Green Synthesis of Silver Nanoparticles Using Acacia

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Abstract In the present study, Acacia farnesiana (Sweet acacia) seed extract is ... The metal NPs obtained from bio-route plays an important role in the emerging ... In order to explore the applicability of new medicinal plant (Acacia farnesiana) .... be indexed to face centered cubic structure of Ag metal (JCPDS 89-3722) [23].
J Clust Sci DOI 10.1007/s10876-013-0599-7 ORIGINAL PAPER

Green Synthesis of Silver Nanoparticles Using Acacia farnesiana (Sweet Acacia) Seed Extract Under Microwave Irradiation and Their Biological Assessment S. Yallappa • J. Manjanna • S. K. Peethambar A. N. Rajeshwara • N. D. Satyanarayan



Received: 31 August 2012 Ó Springer Science+Business Media New York 2013

Abstract In the present study, Acacia farnesiana (Sweet acacia) seed extract is used to reduce Ag? ? Ag0 under microwave irradiation. The formation of silver nanoparticles (AgNPs) is monitored by recording the UV–Vis absorption spectra for surface plasmon resonance (SPR) peak at *450 nm. The absorbance of SPR increases linearly with increasing temperature of the reaction mixture. Rapid reduction of silver ions occurred to form AgNPs, 80–90 % yield in about 150 s. A marginal decrease in pH and increase in solution potential (E) of the reaction mixture during the formation of AgNPs are in agreement with the proposed mechanism. XRD pattern of the AgNPs agree with the fcc structure of Ag metal, and the calculated crystallite size is *17 nm. FT-IR and solid-state 13C NMR spectra indicate the functional groups of flavonones and terpenoids (biomolecules from plant extract) which are adsorbed on AgNPs, thereby the present method led to in situ biofunctionalization/bio-capping of AgNPs. TG analysis shows the thermal decomposition of these plant residues present on AgNPs at about 250 °C. The spherical shape of the particles with a diameter (/) in the range of *15–20 nm is evident from FE-SEM image. Elemental analysis by EDX analysis confirms the presence of Ag as the only major element. The in vitro antibacterial screening of AgNPs shows that these bio-capped AgNPs have higher inhibitory action for E. coli and S. aureus followed by B. subtilis and P. aeruginosa. In addition, AgNPs show very good antioxidant property. S. Yallappa  J. Manjanna (&) Department of Industrial Chemistry, Kuvempu University, Shankaraghatta 577451, Karnataka, India e-mail: [email protected] S. K. Peethambar  A. N. Rajeshwara Department of Biochemistry, Kuvempu University, Shankaraghatta 577451, Karnataka, India N. D. Satyanarayan Department of Pharmaceutical Chemistry, P.G. Centre, Kuvempu University, Kadur 577548, Karnataka, India

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Keywords Acacia farnesiana  Silver nanoparticles  Bioreduction  Antioxidant property  Antibacterial activity

