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Green synthesis of silver nanoparticles using Terminalia chebula extract at room temperature and their antimicrobial studies Kesarla Mohan Kumar a , Madhulika Sinha a , Badal Kumar Mandal a,∗ , Asit Ranjan Ghosh b , Koppala Siva Kumar c , Pamanji Sreedhara Reddy c a b c
Trace Elements Speciation Research Laboratory, Environmental and Analytical Chemistry Division, School of Advanced Sciences, VIT University, Vellore 632014, India Medical Biotechnology Division, School of Bio Sciences and Technology, VIT University, Vellore 632014, India Department of Physics, Sri Venkateswara University, Tirupati, India
a r t i c l e
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Article history: Received 6 October 2011 Received in revised form 25 January 2012 Accepted 2 February 2012 Keywords: Silver nanoparticles Terminalia chebula Di/tri-galloyl glucose Gallic acid Glucose
a b s t r a c t A green rapid biogenic synthesis of silver nanoparticles (Ag NPs) using Terminalia chebula (T. chebula) aqueous extract was demonstrated in this present study. The formation of silver nanoparticles was confirmed by Surface Plasmon Resonance (SPR) at 452 nm using UV–visible spectrophotometer. The reduction of silver ions to silver nanoparticles by T. chebula extract was completed within 20 min which was evidenced potentiometrically. Synthesised nanoparticles were characterised using UV–vis spectroscopy, Fourier transformed infrared spectroscopy (FT-IR), powder X-ray diffraction (XRD), transmission electron microscopy (TEM) and atomic force microscopy (AFM). The hydrolysable tannins such as di/tri-galloyl-glucose present in the extract were hydrolyzed to gallic acid and glucose that served as reductant while oxidised polyphenols acted as stabilizers. In addition, it showed good antimicrobial activity towards both Gram-positive bacteria (S. aureus ATCC 25923) and Gram-negative bacteria (E. coli ATCC 25922). Industrially it may be a smart option for the preparation of silver nanoparticles. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Many researchers have widely used noble nanoparticles (NPs) in various technological applications because of their unique properties. So, synthetic methods of such noble NPs are of great interest. Among noble metal NPs, silver NPs (Ag NPs) in particular are known for their versatile applications in medical industries [1] (in ointments to prevent infection of wounds and burns), in food processing industries [2], in textile industries [3] (Ag impregnated fabrics) and in consumer goods [4] being an efficient antimicrobial agent [5]. Many chemical and physical methods are available in literature to synthesise Ag NPs which includes chemical reduction [6], microwave assisted process [7], polyol process [8], pulse sonoelectrochemcial method [9], etc. Among them, chemical reduction was the most popular method in synthesising NPs where toxic reducing agents were used but most size-selective many physical methods were very expensive. Since Ag NPs were used widely in human contacting ointments, it is necessary to develop environmental friendly green protocols in manufacturing Ag NPs avoiding uses of toxic chemicals at any stage of the process.
∗ Corresponding author. Tel.: +91 0416 2202339; fax: +91 0416 2243092. E-mail addresses:
[email protected],
[email protected] (B.K. Mandal). 1386-1425/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.saa.2012.02.001
Green chemistry principles drive researchers to develop synthetic strategies using biological methods where enzymes [10], microorganisms [11] and plant extracts [12–14] play a major role in the formation of NPs. Among biological methods, plant extract based synthetic method of NPs is the best eco-friendly alternative to available traditional chemical and physical methods [6–9]. Conversely enzyme and microorganism based synthetic systems involve lengthy process in maintaining cultures [12]. Eventually, it is necessary to minimize the time required to reduce Ag+ ion using plant extract to compete with chemical approaches to fulfil its high demand in consumers industries. In recent years this green bio-reduction methods were adapting by many researchers especially in the synthesis of Ag NPs using plant extracts such as Macrotyloma uniflorum [15], Anacardium occidentale [16], Cinnamon zeylancium bark [17], Allium sativum [18], Murraya koenigii leaf [19], Magnifer indica [20], Hibiscusrosa sinensis[14], Mushroom extract [21], Coleus amboinicus lour [22], Medicago sativa [23], and Citrus sinensis peel [24]. Out of the above mentioned methods, synthesis using Muraraya koenigii leaf [19] and Citrus sinensis peel [24] were found to be rapid at room temperature and Allium sativum mediated synthesis in presence of sunlight where light was used as a constructive agent for the synthesis of Ag NPs in the aqueous medium, completed within seconds [18]. The main objectives of the present study were (i) to synthesise Ag NPs using aqueous extract of Terminalia chebula fruits, (ii) to
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2. Experimental
2.3.5. Potentiometric study Variation in oxidation–reduction potential of Ag NPs in broth during the conversion of Ag–salt to Ag NPs was studied with time using a potentiometer (Equip-Tronics dual channel potentiometer model EQ-603) which was equipped with platinum electrode and saturated calomel electrodes.
