Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 132 (2014) 743–750
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Catalytically and biologically active silver nanoparticles synthesized using essential oil Vidya Vilas a,b, Daizy Philip a,⇑, Joseph Mathew b a b
Department of Physics, Mar Ivanios College, Thiruvananthapuram 695015, India Department of Chemistry, Mar Ivanios College, Thiruvananthapuram 695015, India
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Essential oil is used for the synthesis
of silver nanoparticles. Rapid, cost-effective, environmentally
benign method is suggested. Efficiency of the nanocatalyst is
portrayed in the degradation of a cationic dye and an organic pollutant. The synthesized biogenic silver nanoparticles act as potent free radical scavengers and antibacterial agent.
a r t i c l e
i n f o
Article history: Received 21 January 2014 Received in revised form 9 May 2014 Accepted 14 May 2014 Available online 13 June 2014 Keywords: Silver nanoparticles Myristica fragrans Catalysis Antibacterial activity Antioxidant activity
a b s t r a c t There are numerous reports on phytosynthesis of silver nanoparticles and various phytochemicals are involved in the reduction and stabilization. Pure explicit phytosynthetic protocol for catalytically and biologically active silver nanoparticles is of importance as it is an environmentally benign green method. This paper reports the use of essential oil of Myristica fragrans enriched in terpenes and phenyl propenes in the reduction and stabilization. FTIR spectra of the essential oil and the synthesized biogenic silver nanoparticles are in accordance with the GC–MS spectral analysis reports. Nanosilver is initially characterized by an intense SPR band around 420 nm, followed by XRD and TEM analysis revealing the formation of 12–26 nm sized, highly pure, crystalline silver nanoparticles. Excellent catalytic and bioactive potential of the silver nanoparticles is due to the surface modification. The chemocatalytic potential of nanosilver is exhibited by the rapid reduction of the organic pollutant, para nitro phenol and by the degradation of the thiazine dye, methylene blue. Significant antibacterial activity of the silver colloid against Gram positive, Staphylococcus aureus (inhibition zone – 12 mm) and Gram negative, Escherichia coli (inhibition zone – 14 mm) is demonstrated by Agar-well diffusion method. Strong antioxidant activity of the biogenic silver nanoparticles is depicted through NO scavenging, hydrogen peroxide scavenging, reducing power, DPPH and total antioxidant activity assays. Ó 2014 Elsevier B.V. All rights reserved.
Introduction
⇑ Corresponding author. Tel.: +91 471 2530887; fax: +91 471 2530023. E-mail addresses: [email protected], [email protected] (D. Philip). http://dx.doi.org/10.1016/j.saa.2014.05.046 1386-1425/Ó 2014 Elsevier B.V. All rights reserved.
Molecular manipulation at the nanoscale has undergone progressive changes ever since its dawn before 4th century AD. Though the methods of nanoparticle (NP) synthesis extends over
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V. Vilas et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 132 (2014) 743–750
a wide range, green synthesis methods has gained wider acclaim owing to the increasing awareness on the undesirable changes of the environment posed by several synthesis strategies. Phytosynthesis of nanoparticles uses phytoconstituents of the plant parts as reducing and capping agents. This ecofriendly method does not demand the use of high temperature, pressure or energy [1]. Development of a facile, pure, specific phytosynthetic route leads to the use of volatile aromatic oil. Kumar et al. [2] have synthesized metal NPs embedded paints based on vegetable oil. The auto oxidation of drying oils have been used for the reduction of metal salts to synthesize metal NPs. Castor oil, palm oil and coconut oil have been used for the synthesis of metal NPs [3–6]. In the first report on essential oil mediated synthesis of metal NPs, Sheny et al. [7] have used Anacardium occidentale as a reducing and capping agent for the synthesis of hexagonal gold NPs. The synthesis of silver (Ag) on a nanoscale is of importance, due to its thermal conductivity, chemical stability, catalytic and antibacterial activity [8,9]. The surface effects and quantum effects of nanoAg affects their chemical reactivity [10] and gives them unique mechanical, optical and electric properties [11]. Recent reports [12–17] suggest biological method for synthesis of antifungal, antibacterial, antioxidant, robust, biocompatible Ag NPs. Size, shape, surface area to volume ratio and the nature of the surface modifier act as limiting factors of the synthesized Ag NPs imparting adequate therapeutic potency. Exploitation of the antibacterial potential of nanoAg can be traced back to ancient times. Oligodynamic action coupled with a broad spectrum of targeted bacteria prompts the use of Ag based nanostructured materials as efficient antibacterial agents [18]. The rapid multiplication and proliferation of drug resistant bacteria is curbed by Ag NPs fortifying its use in burn treatments, water filters and as an antimicrobial finish on fabrics [15]. Antioxidant nanoAg repairs reactive oxygen species (ROS) damage to cell components and cell function disruption enhancing the immune system. The conjoint application of nanoAg as an antibacterial and an antioxidant agent ensures its use as a biomedical nanoproduct. NanoAg extends its aptness as a catalyst in pollution treatment, due to its small size, larger surface area to volume ratio and greater accessibility of the surface atoms. Ag NPs have attracted the interests of scientists in its use as a competent catalyst in the reduction of major pollutants of the dye industry that pose serious health risks. Myristica fragrans (M. fragrans) is an evergreen tree, successfully cultivated in South India. There are reports [19,20] on the hepatoprotective and anti cancer activity of Myristicin and antiinflammatory property of t – Caryophyllene; the essential oil constituents. The leaf oil exhibit significant antimicrobial and larvicide activity and cytotoxic activity against MCF-7 breast cancer cell line and A 357 epidermal skin cancer line [19]. In the present study, Ag NPs are synthesized using the essential oil extracted from leaves of M. fragrans. The catalytic potential of the synthesized Ag NPs is tested by studying the degradation of para nitro phenol (4NP), an anthropogenic pollutant and methylene blue (MB), a thiazine dye. The antibacterial activity of the biogenic Ag NPs against the pathogenic Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) is studied using Agar well diffusion method. The in vitro antioxidant activity of Ag NPs is investigated through a series of assays.
