Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 129 (2014) 121–124
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Biosynthesis, characterization and antibacterial studies of silver nanoparticles using pods extract of Acacia auriculiformis Pradnya Nalawade a, Poulomi Mukherjee b, Sudhir Kapoor a,⇑ a b
Radiation & Photochemistry Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400 085, India Nuclear Agriculture & Bio-Technology Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400 085, 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
Formation of Ag nanoparticles
Effect of pH on the yield, absorption spectrum and size of Ag nanoparticles.
without using any reductant and stabilizer. Growth mechanism shows dependence on pH of the solution. The formed nanoparticles show good bactericidal activity.
a r t i c l e
i n f o
Article history: Received 29 November 2013 Received in revised form 3 March 2014 Accepted 18 March 2014 Available online 27 March 2014 Keywords: Metals Nanostuctures Optical properties Chemical synthesis Antibacterial activity
a b s t r a c t The present study reports an environmental friendly method for the synthesis of silver nanoparticles (Ag NPs) using an aqueous extract of Acacia auriculiformis that acts as reducing agent as well as capping agent. The obtained NPs were characterized by UV–vis absorption spectroscopy and showed a sharp surface plasmon absorption band at 400 nm. Fourier transform infrared spectroscopy (FTIR) showed nanoparticles were capped with plant compounds. Transmission electron microscopy (TEM) showed that the particles were spherical in nature with diameter ranging from 20 to 150 nm depending on the pH of the solution. The as-synthesized Ag NPs showed antibacterial activity against both Gram negative and Gram positive bacteria with more efficacy against Gram negative bacteria. Ó 2014 Elsevier B.V. All rights reserved.
Introduction Synthesis of metal nanoparticles (NPs) has received increased attention due to their novel optical, chemical, photo-electrochemical and electronic properties which are different from that of bulk [1]. Generally, in chemical reduction methods reducing agents are
⇑ Corresponding author. Tel.: +91 22 25590298; fax: +91 22 25505151. E-mail address: [email protected] (S. Kapoor). http://dx.doi.org/10.1016/j.saa.2014.03.032 1386-1425/Ó 2014 Elsevier B.V. All rights reserved.
added to reduce metal ions. These chemicals can be toxic and highly reactive to the environment and humans, or the procedures are too expensive to be reasonable at an industrial scale [1,2]. Therefore, efforts have been taken by various scientists for the search of inexpensive, reliable, safe, and ‘‘green’’ approach to the synthesis of stable metal NPs [3]. In this respect, an environmental acceptable solvent and an eco-friendly reducing and capping agent are essential elements for a complete ‘‘green’’ NPs synthesis [3]. Recently, the use of NPs in biomedical applications has received great interest [3]. In particular, the biomedical applications of Ag
P. Nalawade et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 129 (2014) 121–124
Materials and methods Dry pods of Acacia auriculiformis leaves were collected from the wild growing trees in Mumbai – Maharashtra, India, as and when required. The pods were washed several times with Millipore purified water to remove the dust particles. Silver nitrate (s.d. fine chem.) and sodium hydroxide (Sigma) were used as received. The experiments were performed using Millipore purified water having electrical resistivity 18.2 MX. The glasswares were washed with chromic acid/aqua regia to remove the traces of metal contaminant and rinsed with Millipore water and dried in an oven at 107 °C. Preparation of aqueous extract of plants Aqueous extract of Acacia auriculiformis was prepared by boiling about 10 g of the washed pods of Acacia auriculiformis (after removing the seeds) in 100 ml of Millipore water for 10 min. After cooling, the extract was filtered using Whatman No. 1 filter paper and kept at 4 °C until used.
1.2
6 hrs 0.8
The antibacterial activity of the Ag NPs was tested using minimum inhibitory concentration (MIC) method. The Acacia auriculiformis extract synthesized Ag NPs sol was subjected to low temperature evaporation in a dry bath at 70 °C. The volume was reduced to varying amounts to increase the concentration of Ag NPs in the sol. Accordingly 4 different concentrations of Ag NPs were obtained which were 10.8 lg/ml, 14.4 lg/ml, 21.6 lg/ml, 31.8 lg/ ml. The antimicrobial effect of the Ag NPs sols were tested on several Gram positive and Gram negative organisms. Among the Gram
1.2 0.9 0.6 0.3 0.0 0
3 hrs
2
4
6
8
10
Time (hrs)
0.6
2 hrs 0.4
1 hr 0.2
5 min 0.0 300
400
500
600
700
Wavelength, nm Fig. 1. Time dependant UV–Vis spectrum of Ag NPs obtained by adding 1 ml of aqueous extract to 1 10 3 M AgNO3 at inherent pH 2.3. Inset: Plot of absorbance vs. time for the growth of Ag NPs.