2.1. Preparation of T. chebula extract
2.4. Antimicrobial study
In order to prepare T. chebula extract, 1 g of finely grinded and meshed T. chebula fruit powder was mixed with 100 mL of deionised water and heated at 90 ◦ C on temperature controlled water bath for 1 h and cooled, passed through 0.2 m cellulose nitrate membrane filter paper. Freshly prepared aqueous extract was used immediately after filtration. No old extract was used for this study at any stage.
The bactericidal activity tests were carried out with Gramnegative bacteria E. coli (strain ATCC 25922) and Gram-positive bacteria S. aureus (strain ATCC 25923) in Muller Hinton Broth (MHB) and Agar using 1% Agar for easy diffusion of the nanoparticles. Throughout this study the same medium was used for all strains. Luria Bertini broth was used as a medium for preparing fresh cultures of organisms. The activity of Ag NPs towards both the bacterial cultures was done using the standard minimum inhibitory concentration (MIC)/minimum bactericidal concentration (MBC) method and well diffusion method.
characterise Ag NPs using UV–vis spectroscopy, FT-IR, XRD, AFM and TEM, and (iii) to check its antimicrobial, i.e., especially antibacterial activity towards both Gram-positive and Gram-negative bacteria.
2.2. Synthesis of Ag NPs 2 mL of 0.01 M Ag2 SO4 (S.D. fine, India) solution was added to 10 mL aqueous extract of T. chebula and mixed thoroughly by manual shaking. The colour change from yellow to reddish brown indicated the formation of Ag NPs. 2.3. Characterisation 2.3.1. UV–visible spectroscopy The initial characterisation of synthesised Ag nanocolloids was carried out using UV–visible spectroscopy. The reduction of silver ions was monitored from 400 to 1200 nm by Jasco V-670 UV-Vis double beam spectrophotometer after 10-fold diluting the sample with deionised water against deionised water as blank. The spectral data recorded were then plotted using Origin 6.1. 2.3.2. X-ray diffraction After mixing of extract and Ag–salt solution the mixture was allowed to complete conversion and Ag NPs were collected after centrifugation followed by filtration. The obtained purified Ag NPs were subjected to X-ray diffraction analysis (Bruker D8 Advance ˚ The scanning range diffractometer with Cu K␣ radiation, = 1.54 A). was done between 10◦ and 90◦ . The instrument was calibrated using lanthanum hexaboride (LaB6) before analysis. 2.3.3. Fourier transformed infrared spectroscopy (FTIR) Purified Ag NPs in the form of powder were analysed using FT-IR spectral measurements. The measurements were carried out on a JASCO FT-IR 4100 instrument in the diffuse reflectance mode at a resolution of 4 cm−1 in KBr pellets. For comparison, T. chebula fruit powder was pelletised and used as control.