Materials and methods Fresh green leaves of M. fragrans were collected from Thiruvananthapuram. Silver nitrate (AgNO3), 4NP and sodium borohydride (NaBH4) were procured from Sigma–Aldrich; MB and acetone from Merck were used. Demineralised water has been used throughout the experiment.
300 g fresh leaves of M. fragrans are hydro distilled in a Clevenger apparatus, yielding 3 mL essential oil (yield 1%). The aromatic oil, diluted using acetone (1:170) is used for the synthesis of Ag NPs. 1 mL of diluted oil is added, with vigorous stirring to 30 mL of 2.14 104 M AgNO3 at 100 °C and pH 7 to get the colloid a1. The experiment is repeated with 2, 3, 4, 5 mL diluted oil to get colloids a2, a3, a4 and a5, respectively. Colour change from yellow to golden yellow and to red, indicated the enhanced formation of Ag NPs, with quantity of diluted oil. 2 mL diluted oil is added with vigorous stirring to 30 mL boiling solution of AgNO3 (2.14 104 M) at varying pH conditions of 7, 8, 9 and 10 to obtain colloids a2, a6, a7 and a8, respectively. Catalytic activity of Ag NPs The efficiency of Ag NPs as heterogeneous catalyst in the reduction of 4NP and MB using NaBH4 is investigated. The stock solutions, 7.1 103 M 4NP and 0.25 M NaBH4 are prepared. After stirring 1 mL each of 4NP and NaBH4 in 23 mL aqueous medium, 1 mL of Ag nanocatalyst is added and vigorously stirred. UV–vis spectra are recorded after regular intervals of time to study the degradation of 4NP. The procedure is repeated with 9.9 102 M NaBH4 and 103 M MB. The progress of the rapid degradation of the cationic dye is studied through UV–vis spectra taken at regular intervals. Antibacterial and antioxidant potential of Ag NPs Agar-well diffusion method is used for the antibacterial studies of green synthesized Ag NPs. Mueller–Hinton plates are seeded with S. aureus and E. coli. Wells of 10 mm are bored and samples of 25, 50,100-lL concentration are added. After incubating at 37 °C for 24 h, the antibacterial activity is assayed by measuring the diameter of the zone of inhibition formed around the well. Experiments are done in triplicate and mean values are presented. Gentamycin is used as the positive control [21,22]. Free radical scavenging activity, reducing power, total antioxidant activity of the green synthesized Ag NPs is exemplified through nitric oxide scavenging activity, hydrogen peroxide scavenging activity, reducing power activity, 2,2-diphenyl -1-picryl hydrazyl (DPPH)) assay and total antioxidant activity (description of the procedure of antioxidant assays is given in Supplemental information). Absorbance of the Ag NP bound coloured complexes of radicals or ions are determined (Atest) in addition to the absorbance of a similar mixture without the test solution (Acontrol). Percentage inhibition (%inhibition) is ascertained using the equation,
%inhibition ¼ ½ðAcontrol Atest Þ=Acontrol 100
ð1Þ
Instrumentation UV–vis spectra are recorded using Perkin–Elmer Lambda-35 spectrophotometer with a scanning speed of 480 nm/min and a slit width of 1 nm. TEM samples are prepared by dropping the Ag colloid on carbon coated copper grids. The images are taken using TecnaiG2 30 TEM after the evaporation of the solvents. XRD pattern is obtained using XPERT-Pro diffractometer operating at 30 mA current and 40 kV voltage for the scanning angle 2h from 20° to 100°. The diffraction pattern is obtained by irradiating with Cu Ka radiation with k of 1.5406 Å. FTIR spectra are recorded using IR Prestige-21 Shimadzu spectrophotometer. GC–MS analysis was carried out on a HP Chem Gas Chromatogram fitted with a DB5 silica column coupled with a model 5973 mass detector. Essential oil components were determined on comparison of the mass spectra with Wiley 275.L database. (GC–MS spectra given in Fig. S1 of Supplemental information).
V. Vilas et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 132 (2014) 743–750
Results and discussion The essential oil of M. fragrans is used for the reduction of Ag+ to Ag . GC–MS spectral analysis reports reveal the relative percentage composition of the leaf essential oil of M. fragrans. Amidst the 92.51% of identified components, the essential oil is primarily comprised of Myristicin (22.76%), (1S) b (-) Pinene (18.92%), Limonene (10.94%), Sabinene (9.14%) and Terpinolene (6.64%). (% Composition given in Table S1 of Supplemental information). The formation and growth of Ag NPs is assumed to be in accordance with ‘LaMer Model’ [23]. The reduction of Ag+ ions is followed by condensation and surface reduction to Ag NPs. Though, the optimum conditions for the synthesis of nanoAg are found out, factors that affect the formation like change in volume of diluted oil and change in pH are studied with in experimental limitations. When metal NPs are subjected to light excitation, the conduction electrons on the metal surface, are set into oscillations. The resonance attained between the frequencies of surface electron oscillations and light photons is localised surface plasmon resonance (LSPR) [24] depicted through UV–vis spectral studies. 0
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The wavelength for maximum absorption (kmax) and band width yield a unique spectral fingerprint for a plasmonic NP with a specific size and shape [25]. The increase in intensity of the SPR band with quantity of diluted oil shows the enhanced formation of Ag NPs. Fig. 1a shows symmetric curves with kmax varying from 420 to 424 nm, with change in oil quantity. SPR band shows an increase in intensity with pH (Fig. 1b) indicating rapid formation of Ag NPs. LSPR is sensitive to size, shape, material and dielectric environment of NPs. Surface morphology and size of the green synthesized Ag NPs is determined through TEM analysis. TEM images of the colloids a2 and a5 are shown in Figs. 2(a and b) and 3(a and b) respectively at different magnifications. TEM images reveal the presence of 12–26 nm sized Ag NPs in the colloids. TEM images further depict the enclosure of nanoAg aggregates by a thin organic layer (Fig. S2 of Supplemental information). SAED pattern shows bright circular diffraction rings which can be indexed from inner to outer as (1 1 1), (2 0 0), (2 2 0), (3 1 1) and (2 2 2) confirming the face centred cubic crystalline structure of nanoAg. The peaks in the X-ray diffraction pattern (Fig. 4a) are due to reflections from the (1 1 1), (2 0 0), (2 2 0), (3 1 1), and (2 2 2) planes confirming the face centred cubic crystalline structure of the Ag
Fig. 1. UV–vis spectra of (a) colloids a1–a5 showing the enhanced formation of silver Ag NPs with volume of diluted oil, (b) colloids a2, a6, a7, a8 showing the enhanced formation of Ag NPs with increase in pH of the medium.