Introduction Metal nanoparticles (NPs) are of great interest due to their distinctive optical, magnetic, electrical and medicinal properties [1–3]. Noble NPs such as silver and gold have been extensively used in biomedical applications viz., antimicrobial coatings on medical instruments and devices, antimicrobial deodorant fibers, biolabelling, drinking water filters etc. [4–7]. As the surface area of NPs is increased, their biological efficiency (antimicrobial, antioxidant and anticancer activity) is also increased due to the increase in surface energy [8]. In addition, silver nanoparticles (AgNPs) have been used as superior disinfectants in water treatment plants, food packaging, wound healing ointments, cosmetics, bandages etc. because of their mutation-resistant antibacterial, antiviral and anti-inflammatory properties [6, 9]. The biological activity of these NPs depends also on their formation history leading to difference in morphological/surface area, crystallinity and surface activation/stability. AgNPs have been synthesized by different physical and chemical approaches viz., chemical reduction, micro-emulsion/reverse micelles, electrochemical reduction, photochemical reduction etc. [10, 11]. Studies have shown that the morphology, stability and properties (physical, chemical and biological) of the nanomaterials are strongly influenced by the experimental conditions viz., concentration of precursors, temperature, nature of solvent, reducing agent and stabilizing/capping agent [12, 13]. The metal NPs obtained from bio-route plays an important role in the emerging stream of nanobiotechnology. For biomedical applications, bio-synthesized AgNPs are advantageous over chemical and physical method, especially because they were environmentally benign even for large scale synthesis [14, 15]. AgNPs have been synthesized by using reducing agents present in medicinal plant extracts like green tea (Camellia sinensis) [16], Neem (Azadirachta indica) leaf broth [17], natural rubber [18], Aloe vera [19], Jatropha curcas [20], guava (Psidium guajava) [21], lemon (Citrus limon) [22], Prosopis juliflora [23] etc. The plant extracts contain metabolites such as polyphenols/flavonoids, proteins, terpenoids, tannins, etc. [21]. These metabolites not only acts as reducing agents for metal ion but also (remains on the metal NPs) as capping agents, which helps to minimize the agglomeration of NPs. Thus, it is possible to control the morphology and protect/stabilize the NPs to improve their biological properties. Further, they offer non-pathogenicity, low or nil toxicity and are easy to adopt [24]. Hence, the use of these medicinal plants has gained much importance, soon after the first reports by Gardea-Torresdey et al. [25, 26] on the formation of Ag and Au NPs by using living plants. The synthetic procedure using plant extract exemplifies a green approach. In order to explore the applicability of new medicinal plant (Acacia farnesiana) in our locality, we have demonstrated the synthesis of AgNPs and their biological assessment. A. farnesiana is commonly known as Sweet acacia, which belongs to the family of Fabaceae. The seeds, leaves and bark of this plant have been used in

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malarial drugs, wound healing ointments, skin disease etc. [27]. We have used the seed extract of A. farnesiana as a reducing agent for Ag? ? Ag0. The rapid synthesis of uniform spherical shaped AgNPs is achieved here through microwave irradiation. The antibacterial and antioxidant properties of these bio-capped AgNPs have also been reported.

Experimental Materials Acacia farnesiana seeds were collected from Kadur town area in Karnataka, India. They were cleaned, dried and ground to fine powder. AgNO3 was procured from Himedia (Bangalore, India). The bacterial strains were procured from Microbial Type Culture Collection (MTCC), Chandigarh, India. Microwave oven (ONIDA-MO 17SJP1W, 2.45 GHz) was used for extracting phytochemicals from A. farnesiana seed in water and subsequently for the synthesis of AgNPs. Synthesis of Stable AgNPs Two grams of finely powdered A. farnesiana seed was added to 200 ml distilled water and kept for microwave irradiation for about 180 s to extract the phytochemicals present in the seeds to the solution. It was filtered through 0.2 lm membrane filter in hot condition to remove fibrous impurities. A 10 ml of this stock-solution was added to 50 ml of 10-3 M AgNO3 solution. The reaction mixture was microwave irradiated to different interval of time. A change in color from light yellow to dark brown indicated the formation of AgNPs. The formation of AgNPs was monitored by recording UV–Vis spectra (Shimadzu, 1650-PC) for surface plasmon resonance (SPR) at around k = *450 nm. The % reduction of Ag? ? Ag0 was calculated on the basis of SPR intensity. For this, we have allowed for complete reduction of 1 mM AgNO3 by microwave irradiation for about 300 s. The optical absorption or extinction coefficient (2.5) at *450 nm corresponding to SPR peak is taken as 100 % reduction. The complete reduction of Ag? ? Ag0 was possible because of the presence of stoichiometrically sufficient concentration of phytochemicals present in plant extract. Unreacted AgNO3 was ruled out here based on the effective AgNO3 test i.e., on adding NaCl to a supernatant solution of reaction mixture after centrifugation, there was no AgCl precipitation. The final washing of the AgNPs with plenty of water also ensures removal of any unreacted AgNO3. Finally, the metal NPs are dried in vacuum oven at 70–80 °C for about 12 h to obtain the product in the powder form. In order to assess the reducing ability of the plant/seed extract, we have measured the solution redox potential (E) and pH in presence of Ag? ions using digital potentiometer and pH meter, respectively. AgNPs obtained here were characterized by X-ray diffraction (XRD, Siemens X-ray diffractometer, Japan) technique with Cu Ka radiation and Ni-filter. Fieldemission scanning electron microscopic (FE-SEM, FEI Nova nano 600, Netherlands) was used to observe the morphology of the bio-capped AgNPs and energy