2.4.1. Evaluation of zone of inhibition (ZOI): well diffusion method The bacterial lawn was prepared on sterile Muller–Hinton agar (1% Agar) plates by using a sterile cotton swab. 108 CFU/mL cultures of S. aureus and 106 CFU/mL culture of E. coli were used to make a lawn culture (by streaking culture using sterile cotton swabs). Wells of approx 8 mm diameter were made. Ag NPs of different concentrations (15–75 g/mL) were placed inside each well with 50 L of sample in each well and a blank well with aqueous extract of T. chebula without Ag NPs. The plates were kept for incubation at 37 ◦ C overnight. Zone diameter was measured and represented graphically. 2.4.2. Determination of MIC and MBC The MIC and MBC were determined by taking nine sterile test tubes separately with 0.9 mL of normal saline (0.84% NaCl), and inoculated with the corresponding microorganism of concentration ∼109 CFU/mL of bacteria. Fresh bacterial culture was made by keeping the freshly inoculated bacterial sample in 5 mL sterilised broth for 6 h of incubation (to reach its Log phase) at 37 ◦ C. 0.1 mL from the fresh culture was taken and added to the first test tube with saline solution and serial dilution was performed for the following seven test tubes, and two tubes maintained as control. To each tube 10 L of Ag NPs was added and the tubes were incubated at 37 ◦ C overnight. A blank tube containing aqueous extract of T. chebula without Ag NPs was also kept to check whether aqueous extract alone kills bacteria or not. A drop from each tube was added on plates. The plates were incubated at 37 ◦ C for 18 to 24 h and the growth of colonies was seen. 3. Results and discussion
2.3.4. Microscopy study 2.3.4.1. Transmission electron microscope. The size and morphology of the synthesised Ag NPs was determined by High Resolution Transmission Electron Microscope (JEOL JEM 2100 HR-TEM). The sample for TEM studies was prepared as follows. 1 mL of reaction mixture containing Ag NPs was diluted to 10 mL, sonicated using ultrasonic bath and a drop of it was placed on Cu grid with Ultrathin Cu on holey C film and allowed to dry it in vacuum. The instrument was operated with an acceleration voltage of 200 kV. 2.3.4.2. Atomic force microscope (AFM). The shape and size of the synthesised Ag NPs was studied using AFM. Sampling for AFM was done by drop coating method on a glass slide and scanning was done at a rate of 100 mV/s with an image size of 1.2 m using AFM (Nanosurf Easysurf2, Switzerland).
In this report aqueous extract of Terminalia chebula (T. chebula) fruits (which is locally known as horitoky or karakkayya in India) to synthesise Ag NPs is presented. The multi functionality of T. chebula aqueous extract (i.e., which acts as both reducing and capping agent) and mechanism of Ag NPs formation are discussed. The antimicrobial activity of synthesised Ag NPs was done and discussed. Recently several approaches have been reported to synthesize Ag NPs in smarter way which includes both chemical and biological approaches [10–14]. According to green chemistry principles, nowadays using bio-based matter in synthesising materials is of great interest to the scientific community. In comparison with chemical approaches bio-based approaches were fast and smart enough against uses of toxic and hazardous chemicals and
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Fig. 1. UV–vis spectrum of Ag nanoparticles.
by-products. In this context the present study used T. chebula aqueous extract to synthesize Ag NPs which is rich in polyphenolic contents (very high levels of ellagitannins and gallotannins [punicalagin, chebulanin, corilagin, chebulagic acid, di-/tri-galloylglucoses], ellagic acid, chebulic acid, gallic acid) [25] with higher antioxidant capacities and with a reduction potential of greater than 0.303 V [26]. On mixing 0.01 M AgSO4 aqueous solution with the extract the appearance of reddish brown colour appeared within 15 min which was quite faster than the previous bio-based reports [27–29]. Rapid formation of Ag NPs was due to higher avail-
potential after 15 min of interaction which indicated the formation of Ag NPs at this stage. The potential was decreased to 0.158 V from initial potential of Ag+ ions (0.303 V) at the end of 15 min. Afterwards potential gradually decreased to 0.101 V at the end of 60 min (Fig. 2). The obtained results were correlated with the results from UV–visible spectroscopic studies which suggested that the desired reaction took place within 15–20 min. After considering all the facts the plausible mechanism of reaction involved during the conversion of Ag+ ions to Ag NPs is proposed as O O