Fig. 2. TEM images of colloid a2 at different magnification. Inset figure depicts the SAED pattern of Ag NPs showing bright diffraction rings corresponding to the indexed planes.
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Fig. 3. TEM images of colloid a5 at different magnifications.
Fig. 4. (a) XRD pattern exhibiting the fcc structure of Ag NPs. (b) FTIR spectra of Ag NPs and diluted essential oil.
Fig. 5. UV–vis spectra showing degradation of (a) para nitro phenol and (b) methylene blue.
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Reduction catalysis using Ag NPs
Fig. 6. Linear correlation between ln[A/A0] and time depicting (a) pseudo first order degradation of 4NP and (b) pseudo first order degradation of MB.
The rapid reduction of 4NP is followed through UV–vis spectral analysis. Fig. 5a shows an intense band at 400 nm due to the formation of sodium nitrophenolate ion [26] which shows a decrease in intensity with time and a concomitant band at 296 nm indicating para amino phenol (4AP) [27–29]. Fig. 5b shows the degradation of the cationic, water soluble dye, MB. Reduction in the intensity of the band at 664 nm is due to the formation of colourless leucomethylene blue [30,31]. In the absence of the nanocatalyst, the reduction reaction proceeds at a very slow rate. The intermediate redox potential of Ag+/Ag system with respect to the donor – acceptor system, E0(A/A) < E0(Ag+/Ag) < E0(D+/D) enables an efficient reaction between the donor and acceptor, owing to the double electron transfer through the metal cluster relay; referred to as the electron relay effect [32]. Analysis of the kinetic data of both the degradation reactions reveals pseudo-first order reaction kinetics, due to the constant concentration of NaBH4 taken in excess. The linear plot of ln[A/A0] versus time (Fig. 6) in both the cases supports the kinetic studies. Antibacterial potential of Ag NPs
NPs (JCPDS file no: 04-0783). Absence of any other peak in the diffraction pattern indicates the high purity of the sample. FTIR spectrum of diluted oil (Fig. 4b) shows prominent bands at 1636 cm1,1510 cm1,1432 cm1and 1130 cm1. Bands at 1636 cm1and 1500 cm1 are due to C@C stretching vibrations of aromatic rings. The band at 1432 cm1 shows the OH deformations in the oil which shifts to 1382 cm1 upon the formation of nanoAg. FTIR spectrum of oil, shows a number of bands between 1300 cm1 and 885 cm1 due to CAO stretching vibrations of alcohols, ethers and esters. A strong broad band at 1055 cm1 is observed in the FTIR spectrum of Ag NPs. FTIR measurements are in accordance with GC–MS results of leaf essential oil of M. fragrans. Weak tertiary alcohols are good reducing agents at 100 °C in the presence of acetone. This is shown by the shift from 1432 cm1 to 1382 cm1. Carbonyl group of the carboxylic acid, with high affinity, then coordinates to Ag, reducing and stabilising them.
The antibacterial activity of Ag NPs against two bacterial strains, S. aureus and E. coli has been investigated. Figs. 7(a–d) and 8(a–d) clearly depicts the enhanced activity of biogenic Ag NPs (a2) against the bacterial strains in comparison to the diluted oil. The marked difference in the diameter of the zone of inhibition of the two samples a2 and a5, is attributed to a thin oil coating around the Ag NPs in a5. However, the zone of inhibition increased on increasing the concentration of a2 (Table 1). The green synthesised Ag NPs showed greater activity against E. coli than S. aureus. The lower efficacy of Ag NPs against S. aureus is attributed to the difference in the membrane structure, distinctively, thickness of the peptidoglycan layer between the two bacteria [33]. During antibacterial action, Ag+ ions, the soft acid interacts strongly with the phosphorus and sulphur compounds (soft base) (Figs. 9 and 10) leading to breakdown of mitochondrial function, DNA damage
Fig. 7. Mueller–Hinton plates showing inhibition zones by (a) diluted essential oil, (b) silver nitrate, (c) colloid a2 and (d) colloid a5 at different concentrations against S. aureus.
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Fig. 8. Mueller–Hinton plates showing inhibition zones by (a) diluted essential oil, (b) silver nitrate, (c) colloid a2 and (d) colloid a5 at different concentrations against E. coli.
Table 1 Inhibition zone diameter (mm) of silver nitrate solution, silver nanoparticles and dilute oil against E. coli and S. aureus using Gentamycin as positive control. Concentration (lg)
Sample
Zone of inhibition (mm) E. coli
S. aureus
25 50 100
23 NZ NZ 11
23 NZ NZ 12
Colloid a2
25 50 100
10 12 14
NZ 10 12
Colloid a5
25 50 100
NZ NZ NZ
NZ NZ NZ
25 50 100
NZ NZ 9
NZ NZ NZ
Gentamycin Silver nitrate
Dilute oil
NZ = no zone of inhibition.