positive organisms Bacillus cereus and Staphylococcus sps. were used. Wild type Escherichia coli BW 25113 and Klebsiella sps. were two representative Gram negative organisms. 100 ll of each culture containing approximately 106 colony forming unit (CFU) per ml was spread on Nutrient agar plates containing 1.4% agar. Wells of diameter 10 mm were bored into the solidified medium and 100 ll of each sample was added. The plates were incubated for 18 h at 37 °C and were examined for the appearance of a clear zone of inhibition as opposed to an uninhibited mat like or dense growth. The diameters of the zones were recorded and mean values for each sample for the respective bacteria are tabulated. Characterization UV–visible absorption spectra were obtained from Chemito spectrascan UV 2600 spectrophotometer. The spectra were recorded at room temperature using a quartz cuvette of path length either 1 cm or 0.2 cm. Zeta potential measurement were carried out on Zetasiezer Nanoseries (Malvern instrument). Samples for transmission electron microscopy (TEM) were prepared by putting a drop of the colloidal solution on a copper grid coated with a thin amorphous carbon film. The excess solvent was removed by using a filter paper, and letting the solvent evaporate under IR lamp. TEM characterization was carried out using a Phillips
Absorbance
2.5
Antibacterial assay
9 hrs
1.0
Biosynthesis of Ag NPs Ag NPs were prepared by adding 1 ml of aqueous extract of Acacia auriculiformis to 5 ml aqueous solution of 1.0 10 3 M AgNO3. The reaction is carried out at room temperature and at inherent pH. To study the effect of pH on the formation of NPs the pH of the solution was varied using 1.0 10 3 M NaOH.
Absorbance
NPs have attracted increasing interest due to its antibacterial and antimicrobial properties [3]. Consequently, Ag NPs have been synthesized by using microorganisms and plant extracts [3–12]. However, the synthesis of NPs using plant extract is potentially advantageous over microorganisms due to the ease of biohazards and the culture of the microorganisms. Synthesis of metal NPs using plant extract is very cost effective, so can be used as an economic and suitable substitute for the largescale production of metal NPs [3–12]. Biomimetic synthesis of NPs using medicinal plants [3–12] and vegetable extracts [13] have also been explored in recent years. Acacia auriculiformis belongs to the family of Fabaceae with medicinal properties [14]. In India, its wood and charcoal are widely used for fuel. It is known to contain tannins and terpenoids along with polyphenols [15]. It has pharmacological properties like antimutagenic, cytotoxic, antihelminthic, antifilarial, microbicidal, antioxidant etc. [15,16]. Recently, it has been suggested that extract of Acacia auriculiformis possesses spermicidal activity (anti-fertility) even at significantly lower concentrations [17]. In the present paper, we demonstrated a hitherto unreported green biological route for the synthesis of Ag NPs using pods extract derived from Acacia auriculiformis. The effect of pH on the formation kinetics of Ag NPs is also demonstrated. The NPs were characterized by UV–visible spectroscopy, FTIR, and TEM analysis. The NPs showed excellent antibacterial activity against both Gram negative and Gram positive bacteria.
Absorbance
122
2.0
a - pH 2.3 inherent b - pH 6.3 e c - pH 7.3 d - pH 9.5 e - pH 11 d c
1.5 b
1.0 0.5
a
0.0 300
350
400
450
500
550
Wavelength, nm Fig. 2. Effect of pH on UV–Vis spectrum of Ag NPs prepared by adding 1 ml of aqueous extract to 1 10 3 M AgNO3 solution. The spectra were recorded after 2 h. Inset: Photographs of (A) aqueous extract, (B) Ag NPs at pH 11 and (C) Ag NPs at pH 2.3 (inherent pH).
P. Nalawade et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 129 (2014) 121–124
450 2.5 440
430 1.5 420
λ max
Absorbance
2.0
1.0 410 0.5 400 0.0 2
4
6
8
10
12
pH Fig. 3. Plot of absorbance and kmax vs. pH of Ag NPs. Other conditions are same as in Fig. 2.