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dispersive X-ray (EDX) for elemental composition. Fourier transform infrared (FTIR, Bruker- TENSOR 27) spectra in KBr pellet and the solid-state 13C NMR (Bruker-DSX 300) spectra were recorded to detect the biomolecules/biomass adsorbed on the surface of AgNPs. Thermo gravimetric (TG, Linseis STA PT1600, Germany) curve was traced under N2 atmosphere with a heating rate of 10 °C min-1. In Vitro Antioxidant Property of AgNPs AgNPs were tested for 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity with butylated hydroxytoluene (BHT) as standard follows [28]. AgNPs were first dispersed in methanol (20–100 lg/mL) and 3 ml of freshly prepared 0.004 % DPPH solution (obtained by dissolving in a 95:5 mixture of methanol and water) was added. It was allowed to react for 20–25 min in the dark and the absorbance of DPPH was measured at 517 nm using UV–Vis spectrophotometer (Shimadzu, 1650-PC) before and after the reaction to know its decay. Radical scavenging activity was calculated using the formula: % of radical scavenging activity ¼ ð½ðAcontrol  Atest Þ = Acontrol Þ  100 where Acontrol is the absorbance of the control sample (DPPH solution without test sample) and Atest is the absorbance of the test sample (DPPH solution ? test compound). The DPPH free radical scavenging activity of BHT was assayed for comparison. Tests were performed in triplicate and the results were normal. In Vitro Antibacterial Activity of AgNPs In vitro antibacterial activity was investigated using 24 h old cultures of bacterial strains by the well plate method using Mueller-Hinton agar [29]. Escherichia coli (E. coli) ATCC 25922, Staphylococcus aureus (S. aureus) ATCC 25923 and Pseudomonas aeruginosa (P. aeruginosa) ATCC 27853 bacterial strains were selected due to their infectious nature. The test samples (AgNPs, seed extract and the standard drug) were dispersed in dimethyl sulfoxide at different concentrations (1.0, 0.5, 0.25 and 0.12 lg/ml). A 20 ml of sterilized agar media was poured to different pre-sterilized petri dishes. Then 60 ll of bacterial culture suspension were added and swabbed with the pre-sterilized cotton swabs. A 6 mm diameter wells were punched carefully using a sterile cork borer and then 60 ll of the above test samples of different concentration were added to each labeled well. The plates were incubated at 37 °C for 24 h. The inhibition zones around the well in each plate were measured in mm. The experiment was performed in triplicate and standard deviation was calculated.

Results and Discussion When the reaction mixture (AgNO3 ? plant seed extract) was irradiated in microwave oven, color change from light yellow to dark-brown occurred within

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Fig. 1 UV–Vis absorption spectra of AgNPs showing the SPR peak from 430 to 450 nm. The inset shows % reduction of Ag? ? Ag0 as a function of microwave irradiation time