Fig. 10. Strong interaction between Ag (soft acid) and sulphur (soft base) of cysteine residues of proteins [HSAB Theory] result in protein denaturation.
or denaturation of protein, ultimately resulting in cell death [34–38]. Also, ESR studies [38] have revealed the role of free radicals generated from Ag NPs as a cause of its bactericidal action. Further, strong association of silver nanoparticles with the cell wall of the bacteria may result in the formation of pits, affecting the permeability and ultimately in cell death [39]. Antibacterial
Fig. 9. Strong interaction between Ag (soft acid) and phosphorus (soft base) of phosphate molecule of DNA [HSAB Theory] result in DNA damage.
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Fig. 11. Antioxidant activity of Ag NPs exhibited through the assays (a) NO scavenging activity, (b) hydrogen peroxide scavenging activity, (c) reducing power activity, (d) DPPH assay and (e) total antioxidant activity.
activity of the silver nanoparticles depends on the available particle surface area. Smaller particles with a larger surface area available for interaction would show a higher antibacterial action than larger particles [1,40]. In vitro antioxidant activity of Ag NPs Interaction of the human system with oxygen generates free radicals, neutralised by glutathione/GSH, the master antioxidant. Oxidative stress induced by the changing environment and life style result in well known and wide spread diseases. Metal NPs are potent antioxidants that can delay the onset or slow the rate of oxidation of autooxidizable materials. Despite its beneficial effects, NO produces nitrite ions on reacting with superoxide, which may lead to tissue damage. Scavengers of NO compete with oxygen, leading to reduced production of nitrite ions. The NO scavenging activity of a2 and a5 increases with increase in concentration of the samples (Fig. 11a.). Hydrogen peroxide can cross cell membranes, rapidly and inside the cell, on reaction with Fe2+ or Cu2+, they may form hydroxy radical, the origin of many toxic effects. Positive hydrogen peroxide tests demonstrate that Ag NPs are competent free radical scavengers (Fig. 11b). Silver colloid a2 readily reduces Fe3+, exhibiting its enhanced reducing power than a5 (Fig. 11c). Silver colloid a2 acts as a better scavenger of the stable DPPH free radical. The power to scavenge DPPH is found to increase with concentration (Fig. 11d). An increase in optical density at 695 nm due to the formation of green phosphomolybdenum complex, exhibits the dose dependent antioxidant activity of the synthesized Ag NPs (Fig. 11e). Conclusion Increasing global concern over environmental protection with the emergence of non-biodegradable pollutants and antibiotic drug resistant bacteria, urge the need for potent chemocatalytic bioactive green synthesized nanoparticles. The study points out the pertinence of using essential oil of M. fragrans for the synthesis of 12–26 nm sized, spherical, crystalline Ag NPs. Terpenes and phenyl propenes in essential oil function as reducing and capping
agents, as revealed by FTIR and GC–MS analysis. Ag NPs of high purity and crystallinity are obtained as depicted by the XRD and SAED patterns. The study demonstrates the strong chemocatalytic potential and antibacterial activity of Ag NPs. The phytosynthesized biogenic Ag NPs exhibit strong electron donating, reducing and free radical scavenging ability. Using a refined phytosynthetic design the study supports the potential of green synthesized nanoAg to be used in diverse fields. Acknowledgements The authors are grateful for the technical supports by NIST and CESS, Thiruvananthapuram. Vidya Vilas (one of the authors) acknowledges KSCSTE, Govt of Kerala for the research fellowship. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.saa.2014.05.046. References [1] M. Vanaja, G. Annadurai, Appl. Nanosci. 3 (2012) 217–223. [2] A. Kumar, V.K. Vamula, P.M. Ajayan, G. John, Nat. Mat. 7 (2008) 236–241. [3] E.C. daSilva, M.G.A. daSilva, S.M.P. Meneghetti, G. Machado, M.A.R.C. Alancar, J.M. Hickmann, M.R. Meneghetti, J. Nanopart. Res. 10 (2008) 201–208. [4] M. MeenaKumari, D. Philip, Spectrochim. Acta, Part A 111 (2013) 154–160. [5] R. Zamiri, A. Zakaria, H.A. Ahargar, A.R. Sadrohosseini, M.A. Mahdi, Int. J. Mol. Sci. 11 (2010) 4764–4770. [6] R. Zamiri, B.Z. Azmi, A.R. Sadrolhosseini, H.A. Ahangar, A.W. Zaidan, M.A. Mahdi, Int. J. Nanomed. 6 (2011) 71–75. [7] D.S. Sheny, J. Mathew, D. Philip, Spectrochim. Acta, Part A 97 (2012) 306–310. [8] J. Pulit, M. Banach, Z. Kowalski, Chem. Eng. Sci. 18 (2011) 185–196. [9] R. Vaidyanathan, K. Kailashwaralal, S. Gopalram, E. Gurunathan, Biotechnol. Adv. 27 (2009) 924–937. [10] E. Roduner, Chem. Soc. Rev. 35 (2006) 583–592. [11] K. Varner, A. El-Badawy, J. Sanford, United States Environmental Protection Agency Scientific, Technical, Research, Engineering and Modelling Support Report, 2010. [12] R. Koyyati, V. Nagati, R. Merugu, P. Manthurpandigya, Int. J. Med. Pharm. Sci. 3 (2013) 89–100. [13] I.R. Bunghez, M.E.B. Patrasue, N. Badea, S.M. Doncea, A. Ponesue, R.M. Ion, J. Optoelec, Adv. Mater. 14 (2012) 1016–1022. [14] J. Banerjee, R.T. Narendhirakannan, J. Nanomater. Biostruct. 6 (2011) 961–968. [15] P.S. Vankar, D. Shukla, Appl. Nanosci. 2 (2012) 163–168.
