH, Lee BC, Lee JC, Kwon YK. 2012. Large-scale synthesis of silver nanoparticles using Ag(I)-S12 polymer through electron beam irradiation. Rad Phys Chem. 81:978–981. Kowaltowski AJ, de Souza-Pinto NC, Castilho RF, Vercesi AE. 2009. Mitochondria and reactive oxygen species. Free Rad Biol Med. 47:333–343. Kowshik M, Ashtaputre S, Kharrazi S, Vogel W, Urban J, Kulkarni SK, Paknikar KM. 2003. Extracellular synthesis of silver nanoparticles by a silver-tolerant yeast strain MKY3. Nanotechnology. 14:95–100. Kumar V, Yadav SK. 2009. Plant-mediated synthesis of silver and gold nanoparticles and their applications. J Chem Technol Biotechnol. 84:151–157. Lukman AI, Gong B, Marjo CE, Roessner U, Harris AT. 2011. Facile synthesis, stabilization, and anti-bacterial performance of discrete Ag nanoparticles using Medicago sativa seed exudates. J Colloid Interface Sci. 353:433–444. Philip D. 2010. Honey mediated green synthesis of silver nanoparticles. Spectrochim Acta A. 75:1078–1081.
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Pourmortazavi SM, Hajimirsadeghi SS. 2007. Supercritical fluid extraction in plant essential and volatile oil analysis. J Chromatogr A. 1163:2–24. Rahimi-Nasrabadi M, Ahmadi F, Batool H. 2012. Essential oil composition of Eucalyptus procera Dehnh. leaves from central Iran. Nat Prod Res. 26:637–642. Rahimi-Nasrabadi M, Gholivand MB, Batooli H. 2009. Chemical composition of the essential oil from leaves and flowering aerial parts of Haplophyllum robustum bge. (Rutaceae). Dig J Nanomater Biosci. 4:819–822. Steinbrenner H, Sies H. 2009. Protection against reactive oxygen species by selenoproteins. Biochim Biophys Acta. 1790:1478–1485. Vilchis-Nestor AR, Sa´nchez-Mendieta V, Camacho-Lo´pez MA, Go´mez-Espinosa RM, Camacho-Lo´pez MA, ArenasAlatorre JA. 2008. Solventless synthesis and optical properties of Au and Ag nanoparticles using Camellia sinensis extract. Mater Lett. 62:3103–3105. Wang SH, Jeong S. 2011. Electrospunnano composites of poly(vinyl pyrrolidone)/nano-silver for antibacterial materials. J Nanosci Nanotechnol. 11:610–613. Wang M, Tian D, Tian P, Yuan L. 2013. Synthesis of micron-SiO2 –nano-Ag particles and their catalytic performance in 4-nitrophenol reduction. Appl Surf Sci. 283:389–395. Zhang Y, Wang J, Yang P. 2013. Convenient synthesis of Ag nanowires with tunable length and morphology. Mater Res Bull. 48:461–468. Zhao X, Xia Y, Li Q, Ma X, Quan F, Geng C, Han Z. 2014. Microwave-assisted synthesis of silver nanoparticles using sodium alginate and their antibacterial activity. Colloids Surf A Physicochem Eng Asp. 444:180–188.
Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
Green synthesis of silver nanoparticles using Eucalyptus leucoxylon leaves extract and evaluating the antioxidant activities of extract a
b
Mehdi Rahimi-Nasrabadi , Seied Mahdi Pourmortazavi , Seyed c
de
Ataollah Sadat Shandiz , Farhad Ahmadi a
f
& Hossein Batooli
Nano-Science Center, Imam Hossein University, Tehran, Iran
b
Faculty of Material and Manufacturing Technologies, Malek Ashtar University of Technology, Tehran, Iran c
Department of Biology, Tehran Science and Research Branch, Islamic Azad University, Tehran, Iran d
Faculty of Pharmacy, Nano Drug Delivery Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran e
Department of Medicinal Chemistry, Faculty of Pharmacy, Kermanshah University of Medical Sciences, Kermanshah, Iran f
Isfahan Research Center of Natural Sources and Agriculture, Kashan Station, Kashan, Iran Published online: 27 May 2014.
To cite this article: Mehdi Rahimi-Nasrabadi, Seied Mahdi Pourmortazavi, Seyed Ataollah Sadat Shandiz, Farhad Ahmadi & Hossein Batooli (2014) Green synthesis of silver nanoparticles using Eucalyptus leucoxylon leaves extract and evaluating the antioxidant activities of extract, Natural Product Research: Formerly Natural Product Letters, 28:22, 1964-1969, DOI: 10.1080/14786419.2014.918124 To link to this article: http://dx.doi.org/10.1080/14786419.2014.918124
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors,
and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content.