CM 200 electron microscope. Particle size was measured from the TEM micrographs and calculated by taking the average of at least 100 particles. Results and discussion Synthesis of Ag NPs On addition of silver nitrate solution to the aqueous pods extracts of Acacia auriculiformis at room temperature and at inherent
123
pH (2.3) the color of the extract changed from clear light brown to cream whitish to light brown and finally to dark clear brown, which indicated the formation of Ag NPs [15]. The formation kinetics of the reduction reaction was studied by recording the UV–visible absorption spectra after different time interval. In Fig. 1, the broad absorption band appears in the region of 380–700 nm is a characteristic of surface plasmon resonance of Ag NPs. The formation and growth of the particles was slow in the beginning but get accelerated once the seed gets formed. Such phenomenon generally shows sigmoidal kinetics as shown in the inset of Fig. 1. After about 21 h of Ag NPs formation brown precipitate was seen. To see the effect of extract concentration, if any, on the stability of the Ag NPs the concentration of the extract was increased by different volumes ranging from 0.5 ml to 3.5 ml. However, it was observed that at all the extract concentrations the Ag NPs precipitated. Effect of pH on the formation and stability of Ag NPs Increase in the pH of the extract caused the formation of the NPs immediately after addition of Ag+ ions. At pH 11 instant formations of Ag NPs was observed. Fig. 2 shows the effect of pH on the UV–visible absorption spectrum of Ag NPs. The color of the silver sol was also found to be changed from brown to yellow when pH of the extract was increased up to pH 11 as shown in photograph (inset Fig. 2). As pH of the solution increased above 2.3, the wavelength of absorption maximum due to Ag NPs also decreased linearly from 432 nm to 403 nm. It can be seen from Fig. 3 that along with blue shift the increase in the absorbance also occurs at definite time which indicates that at higher pH rate of reduction of
Fig. 4. TEM of Ag NPs prepared at (a) inherent pH (b) corresponding diffraction pattern and (c) pH 11. Other conditions are same as in Fig. 2.
124
P. Nalawade et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 129 (2014) 121–124
Table 1 Zone of inhibition of bacterial growth in mm. Bacteria
Concentration of Ag NPs in lg/ml 10.8
14.4
21.6
30.8
E. coli Klebsiella sps. Bacillus cereus Staphylococcus sps.
1.67 1.35 1.37 1.54
1.78 1.43 1.3 1.38
1.67 1.37 1.37 1.49
1.84 1.52 1.37 1.47
Ag+ ions is greater than at inherent pH. The blue shift of absorption maxima of Ag NPs can be correlated to the formation of small size NPs at higher pH. It was also seen that the Ag NPs obtained at pH higher than 9.5 have shown enhanced colloidal stability compared to those obtained at lower pH. To confirm this, zeta potential (f) measurement was carried out. The zeta (f) potential is commonly used to find out the stability of colloidal systems [18,19]. The colloids are normally considered as stable when its zeta potential value is P30 mV [18]. The as-synthesized Ag NPs had f potentials 22.4, 25.1, 26.4, 28.6 and 33.2 for samples prepared at pH 2.3, pH 6.2, pH 7.3, pH 9.5 and pH 11, respectively. Representative f potential plot at pH 11 is given in Fig. S1. The Ag NPs obtained at pH 11 were stable for more than two months at room temperature and above 7 months at 4 °C. It was also seen that the particles formed at higher pH are nearly monodispersed with polygonal morphology. Representative TEM of Ag NPs obtained at inherent pH and pH 11 is shown in Fig. 4. It can be seen that at inherent pH (Fig. 4a) particles tend to aggregate and form aggregates of average diameter of 150 nm, however, at higher pH 11 (Fig. 4c) particles are nearly monodispersed with an average particle size of 20 nm. Role of A. auriculiformis pods extract in formation, stability and growth of NPs The Acacia auriculiformis is known to have tannins and terpenoids along with the polyphenols. It is known that polyphenols and tannins act as reductants and stabilizers for Ag NPs [13,20]. Therefore, the FTIR spectrum of dried biomass of Acacia