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Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Catalytically and biologically active silver nanoparticles synthesized using essential oil Vidya Vilas a,b, Daizy Philip a,⇑, Joseph Mathew b a b
Department of Physics, Mar Ivanios College, Thiruvananthapuram 695015, India Department of Chemistry, Mar Ivanios College, Thiruvananthapuram 695015, India
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Essential oil is used for the synthesis
of silver nanoparticles. Rapid, cost-effective, environmentally
benign method is suggested. Efficiency of the nanocatalyst is
portrayed in the degradation of a cationic dye and an organic pollutant. The synthesized biogenic silver nanoparticles act as potent free radical scavengers and antibacterial agent.
a r t i c l e
i n f o
Article history: Received 21 January 2014 Received in revised form 9 May 2014 Accepted 14 May 2014 Available online 13 June 2014 Keywords: Silver nanoparticles Myristica fragrans Catalysis Antibacterial activity Antioxidant activity
a b s t r a c t There are numerous reports on phytosynthesis of silver nanoparticles and various phytochemicals are involved in the reduction and stabilization. Pure explicit phytosynthetic protocol for catalytically and biologically active silver nanoparticles is of importance as it is an environmentally benign green method. This paper reports the use of essential oil of Myristica fragrans enriched in terpenes and phenyl propenes in the reduction and stabilization. FTIR spectra of the essential oil and the synthesized biogenic silver nanoparticles are in accordance with the GC–MS spectral analysis reports. Nanosilver is initially characterized by an intense SPR band around 420 nm, followed by XRD and TEM analysis revealing the formation of 12–26 nm sized, highly pure, crystalline silver nanoparticles. Excellent catalytic and bioactive potential of the silver nanoparticles is due to the surface modification. The chemocatalytic potential of nanosilver is exhibited by the rapid reduction of the organic pollutant, para nitro phenol and by the degradation of the thiazine dye, methylene blue. Significant antibacterial activity of the silver colloid against Gram positive, Staphylococcus aureus (inhibition zone – 12 mm) and Gram negative, Escherichia coli (inhibition zone – 14 mm) is demonstrated by Agar-well diffusion method. Strong antioxidant activity of the biogenic silver nanoparticles is depicted through NO scavenging, hydrogen peroxide scavenging, reducing power, DPPH and total antioxidant activity assays. Ó 2014 Elsevier B.V. All rights reserved.
Introduction
⇑ Corresponding author. Tel.: +91 471 2530887; fax: +91 471 2530023. E-mail addresses: [email protected], [email protected] (D. Philip). http://dx.doi.org/10.1016/j.saa.2014.05.046 1386-1425/Ó 2014 Elsevier B.V. All rights reserved.
Molecular manipulation at the nanoscale has undergone progressive changes ever since its dawn before 4th century AD. Though the methods of nanoparticle (NP) synthesis extends over
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a wide range, green synthesis methods has gained wider acclaim owing to the increasing awareness on the undesirable changes of the environment posed by several synthesis strategies. Phytosynthesis of nanoparticles uses phytoconstituents of the plant parts as reducing and capping agents. This ecofriendly method does not demand the use of high temperature, pressure or energy [1]. Development of a facile, pure, specific phytosynthetic route leads to the use of volatile aromatic oil. Kumar et al. [2] have synthesized metal NPs embedded paints based on vegetable oil. The auto oxidation of drying oils have been used for the reduction of metal salts to synthesize metal NPs. Castor oil, palm oil and coconut oil have been used for the synthesis of metal NPs [3–6]. In the first report on essential oil mediated synthesis of metal NPs, Sheny et al. [7] have used Anacardium occidentale as a reducing and capping agent for the synthesis of hexagonal gold NPs. The synthesis of silver (Ag) on a nanoscale is of importance, due to its thermal conductivity, chemical stability, catalytic and antibacterial activity [8,9]. The surface effects and quantum effects of nanoAg affects their chemical reactivity [10] and gives them unique mechanical, optical and electric properties [11]. Recent reports [12–17] suggest biological method for synthesis of antifungal, antibacterial, antioxidant, robust, biocompatible Ag NPs. Size, shape, surface area to volume ratio and the nature of the surface modifier act as limiting factors of the synthesized Ag NPs imparting adequate therapeutic potency. Exploitation of the antibacterial potential of nanoAg can be traced back to ancient times. Oligodynamic action coupled with a broad spectrum of targeted bacteria prompts the use of Ag based nanostructured materials as efficient antibacterial agents [18]. The rapid multiplication and proliferation of drug resistant bacteria is curbed by Ag NPs fortifying its use in burn treatments, water filters and as an antimicrobial finish on fabrics [15]. Antioxidant nanoAg repairs reactive oxygen species (ROS) damage to cell components and cell function disruption enhancing the immune system. The conjoint application of nanoAg as an antibacterial and an antioxidant agent ensures its use as a biomedical nanoproduct. NanoAg extends its aptness as a catalyst in pollution treatment, due to its small size, larger surface area to volume ratio and greater accessibility of the surface atoms. Ag NPs have attracted the interests of scientists in its use as a competent catalyst in the reduction of major pollutants of the dye industry that pose serious health risks. Myristica fragrans (M. fragrans) is an evergreen tree, successfully cultivated in South India. There are reports [19,20] on the hepatoprotective and anti cancer activity of Myristicin and antiinflammatory property of t – Caryophyllene; the essential oil constituents. The leaf oil exhibit significant antimicrobial and larvicide activity and cytotoxic activity against MCF-7 breast cancer cell line and A 357 epidermal skin cancer line [19]. In the present study, Ag NPs are synthesized using the essential oil extracted from leaves of M. fragrans. The catalytic potential of the synthesized Ag NPs is tested by studying the degradation of para nitro phenol (4NP), an anthropogenic pollutant and methylene blue (MB), a thiazine dye. The antibacterial activity of the biogenic Ag NPs against the pathogenic Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) is studied using Agar well diffusion method. The in vitro antioxidant activity of Ag NPs is investigated through a series of assays.