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This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Natural Product Research, 2014 Vol. 28, No. 22, 1964–1969, http://dx.doi.org/10.1080/14786419.2014.918124
Green synthesis of silver nanoparticles using Eucalyptus leucoxylon leaves extract and evaluating the antioxidant activities of extract Mehdi Rahimi-Nasrabadia*, Seied Mahdi Pourmortazavib, Seyed Ataollah Sadat Shandizc, Farhad Ahmadid,e and Hossein Batoolif
Downloaded by [University of Maastricht] at 01:14 28 October 2014
a
Nano-Science Center, Imam Hossein University, Tehran, Iran; bFaculty of Material and Manufacturing Technologies, Malek Ashtar University of Technology, Tehran, Iran; cDepartment of Biology, Tehran Science and Research Branch, Islamic Azad University, Tehran, Iran; dFaculty of Pharmacy, Nano Drug Delivery Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran; eDepartment of Medicinal Chemistry, Faculty of Pharmacy, Kermanshah University of Medical Sciences, Kermanshah, Iran; fIsfahan Research Center of Natural Sources and Agriculture, Kashan Station, Kashan, Iran (Received 5 March 2014; final version received 22 April 2014) This study was designed to examine the in vitro antioxidant activity of essential oil and methanol extracts of Eucalyptus leucoxylon. Furthermore, the polar fraction of the extract was used as a reducing agent for the green synthesis of silver nanoparticles (Ag NPs). Antioxidant activities of the samples were determined by using three different test systems, namely DPPH and b-carotene/linoleic acid and reducing power. The structure and composition of the prepared Ag NPs were characterised by X-ray diffraction, scanning electron microscopy, transmission electron microscopy and UV – vis spectroscopy. Synthesised Ag NPs were almost spherical in shape with an average diameter of about , 50 nm and synthesised within 120 min reaction time at room temperature. Keywords: Eucalyptus leucoxylon; antioxidant activity; silver nanoparticles; green synthesis
1. Introduction Reactive oxygen species (ROS), including singlet oxygen, superoxide anion, hydroxyl radical and so on, are highly toxic and are generated in cells under normal metabolic activities (Bahramikia et al. 2009). The presence of ROS is necessary for some living process such as signal transduction and production of energy to fuel biological process; but at high concentrations, ROS can damage macromolecules such as DNA, proteins and lipids (Kowaltowski et al. 2009). Researchers showed that oxidative damage is linked to the pathogenesis of oxidative diseases such as cancer, vascular problems and diabetes. It has been proved that antioxidants can play an important role in protection against these disorders. Antioxidants can delay or retard the initiation or propagation steps in oxidative chain reaction and thus prevent the damage mediated by ROS (Steinbrenner & Sies 2009). Furthermore, lipid oxidation, which is caused by free radicals, is an important factor in food deterioration during its processing and storage. It decreases the quality of food products (Gachkar et al. 2007; Ebrahimzadeh et al. 2009). Therefore, synthetic antioxidants such as butylated hydroxyl toluene (BHT), butylated hydroxyl anisole (BHA), gallic acid (GA) and some other synthetic antioxidants are widely used for food
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
Downloaded by [University of Maastricht] at 01:14 28 October 2014
Natural Product Research
1965
preservation and to prolong the storage period. Some disadvantages of the compounds such as BHT and BHA have been proved. Thus, there is an increasing interest in the extraction of antioxidants from natural sources by using various techniques (Pourmortazavi & Hajimirsadeghi 2007; Gholivand et al. 2009; Rahimi-Nasrabadi et al. 2009), and efforts have been made into identifying compounds that can act as appropriate antioxidants to replace synthetic food additives (Rahimi-Nasrabadi et al. 2012). In addition to the above-mentioned benefits of natural antioxidants, the extract of plants with antioxidant activity can be used as a reducing agent in the production of metal nanoparticles. Metal nanoparticles have been shown to possess enormous application potential in areas such as photography, catalysis, biological labelling, photonics, optoelectronics and surface-enhanced Raman scattering (Huang et al. 2010; Zhao et al. 2014). Various physical or chemical methods (Kim et al. 2012; Bera & Raj 2013; Zhang et al. 2013) have been suggested for the synthesis of silver nanoparticles (Ag NPs). However, all these methods suffer from some disadvantages such as need to a strictly controlled synthesis media, use of toxic reagent, high synthesis costs and complexities in synthesis reaction. More investigations are hence directed towards finding other easy-to-handle and economical methods for the synthesis of Ag NPs. Methods that are environmentally friendly, possess potentials for scaling-up and do not require the use of high pressure, energy, temperature and toxic chemicals are also preferred. From