30 s. This is an indication for the reduction of Ag? ? Ag0. The color intensity or optical density of the reaction mixture increased with irradiation time up to about 150 s. The wavelength of the maximum absorption varied from 430 to 450 nm (Fig. 1), depending on the exposure time, is attributed to the surface plasmon resonance of Ag0 particles [30]. Such a red shift of SPR from 430 to 450 nm is probably due to increase in particle size [20]. The % reduction of Ag? ? Ag0 versus time (inset of Fig. 1) was calculated based on SPR intensity. The formation of AgNPs versus time shows a semi-sigmoidal behavior i.e., a gradual increase in formation of AgNPs until about 90 s (35 % reduction) and then a rapid formation until about 150 s (88 % reduction) followed by a gradual increase until completion in about 300 s. A similar behavior was seen for the reduction of Ag? ions by using guava leaf extract [21]. This semi-sigmoidal behavior may be explained based on the temperature attained during the irradiation time viz., 30 °C at 10 s, 45 °C at 30 s, 60 °C at 50 s, 80 °C at 70 s, 95 °C at 90 s and 95–98 °C at 90–300 s. It is well known that the activation energy of the reactants increases with increasing temperature, which in turn increased AgNPs formation. It is clear that the formation (yield) of AgNPs increased sharply when the irradiation time is about 90–150 s. Further irradiation led to gradual increase in % yield of AgNPs as the concentration of reactants depletes towards the end of reaction. The prominent peaks in the XRD pattern (Fig. 2) are at 2h = 38.1°, 44.3°, 64.4° and 77.4° corresponding to (111), (200) (220) and (311), respectively, which could be indexed to face centered cubic structure of Ag metal (JCPDS 89-3722) [23]. The average crystallite size d of AgNPs was calculated to be about 17 nm using Scherer formula, d = Kk/bcosh where, K—shape factor is between 0.9 and 1.1, k—incident ˚ ), b—full width half maximum in radians of X-ray wavelength (Cu Ka = 1.542 A

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Fig. 2 XRD pattern of vacuum dried AgNPs

Fig. 3 Redox potential (E) and pH of the reaction mixture as function of microwave irradiation time

the prominent line (111) and h—position of that line in the pattern. Thus, the green synthesis of AgNPs was achieved here using A. farnesiana seed extract as a bioreluctant through microwave irradiation. We have measured the pH and redox potential (E) of the reaction mixture to understand the formation of AgNPs. This procedure is useful for choosing the suitable plant extracts instead of arbitrary selection. The initial E = 0.017 V increased to 0.033 V after 300 s of microwave irradiation (Fig. 3). Such an increase

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in E to a more positive value, although marginally, indicates the depletion of bioreductant in the seed extract for Ag? ? Ag0. Furthermore, the initial pH 4.8 of the reaction mixture decreased to pH 2.0 due to the release of H? ions during the oxidation of bio-reductant. A similar observation is made in our previous report for CuNPs using T. arjuna bark extract [31]. The uniform spherical shaped particles with a diameter (/) in the range of *15–20 nm can be seen from the FESEM image, Fig. 4a. On careful observation, we see some kind of scum on the particles, which is due to adsorption (bio-capping) of organic moieties from the plant extract [17]. EDX spectrum (Fig. 4b) here 75 wt% of the product is Ag and the remaining is C (10 %) and O (14 %) with a small contamination of Cl (\1 wt%). The significant amount of C and O is an evidence for the organic substances (phytochemicals) attached to the AgNPs in the form of bio-capping. Song et al. [32] have also reported the significant amounts of C and O in their AgNPs obtained using different plant leaf extracts (Pine, Persimmon, Ginkgo, Magnolia and Platanus). Figure 5 shows the TG curve of bio-capped AgNPs under N2 atmosphere. The initial wt loss of *5 % at T \ 100 °C is due to loss of water molecule adsorbed on AgNPs. A wt loss of *10 % occurred between 100 and 250 °C and thereafter we see a gradual wt loss up to 500 °C leading to overall wt loss of *45 %. This is attributed to thermal degradation of plant residue (phytochemicals) on the AgNPs. In fact, it is an important observation supporting our claim that the AgNPs here have been bio-capped with plant residue. This is in corroboration with the FT-IR and solid state 13C NMR spectra, discussed below. Such a bio-capping can play an important role in biomedical application of these AgNPs and their stability. The fact that there was no weight gain is an indication that the AgNPs remained intact without any oxidation because of the N2 environment. FT-IR (Fig. 6a) was recorded to detect any biomolecules/biomass adsorbed on the AgNPs. The band appearing at 1,626 cm-1 corresponds to amide group from carbonyl stretch in proteins present in the seed extract. The peaks at 1440, 1058 cm-1 are assigned to geminal methyl group and C–N stretching vibration of amine, respectively. All these prominent peaks correspond to flavonones and terpenoids present on AgNPs. The same set of biomolecules (from seed extract) were responsible for the Ag? reduction and their bio-capping [17, 23].