Materials and methods Fresh green leaves of M. fragrans were collected from Thiruvananthapuram. Silver nitrate (AgNO3), 4NP and sodium borohydride (NaBH4) were procured from Sigma–Aldrich; MB and acetone from Merck were used. Demineralised water has been used throughout the experiment.
300 g fresh leaves of M. fragrans are hydro distilled in a Clevenger apparatus, yielding 3 mL essential oil (yield 1%). The aromatic oil, diluted using acetone (1:170) is used for the synthesis of Ag NPs. 1 mL of diluted oil is added, with vigorous stirring to 30 mL of 2.14 104 M AgNO3 at 100 °C and pH 7 to get the colloid a1. The experiment is repeated with 2, 3, 4, 5 mL diluted oil to get colloids a2, a3, a4 and a5, respectively. Colour change from yellow to golden yellow and to red, indicated the enhanced formation of Ag NPs, with quantity of diluted oil. 2 mL diluted oil is added with vigorous stirring to 30 mL boiling solution of AgNO3 (2.14 104 M) at varying pH conditions of 7, 8, 9 and 10 to obtain colloids a2, a6, a7 and a8, respectively. Catalytic activity of Ag NPs The efficiency of Ag NPs as heterogeneous catalyst in the reduction of 4NP and MB using NaBH4 is investigated. The stock solutions, 7.1 103 M 4NP and 0.25 M NaBH4 are prepared. After stirring 1 mL each of 4NP and NaBH4 in 23 mL aqueous medium, 1 mL of Ag nanocatalyst is added and vigorously stirred. UV–vis spectra are recorded after regular intervals of time to study the degradation of 4NP. The procedure is repeated with 9.9 102 M NaBH4 and 103 M MB. The progress of the rapid degradation of the cationic dye is studied through UV–vis spectra taken at regular intervals. Antibacterial and antioxidant potential of Ag NPs Agar-well diffusion method is used for the antibacterial studies of green synthesized Ag NPs. Mueller–Hinton plates are seeded with S. aureus and E. coli. Wells of 10 mm are bored and samples of 25, 50,100-lL concentration are added. After incubating at 37 °C for 24 h, the antibacterial activity is assayed by measuring the diameter of the zone of inhibition formed around the well. Experiments are done in triplicate and mean values are presented. Gentamycin is used as the positive control [21,22]. Free radical scavenging activity, reducing power, total antioxidant activity of the green synthesized Ag NPs is exemplified through nitric oxide scavenging activity, hydrogen peroxide scavenging activity, reducing power activity, 2,2-diphenyl -1-picryl hydrazyl (DPPH)) assay and total antioxidant activity (description of the procedure of antioxidant assays is given in Supplemental information). Absorbance of the Ag NP bound coloured complexes of radicals or ions are determined (Atest) in addition to the absorbance of a similar mixture without the test solution (Acontrol). Percentage inhibition (%inhibition) is ascertained using the equation,
%inhibition ¼ ½ðAcontrol Atest Þ=Acontrol 100
ð1Þ
Instrumentation UV–vis spectra are recorded using Perkin–Elmer Lambda-35 spectrophotometer with a scanning speed of 480 nm/min and a slit width of 1 nm. TEM samples are prepared by dropping the Ag colloid on carbon coated copper grids. The images are taken using TecnaiG2 30 TEM after the evaporation of the solvents. XRD pattern is obtained using XPERT-Pro diffractometer operating at 30 mA current and 40 kV voltage for the scanning angle 2h from 20° to 100°. The diffraction pattern is obtained by irradiating with Cu Ka radiation with k of 1.5406 Å. FTIR spectra are recorded using IR Prestige-21 Shimadzu spectrophotometer. GC–MS analysis was carried out on a HP Chem Gas Chromatogram fitted with a DB5 silica column coupled with a model 5973 mass detector. Essential oil components were determined on comparison of the mass spectra with Wiley 275.L database. (GC–MS spectra given in Fig. S1 of Supplemental information).
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Results and discussion The essential oil of M. fragrans is used for the reduction of Ag+ to Ag . GC–MS spectral analysis reports reveal the relative percentage composition of the leaf essential oil of M. fragrans. Amidst the 92.51% of identified components, the essential oil is primarily comprised of Myristicin (22.76%), (1S) b (-) Pinene (18.92%), Limonene (10.94%), Sabinene (9.14%) and Terpinolene (6.64%). (% Composition given in Table S1 of Supplemental information). The formation and growth of Ag NPs is assumed to be in accordance with ‘LaMer Model’ [23]. The reduction of Ag+ ions is followed by condensation and surface reduction to Ag NPs. Though, the optimum conditions for the synthesis of nanoAg are found out, factors that affect the formation like change in volume of diluted oil and change in pH are studied with in experimental limitations. When metal NPs are subjected to light excitation, the conduction electrons on the metal surface, are set into oscillations. The resonance attained between the frequencies of surface electron oscillations and light photons is localised surface plasmon resonance (LSPR) [24] depicted through UV–vis spectral studies. 0
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The wavelength for maximum absorption (kmax) and band width yield a unique spectral fingerprint for a plasmonic NP with a specific size and shape [25]. The increase in intensity of the SPR band with quantity of diluted oil shows the enhanced formation of Ag NPs. Fig. 1a shows symmetric curves with kmax varying from 420 to 424 nm, with change in oil quantity. SPR band shows an increase in intensity with pH (Fig. 1b) indicating rapid formation of Ag NPs. LSPR is sensitive to size, shape, material and dielectric environment of NPs. Surface morphology and size of the green synthesized Ag NPs is determined through TEM analysis. TEM images of the colloids a2 and a5 are shown in Figs. 2(a and b) and 3(a and b) respectively at different magnifications. TEM images reveal the presence of 12–26 nm sized Ag NPs in the colloids. TEM images further depict the enclosure of nanoAg aggregates by a thin organic layer (Fig. S2 of Supplemental information). SAED pattern shows bright circular diffraction rings which can be indexed from inner to outer as (1 1 1), (2 0 0), (2 2 0), (3 1 1) and (2 2 2) confirming the face centred cubic crystalline structure of nanoAg. The peaks in the X-ray diffraction pattern (Fig. 4a) are due to reflections from the (1 1 1), (2 0 0), (2 2 0), (3 1 1), and (2 2 2) planes confirming the face centred cubic crystalline structure of the Ag
Fig. 1. UV–vis spectra of (a) colloids a1–a5 showing the enhanced formation of silver Ag NPs with volume of diluted oil, (b) colloids a2, a6, a7, a8 showing the enhanced formation of Ag NPs with increase in pH of the medium.