this point of view, the synthesis of Ag NPs using non-toxic and environmentally friendly green synthesis methods is attractive especially if it is intended for biomedical applications. Lately, methods using biological micro-organisms such as bacteria (Joerger et al. 2000) or fungus (Ahmad et al. 2003), yeast (Kowshik et al. 2003), honey (Philip 2010) and plant extracts (Dhar Dwivedi & Gopal 2010; Lukman et al. 2011; Kaviya et al. 2012) have appeared as a simple and practical option to more complex chemical synthetic procedures to produce nanomaterials; thus, much research and progress have been made in this field in the last few years. Utilisation of plant extracts for the synthesis of nanoparticles can be beneficial over other biological methods, due to its elimination of developing process of preserving cell cultures. Plant extracts can act both as reducing and stabilising agents in the synthesis of nanoparticles (VilchisNestor et al. 2008). Different extracts contain different concentrations and combinations of organic reducing agents and thus, the type of the plant extract is known to influence the characteristics of the produced nanoparticles (Kumar & Yadav 2009). Usually, a plant extractmediated reduction will be accomplished by mixing the aqueous extract with the solution of the relevant metal salt. This reaction will be carried out at room temperature within a few minutes. To the best of our knowledge, there is no information on the antioxidant properties of E. leucoxylon in the literature. The aim of this work is to evaluate in vitro antioxidant properties of the essential oil and methanol extracts of E. leucoxylon by using DPPH, b-carotene/linoleic acid and reducing power assays. Furthermore, the easy synthesis of Ag NPs by a simple and environmentally friendly procedure involving the reduction of aqueous Ag (I) by E. leucoxylon extract is reported. 2. Results and discussion 2.1. Antioxidant properties DPPH-scavenging activity is usually presented by IC50 value, defined as the concentration of the antioxidant needed to scavenge 50% of DPPH present in the test solution (Table 1). The presence of antioxidant in the sample leads to the disappearance of DPPH radical chromogens which can be detected by spectrophotometry at 517 nm. Lower IC50 value indicates higher antioxidant activity. The extract and the essential oil of E. leucoxylon exhibited remarkable
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Table 1. Antioxidant activities of E. leucoxylon essential oil and methanol extract. Sample Essential oil Polar subfraction Non-polar subfraction Ascorbic acid BHT
DPPH IC50 (mg/mL)
b-Carotene bleaching (RAA) (%)
Total phenol contents GAEs (mg/mg)
– 21.0 ^ 1.4 82.4 ^ 2.1 5.8 ^ 0.3 25.4 ^ 1.5
41.5 ^ 0.9 49.2 ^ 1.0 85.6 ^ 2.1 n.d. 100.0
n.d. 256.6 ^ 3.9 128.3 ^ 1.2 n.d. n.d.
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Note: n.d., not determined.
antioxidant activities. In DPPH system, polar subfraction of methanol extract showed the highest radical-scavenging activity (21.0 ^ 1.4 mg/mL). The antioxidant activities of the plant extract and the oil were evaluated by using the spectrophotometric b-carotene bleaching test. The relative antioxidative activities (RAAs) of the extracts were calculated from the equation, RAA ¼ Asample/ABHT, where ABHT is the absorbance of the control (BHT) and Asample is the absorbance of the extract or oil. The calculated RAAs of the extract and the oil are given in Table 1. The inhibition values of linoleic acid oxidation were estimated as 49.2 ^ 1.0%, 85.6 ^ 2.1% and 41.5 ^ 0.9% in the presence of the polar subfraction (PS) of methanol extract, non-polar subfraction (NS) and the essential oil, respectively. Different studies have indicated the electron donation capacity, reflecting the reducing power of bioactive compounds in association with antioxidant activity. Figure S1 (Supplementary material) shows the reducing power of the E. leucoxylon essential oil and methanol extracts as a function of their concentrations. In this assay, the yellow colour of the test solution changes to various shades of green and blue, depending on the reducing power of each compound. The presence of reducers causes the conversion of Fe3þ/ferric cyanide complex, used in this method, to ferrous form. By measuring the formation of Perl’s Prussian blue (Fe4[Fe (CN)6]3) at 700 nm, it is possible to determine the Fe2þ concentration. The reducing power of the essential oil and the methanol extract increased with their concentrations. At 100, 250, 500 and 1000 mg/mL, reducing powers were around 0.081, 0.205, 0.399 and 0.711 for essential oil and 0.487, 0.876, 0.926 and 0.932 for PS, respectively, while a solution of 250 mg/mL of ascorbic acid, as positive control used in this test, had a reducing power value of 0.929. The reducing power of other concentrations of extracts and essential oil are presented in Figure S1 (Supplementary material). Moreover, as can be seen from Figure S1 (Supplementary material), at higher concentrations the reducing power of extracts and essential oil are closer