Fig. 4 a FESEM image and b EDX spectra of AgNPs

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Fig. 5 TG spectra of bio-capped AgNPs

A solid-state 13C NMR spectrum (Fig. 6b) also indicates the presence of biomolecules on AgNPs. We have observed prominent peaks at 195 ppm corresponding to ketones and aldehydes (flavonones, terpenoid aldehydes) from the plant extract. The peak at 184 ppm indicates the presence of amide group while the peaks at 156, 145 and 76 ppm were ascribed to unsaturated compounds, reducing sugars and carbonyl groups, respectively. A similar peak pattern was observed in our previous study for the bio-capped CuNPs obtained by using T. arjuna bark extract [31]. In fact, detailed studies are necessary to identify the exact biomolecule/s responsible for the reduction of these metal ions, as there are large no of phytochemicals present in plant extracts. DPPH is a stable nitrogen-centered free radical and shows a characteristic absorption at 517 nm, whose color changes from violet to yellow upon reduction [33]. As shown in Fig. 7, the antioxidant property of bio-capped AgNPs is comparable to that of BHT. The antioxidant property of A. farnesiana seed extract alone was found to be less effective. Our particles have shown much better activity when compared to a recent study [34] wherein Syzygium cumini seed extract is used as reducing agent to obtain AgNPs. We think, the potential antioxidant property of AgNPs here is due to their quantitative oxidation (Ag0 ? DPPH ? Ag? ? 1, 1-diphenyl-2-picryl hydrazine) probably due to thin layer and/or porous nature of bio-capped molecules. We have investigated the antibacterial activity against viz., B. subtilis, E. coli, S. aureus and P. aeruginosa under well-plate method. The activity of the AgNPs differs due to the concentration used against tested human pathogens. The antibacterial efficiency is increased with higher concentration (i.e., 1.0 [ 0.5 [ 0.25 [ 0.12 lg/ ml) of test samples (AgNPs/seed extract alone). As shown in Fig. 8, the AgNPs showed higher inhibition zone when compared to A. farnesiana seed extract against all the tested bacteria and it is more pronounced for E. coli.

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Fig. 6 a FT-IR and b solid-state

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C NMR spectra of bio-capped AgNPs

The precise mechanism of antibacterial effect of Ag0 is not clear; many mechanisms are proposed [35–38]. As the AgNPs have a positive surface charge and the cell membrane is negatively charged, electrostatic attraction leading to the diffusion of Ag0 into the cell followed by redox reaction results in antibacterial property [35, 36]. However, Sondi and Salopek-Sondi [37] have proposed the formation of pits in the cell wall, leading cell deaths due to increased membrane permeability. For thiols-functionalized Ag? ions, Holt and Bard [38] have shown the generation of reactive oxygen species in the respiratory chain of E. Coli, which damaged the cell membrane, protein, RNA/DNA molecule and finally leading to cell death.

Conclusion A rapid and quantitative formation of uniform spherical shaped (*15–20 nm) AgNPs is achieved by using A. farnesiana (Sweet acacia) seed extract as a bio-

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Fig. 7 Antioxidant property of AgNPs

Fig. 8 Antibacterial activity of AgNPs

reductant for Ag? ions under microwave irradiation. The marginal increase in redox potential and decreasing pH of the reaction mixture indicate the involvement of biomolecules in the reduction process. The formation of highly crystalline fcc structured AgNPs have been confirmed by XRD and EDX analysis. FT-IR and solid-state 13C NMR spectra have shown the bio-capping present in AgNPs. These AgNPs have shown moderate antibacterial and very good antioxidant properties.

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This study indicates that in situ bio-capped AgNPs can be used in many biomedical applications. Acknowledgments The authors are thankful to Dr. Michel Raj of St. Joseph College, Bangalore and Dr. Harish C Barshilia of NAL, Bangalore for providing XRD and FE-SEM data, respectively.

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