Fig. 2. TEM images of colloid a2 at different magnification. Inset figure depicts the SAED pattern of Ag NPs showing bright diffraction rings corresponding to the indexed planes.
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Fig. 3. TEM images of colloid a5 at different magnifications.
Fig. 4. (a) XRD pattern exhibiting the fcc structure of Ag NPs. (b) FTIR spectra of Ag NPs and diluted essential oil.
Fig. 5. UV–vis spectra showing degradation of (a) para nitro phenol and (b) methylene blue.
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Reduction catalysis using Ag NPs
Fig. 6. Linear correlation between ln[A/A0] and time depicting (a) pseudo first order degradation of 4NP and (b) pseudo first order degradation of MB.
The rapid reduction of 4NP is followed through UV–vis spectral analysis. Fig. 5a shows an intense band at 400 nm due to the formation of sodium nitrophenolate ion [26] which shows a decrease in intensity with time and a concomitant band at 296 nm indicating para amino phenol (4AP) [27–29]. Fig. 5b shows the degradation of the cationic, water soluble dye, MB. Reduction in the intensity of the band at 664 nm is due to the formation of colourless leucomethylene blue [30,31]. In the absence of the nanocatalyst, the reduction reaction proceeds at a very slow rate. The intermediate redox potential of Ag+/Ag system with respect to the donor – acceptor system, E0(A/A) < E0(Ag+/Ag) < E0(D+/D) enables an efficient reaction between the donor and acceptor, owing to the double electron transfer through the metal cluster relay; referred to as the electron relay effect [32]. Analysis of the kinetic data of both the degradation reactions reveals pseudo-first order reaction kinetics, due to the constant concentration of NaBH4 taken in excess. The linear plot of ln[A/A0] versus time (Fig. 6) in both the cases supports the kinetic studies. Antibacterial potential of Ag NPs
NPs (JCPDS file no: 04-0783). Absence of any other peak in the diffraction pattern indicates the high purity of the sample. FTIR spectrum of diluted oil (Fig. 4b) shows prominent bands at 1636 cm1,1510 cm1,1432 cm1and 1130 cm1. Bands at 1636 cm1and 1500 cm1 are due to C@C stretching vibrations of aromatic rings. The band at 1432 cm1 shows the OH deformations in the oil which shifts to 1382 cm1 upon the formation of nanoAg. FTIR spectrum of oil, shows a number of bands between 1300 cm1 and 885 cm1 due to CAO stretching vibrations of alcohols, ethers and esters. A strong broad band at 1055 cm1 is observed in the FTIR spectrum of Ag NPs. FTIR measurements are in accordance with GC–MS results of leaf essential oil of M. fragrans. Weak tertiary alcohols are good reducing agents at 100 °C in the presence of acetone. This is shown by the shift from 1432 cm1 to 1382 cm1. Carbonyl group of the carboxylic acid, with high affinity, then coordinates to Ag, reducing and stabilising them.
The antibacterial activity of Ag NPs against two bacterial strains, S. aureus and E. coli has been investigated. Figs. 7(a–d) and 8(a–d) clearly depicts the enhanced activity of biogenic Ag NPs (a2) against the bacterial strains in comparison to the diluted oil. The marked difference in the diameter of the zone of inhibition of the two samples a2 and a5, is attributed to a thin oil coating around the Ag NPs in a5. However, the zone of inhibition increased on increasing the concentration of a2 (Table 1). The green synthesised Ag NPs showed greater activity against E. coli than S. aureus. The lower efficacy of Ag NPs against S. aureus is attributed to the difference in the membrane structure, distinctively, thickness of the peptidoglycan layer between the two bacteria [33]. During antibacterial action, Ag+ ions, the soft acid interacts strongly with the phosphorus and sulphur compounds (soft base) (Figs. 9 and 10) leading to breakdown of mitochondrial function, DNA damage
Fig. 7. Mueller–Hinton plates showing inhibition zones by (a) diluted essential oil, (b) silver nitrate, (c) colloid a2 and (d) colloid a5 at different concentrations against S. aureus.
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Fig. 8. Mueller–Hinton plates showing inhibition zones by (a) diluted essential oil, (b) silver nitrate, (c) colloid a2 and (d) colloid a5 at different concentrations against E. coli.
Table 1 Inhibition zone diameter (mm) of silver nitrate solution, silver nanoparticles and dilute oil against E. coli and S. aureus using Gentamycin as positive control. Concentration (lg)
Sample
Zone of inhibition (mm) E. coli
S. aureus
25 50 100
23 NZ NZ 11
23 NZ NZ 12
Colloid a2
25 50 100
10 12 14
NZ 10 12
Colloid a5
25 50 100
NZ NZ NZ
NZ NZ NZ
25 50 100
NZ NZ 9
NZ NZ NZ
Gentamycin Silver nitrate
Dilute oil
NZ = no zone of inhibition.