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Ag+ + e− → Ag0 ability of polyphenols and hence higher reduction potential of T. chebula extract. Moreover, T. Chebula extracts not only reduced Ag+ ions to Ag NPs, but acted as capping/stabilising agents. In addition, hydrolysable tannins such as tri/di-gallolyl glucose present T. Chebula fruits were known to hydrolyse in mild acid/basic conditions into glucose and gallic acid [30]. Consequently gallic acid reduced Ag+ ions into Ag NPs, but it is a poor stabilising agent which does not prevent aggregation of NPs. On the other hand, glucose is a poor reducing agent but is a good stabilizer and hence it may take a critical role in stabilising Ag NPs. This multifunctionality of T. Chebula extract where reduction and stabilisation occurred simultaneously could be taken care of stability and aggregation problems during chemical synthesis. Due to excitation of surface plasmon vibrations in Ag NPs, its aqueous suspension appeared as reddish brown colour. The reduction of Ag+ ions into Ag NPs was monitored using UV–vis spectroscopy. Fig. 1 shows the UV–vis spectrum which confirms the formation of Ag NPs. It was observed that a broad plasma resonance appeared at max 452 nm. The absorption band at 452 nm was indicative of anisotropic nanoparticles formation [22]. The reduction process where Ag+ ions converted to Ag NPs was monitored potentiometrically. There was a sharp decrease in
Crystal behaviour of the purified solid Ag NPs was evaluated using Powder XRD. Powder XRD pattern of the synthesised Ag NPs showed four distinct diffraction peaks at 38.26, 44.25, 64.53 and 77.52 which could be assigned to (1 1 1), (2 0 0), (2 2 0) and (3 1 1) of face centred cubic (fcc) Ag NPs respectively (Fig. 3). The lattice constant was in agreement with the database of Joint Committee on Powder Diffraction Standards (JCPDS. No. 01-087-0597). Morphology and particle size of Ag NPs were characterised using HR-TEM. Typical HR-TEM micrographs at different magnifications were shown in Fig. 4. It is clear from the TEM images the formation of anisotropic nanostructures of pentagons, spherical and triangular shaped NPs with size less than 100 nm. This anisotropy was due to lack of protective bio-molecules for lateral formed nascent nanocrystals. To attain thermodynamic stability, these fresh nanocrystals under lack of protective bio-molecules developed triangles and hexagon crystals [22]. The distance of 2.36 A˚ between the lattice planes is in agreement with the (1 1 1) lattice spacing of face centred cubic (fcc) Ag nanocrystals (Fig. 4c). Crystalline nature of Ag NPs was evidenced from the selected area electron diffraction pattern (SAED) pattern and the pattern can be indexed to (1 1 1), (2 0 0), (2 2 0) and (3 1 1) Bragg reflection planes (Fig. 4d). The EDAX spectrum showed the signal for silver
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and no other signals were observed which suggested that synthesised Ag NPs were basically free from other impurities. The appearance of Cu peaks was aroused from copper grid used to coat samples in TEM-EDAX studies (Fig. 5). Furthermore, AFM
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of the same dried Ag NPs sample illustrated that the coating of oxidised polyphenols and glucose material remained and encapsulated on the surfaces of Ag NPs indicating a tribute role of bio-oxidised materials of T. chebula extract in stabilisation of Ag NPs (Fig. S1). Furthermore capping was evidenced by FT-IR measurements of T. chebula and purified Ag NPs (Fig. 6). FT-IR analysis shows IR bands at 3557, 2922, 1696, 1617, 1447, 2336, 1196 and 1053 cm−1 . The band at 3557 cm−1 corresponds to –C O overtone, the peak at 2922 corresponds to aliphatic –C–H stretching. The bands appeared at 1696 and 1617 cm−1 are due to –C O and–C C stretching respectively. The bands appeared at 1447, 1336 and 1053 cm−1 can be assigned to –C–N, –C–O and –C–O–C stretching respectively. The peak at 1196 cm−1 is due to –C–O–H bending. The absence of –OH stretching and appearance of –C O overtone in the FT-IR spectrum of purified Ag NPs indicate that the polyphenols present in the aqueous extract were responsible for reduction of Ag2+ to Ag0 and the oxidised form of polyphenols binds the Ag nanoparticles via –C O coordination (see graphical extract). Although glucose contains carbonyl group, it may not take part in stabilisation process of Ag NPs, because FT-IR spectrum of purified Ag NPs powder did not show any –OH stretching except –C O overtone at 3557 cm−1 (Fig. 6). Hence FT-IR study reveals the multi-functionality of the aqueous extract of T. chebula where reduction and stabilisation occurs simultaneously.
Fig. 4. Study of morphology of Ag NPs: (a) and (b) TEM, (c) HRTEM, and (d) SAED pattern of Ag NPs.
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Fig. 5. EDAX pattern of Ag Nps.