to the reducing power of ascorbic acid. The total phenolic content of extracts from E. leucoxylon was determined by the Folin and Ciocalteu method and calculated as gallic acid equivalents (GAEs). The data presented in Table 1 indicate that the amount of total phenol in PS (256.6 ^ 3.9 mg/mg as GAEs) was higher than NS (128.3 ^ 1.2 mg/mg). The high antioxidant activity in PS in comparison to NS is due to its high phenolic content. As can be seen from the results, there is a good agreement between the amounts of phenolic compound with antioxidant activity. The results indicated that the methanolic extract has higher total phenolic compounds than the oil. 2.2. Synthesis of Ag NPs Being a material of great technological importance, especially in medical applications, nanostructured silver is a subject of widespread investigations. This is also reflected in the field of polymer nanocomposites, where Ag NPs have been fabricated in various synthetic (Wang &
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Jeong 2011) and biopolymer matrices (Bozanic et al. 2007). Besides the well-known antimicrobial activity, the nano-sized silver has led to significant advancement in areas such as photonics, microelectronics, lithography, optics and catalysis (Wang et al. 2013). Therefore, the use of green compound for synthesising Ag NPs is of interest. As can be seen in antioxidant assays especially in reducing power assay, the reducing power of polar subfraction of extract used in the synthesis of Ag NPs is very high and comparable with ascorbic acid, and thus can be used as an efficient reducer in the production of Ag NPs. It was observed that upon addition of the extract into the reactor containing the aqueous silver nitrate solution, the colour of the medium changed to yellowish brown within 10 min. This indicates the formation of Ag NPs (Dhar Dwivedi & Gopal 2010; Lukman et al. 2011). The spectra were recorded after 10, 20, 30 and 60 min. By utilising the UV –vis spectroscopy and acquiring the spectra, the influence of the reaction time on synthesis of Ag NPs was appraised, and it is observed that the peak becomes sharper with an increase in time. The surface plasmon resonance band of Ag NPs emerged at 440 nm and even after 1 h of incubation only small change can be observed (Figure S2 (Supplementary material)). The increase in intensity could be due to the increasing number of nanoparticles produced as a result of reduction of silver ions present in the aqueous solution (Lukman et al. 2011). It was announced earlier that the absorbance at around 440 nm for silver is a distinctive trait of these metal particles (Dhar Dwivedi & Gopal 2010).
2.3. Characterisation of Ag NPs The synthesised Ag NPs were characterised by X-ray powder diffraction for the determination of their composition and purity. Figure S3 (Supplementary material) shows the X-ray difrraction (XRD) pattern for the synthesised Ag NPs. Four major peaks with 2u values of 38.1758, 44.1758, 63.5258 and 78.3258 appeared in the XRD pattern of prepared Ag NPs. These peaks correspond to the (111), (200), (220) and (311) planes of face centred-cubic geometry of Ag NPs, which is in agreement with peaks from PC-APD Diffraction software No. 01-087-0719. The prepared Ag NPs were characterised by using scanning electron microscopy (SEM) for the determination of their morphology. As shown in Figure S4 (Supplementary material), the SEM result revealed that the prepared Ag NPs have about 50 nm average diameter under optimum conditions. Furthermore, the transmission electron microscopy (TEM) image of Ag NPs presents a cross-sectional view of the synthesised nanoparticles (Figure 1).
Figure 1. TEM image of synthesised Ag NPs via green synthesis method.
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3. Conclusions Methanolic extracts were found to be effective antioxidants in different in vitro assays including b-carotene bleaching and DPPH radical scavenging and can be suggested as a natural additive in food and pharmaceutical industries. E. leucoxylon extracts appear to contain compounds with antioxidant activities. However, the components responsible for the antioxidant activities of its extracts are currently unclear. Further works should be performed on the isolation and identification of the components in the extracts. In addition, the in vivo safety needs to be thoroughly investigated in experimental rodent models prior to its possible application. Furthermore, Ag NPs have been synthesised via simple and green procedure using the polar fraction of E. leucoxylon extract as a reducing agent. No toxic reagent has been used in this method. Thus, synthesised Ag NPs possess a great potential for biomedical application. The composition and structural characterisation of the produced Ag NPs were evaluated by using SEM, TEM, XRD and UV – vis proving the formation of Ag NPs with an average size of 50 nm.
Supplementary material Experimental details relating to this article are available online, alongside Figures S1– S4.
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