Fig. 10. Strong interaction between Ag (soft acid) and sulphur (soft base) of cysteine residues of proteins [HSAB Theory] result in protein denaturation.
or denaturation of protein, ultimately resulting in cell death [34–38]. Also, ESR studies [38] have revealed the role of free radicals generated from Ag NPs as a cause of its bactericidal action. Further, strong association of silver nanoparticles with the cell wall of the bacteria may result in the formation of pits, affecting the permeability and ultimately in cell death [39]. Antibacterial
Fig. 9. Strong interaction between Ag (soft acid) and phosphorus (soft base) of phosphate molecule of DNA [HSAB Theory] result in DNA damage.
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Fig. 11. Antioxidant activity of Ag NPs exhibited through the assays (a) NO scavenging activity, (b) hydrogen peroxide scavenging activity, (c) reducing power activity, (d) DPPH assay and (e) total antioxidant activity.
activity of the silver nanoparticles depends on the available particle surface area. Smaller particles with a larger surface area available for interaction would show a higher antibacterial action than larger particles [1,40]. In vitro antioxidant activity of Ag NPs Interaction of the human system with oxygen generates free radicals, neutralised by glutathione/GSH, the master antioxidant. Oxidative stress induced by the changing environment and life style result in well known and wide spread diseases. Metal NPs are potent antioxidants that can delay the onset or slow the rate of oxidation of autooxidizable materials. Despite its beneficial effects, NO produces nitrite ions on reacting with superoxide, which may lead to tissue damage. Scavengers of NO compete with oxygen, leading to reduced production of nitrite ions. The NO scavenging activity of a2 and a5 increases with increase in concentration of the samples (Fig. 11a.). Hydrogen peroxide can cross cell membranes, rapidly and inside the cell, on reaction with Fe2+ or Cu2+, they may form hydroxy radical, the origin of many toxic effects. Positive hydrogen peroxide tests demonstrate that Ag NPs are competent free radical scavengers (Fig. 11b). Silver colloid a2 readily reduces Fe3+, exhibiting its enhanced reducing power than a5 (Fig. 11c). Silver colloid a2 acts as a better scavenger of the stable DPPH free radical. The power to scavenge DPPH is found to increase with concentration (Fig. 11d). An increase in optical density at 695 nm due to the formation of green phosphomolybdenum complex, exhibits the dose dependent antioxidant activity of the synthesized Ag NPs (Fig. 11e). Conclusion Increasing global concern over environmental protection with the emergence of non-biodegradable pollutants and antibiotic drug resistant bacteria, urge the need for potent chemocatalytic bioactive green synthesized nanoparticles. The study points out the pertinence of using essential oil of M. fragrans for the synthesis of 12–26 nm sized, spherical, crystalline Ag NPs. Terpenes and phenyl propenes in essential oil function as reducing and capping
agents, as revealed by FTIR and GC–MS analysis. Ag NPs of high purity and crystallinity are obtained as depicted by the XRD and SAED patterns. The study demonstrates the strong chemocatalytic potential and antibacterial activity of Ag NPs. The phytosynthesized biogenic Ag NPs exhibit strong electron donating, reducing and free radical scavenging ability. Using a refined phytosynthetic design the study supports the potential of green synthesized nanoAg to be used in diverse fields. Acknowledgements The authors are grateful for the technical supports by NIST and CESS, Thiruvananthapuram. Vidya Vilas (one of the authors) acknowledges KSCSTE, Govt of Kerala for the research fellowship. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.saa.2014.05.046. References [1] M. Vanaja, G. Annadurai, Appl. Nanosci. 3 (2012) 217–223. [2] A. Kumar, V.K. Vamula, P.M. Ajayan, G. John, Nat. Mat. 7 (2008) 236–241. [3] E.C. daSilva, M.G.A. daSilva, S.M.P. Meneghetti, G. Machado, M.A.R.C. Alancar, J.M. Hickmann, M.R. Meneghetti, J. Nanopart. Res. 10 (2008) 201–208. [4] M. MeenaKumari, D. Philip, Spectrochim. Acta, Part A 111 (2013) 154–160. [5] R. Zamiri, A. Zakaria, H.A. Ahargar, A.R. Sadrohosseini, M.A. Mahdi, Int. J. Mol. Sci. 11 (2010) 4764–4770. [6] R. Zamiri, B.Z. Azmi, A.R. Sadrolhosseini, H.A. Ahangar, A.W. Zaidan, M.A. Mahdi, Int. J. Nanomed. 6 (2011) 71–75. [7] D.S. Sheny, J. Mathew, D. Philip, Spectrochim. Acta, Part A 97 (2012) 306–310. [8] J. Pulit, M. Banach, Z. Kowalski, Chem. Eng. Sci. 18 (2011) 185–196. [9] R. Vaidyanathan, K. Kailashwaralal, S. Gopalram, E. Gurunathan, Biotechnol. Adv. 27 (2009) 924–937. [10] E. Roduner, Chem. Soc. Rev. 35 (2006) 583–592. [11] K. Varner, A. El-Badawy, J. Sanford, United States Environmental Protection Agency Scientific, Technical, Research, Engineering and Modelling Support Report, 2010. [12] R. Koyyati, V. Nagati, R. Merugu, P. Manthurpandigya, Int. J. Med. Pharm. Sci. 3 (2013) 89–100. [13] I.R. Bunghez, M.E.B. Patrasue, N. Badea, S.M. Doncea, A. Ponesue, R.M. Ion, J. Optoelec, Adv. Mater. 14 (2012) 1016–1022. [14] J. Banerjee, R.T. Narendhirakannan, J. Nanomater. Biostruct. 6 (2011) 961–968. [15] P.S. Vankar, D. Shukla, Appl. Nanosci. 2 (2012) 163–168.
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