3.1. Antibacterial activity Ag NPs have shown promising antimicrobial properties explained elaborately elsewhere [31–33]. However, very few research articles have mentioned about antimicrobial properties of Ag NPs synthesised through green route with enhanced antimicrobial activity [34–36]. The present data collected from the green synthesised Ag NPs has shown effective results towards Grampositive bacteria (S. aureus ATCC 25923) and Gram-negative bacteria (E. coli ATCC 25922). The MIC/MBC was determined in batch cultures with varying concentration of Ag NPs. Kim et al. [37] reported that Gram-positive S. aureus was more resistant to Ag NPs compared to Gram-negative E. coli, based on their studies with single strain of each culture. Diameter of Inhibition Zones (DIZ) is measured and shown graphically (Fig. 7). Although the result of E. coli exposed to 90 g/mL (zone no. 6) as shown in Fig. S2, this concentration has not been considered for the construction of histogram (Fig. 7) to make similarity with S. aureas. Blank experiments were performed using aqueous extract and no inhibition zone was found in both the cases (data not shown). Results of antimicrobial studies show higher sensitivity of Ag NPs for S. aureus [Fig. S2(a) and (b)] compared to that of E. coli. [Fig. S2(c) and (d)]. Furthermore, we assumed
that a different activity was observed with E. coli where initially, inhibitory zones were formed which was able to inhibit the growth of bacteria but few resistant colonies were seen growing on the zones formed initially. Thus Ag NPs have shown higher sensitivity towards S. aureus strain than E. coli. Here we have measured the zone diameter only of the cleared zone area excluding the growth of resistant colonies in the zones formed. Ruparelia et al. [38] reported antibacterial activity of NPs based on the strain type and Ag NPs was found to be more sensitive towards E. coli than S. aureus, but this solely depends upon the strain type used and the morphology of NPs incorporated for the studies. Although various hypotheses have been proposed to explain the mechanism of antimicrobial activity of Ag NPs, it is widely believed that Ag NPs are incorporated in the cell membrane, which causes leakage of intracellular substances and eventually causes cell death [39,40]. Antibacterial activity using S. aureus and E. coli was performed by evaluation of MIC/MBC (Fig. S3). The lowest concentration of 15 g/mL (Ag NPs) showed cell growth on plates for 1 × 109 CFU/mL but had shown no growth for concentrations ranging from 1 × 108 to 1 × 104 CFU/mL indicating bactericidal activity. The lowest concentration of 15 g/mL of Ag NPs was found effective in the study
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of MIC/MBC and can kill the bacteria up to 1 × 108 CFU/mL concentrations, as no cell growth can be seen upon plating, followed by incubation at 37 ◦ C for 24 h, which shows that it is responsible in complete killing of bacteria up to this concentration. However the test tube with 109 CFU/mL concentrations of bacteria showed very little turbidity (in the case of E. coli)/and no turbidity (in the case of S. aureus) which showed selective bacteriostatic nature of NPs as colonies might form on plating. Thus we conclude that bio-inspired Ag NPs are a potential bacteriostatic as well as bactericidal agent. 4. Conclusion A facile rapid green eco-friendly one step method to synthesise Ag NPs using aqueous T. chebula extract has been developed. Polyphenols present in the form of hydrolysable tannins serve both as reducing and capping agents. This method is a promising alternative to the traditional reduction routes to avoid usage of toxic chemicals. Ag NPs obtained by this method exhibit antimicrobial activity towards tested bacteria cultures. This green method may find various medicinal as well as technological applications in demands. Acknowledgements Mr. KMK greatly acknowledges the help of VIT University, Vellore 632014, India for the platform given to do this research. Also, the authors greatly acknowledge the help of Prof. A. Senthil Kumar of Chemistry-SAS, Prof. N. Ramani of English-SSL, VIT University. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.saa.2012.02.001. References [1] R.O. Becker, Metal-Based Drugs 6 (1999) 297–300. [2] R. Tankhiwale, S.K. Bajpai, J. Appl. Polym. Sci. 115 (2010) 1894–1900. [3] N. Duran, P.D. Marcato, O.L. Alves, J.P.S. Da Silva, G.I.H. De Souza, F.A. Rodrigues, E. Esposito, J. Nanopart. Res. 12 (2010) 285–292. [4] H. Jiang, S. Manolache, A.C.L. Wong, F.S. Denes, J. Appl. Polym. Sci. 93 (2004) 1411–1422. [5] M. Rai, A. Yadav, A. Gade, Biotechnol. Adv. 27 (2009) 76–83. [6] G.P.C. Rao, J. Yang, Appl. Spectrosc. 64 (2010) 1094–1099.
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