auriculiformis pods extracts before and after bioreduction of silver ions was recorded (Fig. S2). It is seen that the stretching peak intensity due to AOAH (of polyphenol) at 3333 cm 1 and AOAR (ester) at 1021 cm 1 is drastically reduced after bioreduction with silver [21]. In addition, a new band at 1540 cm 1, which is assigned to C@O of amide II band, appeared in the FTIR spectrum of Ag NPs. This clearly indicates the role played by hydroxyl groups of polyphenols and ester groups of terpenoids in the reduction of Ag+ ions. Screening of antibacterial activity by agar well diffusion method Of the four bacteria studied the Acacia auriculiformis pods extract stabilized Ag NPs were most effective against E. coli as evident from the higher values of zone of inhibition while lowest values were obtained for Bacillus cereus (Fig. S3). Increasing the Ag NPs concentration did not result in significant increase of the zone of inhibition in most cases while there were a few exceptions to this trend as can be seen from the Table 1. The absence of drastic increase in the zone of inhibition with increase in concentration of Ag NPs could be due to lesser diffusibility of the concentrated sol in the agar gel. The Ag NPs sol was effective against Gram positive
bacteria as well like Staphylococcus sps., strains which are potential human pathogens. Potentially pathogenic Gram negative Klebsiella sps. was also inhibited by Ag NPs synthesized and stabilized by Acacia auriculiformis pods extract. It is well known that the antibacterial action of Ag NPs by way of cleaving of the cell wall is a process more effective in Gram negative bacterial wall as opposed to thick peptidoglycan layer of Gram positive bacteria. However according to Ruparelia et al. the cell wall and membrane composition and structure is not the sole determinant of NPs mediated toxicity to bacteria [22]. Summarily, the sol is effective against laboratory strains (E. coli) environmental isolates (Bacillus cereus) as well as isolates from pathological laboratories (Staphylococcus sps. and Klebsiella sps.), exhibiting well the versatility of this preparation against many different organisms. Conclusions We have developed a simple eco-friendly and convenient green method for the synthesis of nearly monodispersed nanoparticles of silver using pods extract of medicinal plant Acacia auriculiformis as both reducing and capping agent. Pods extract of Acacia auriculiformis is found suitable for the green synthesis of Ag NPs. Stable and nearly spherical Ag NPs of particle size 20 nm are obtained at high pH. FTIR and UV–visible spectroscopy confirmed that the constituents of extract reduce the Ag+ ions. The as synthesized Ag NPs were found to have antibacterial properties. 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.2014.03.032. References [1] M.-C. Daniel, D. Astruc, Chem. Rev. 104 (2004) 293–346. [2] M. Kowshik, S. Ashtaputre, S. Kharrazi, W. Vogel, J. Urban, S.K. Kulkarni, K.M. Paknikar, Nanotechnology 14 (2003) 95–100. [3] J. Xie, J.Y. Lee, D.I.C. Wang, Y.P. Ting, ACS Nano 1 (2007) 429–439. [4] R. Iravani, Green Chem. 13 (2011) 2638–2650. [5] S.P. Chandran, M. Chaudhary, R. Pasrichia, A. Ahmad, M. Sastry, Biotechnol. Prog. 22 (2006) 577–583. [6] S.S. Shankar, A. Rai, A. Ahmad, M. Sastry, J. Colloid Interface Sci. 275 (2004) 496–502. [7] S.S. Shankar, A. Rai, A. Ahmad, M. Sastry, Chem. Mater. 17 (2005) 566–572. [8] V. Armendariz, I. Herrera, J.R. Peralta-Videa, M. Jose-Yacaman, H. Troiani, P. Santiago, J.L. Gardea-Torresdey, J. Nanopart. Res. 6 (2004) 377–382. [9] B. Ankamwar, C. Damle, A. Ahmad, M. Sastry, J. Nanosci. Nanotechnol 5 (2005) 1665–1671. [10] A. Rai, A. Singh, A. Ahmad, M. Sastry, Langmuir 22 (2006) 736–741. [11] A. Kanchana, I. Agarwal, S. Sunkar, J. Nellore, K. Namasivayam, Dig. J. Nanomater. Bios. 6 (2011) 1741–1750. [12] A.K. Jha, K. Prasad, Dig. J. Nanomater. Bios. 6 (2011) 1717–1723. [13] J. Jacob, T. Mukherjee, S. Kapoor, Mater. Sci. Eng. C 32 (2012) 1827–1834. [14] en.wikipedia.org/wiki/Acacia_auriculiformis. [15] R. Singh, S. Singh, S. Kumar, S. Arora, Food Chem. 103 (2007) 505–511. [16] R. Singh, S. Singh, S. Kumar, S. Arora, Food Chem. 103 (2007) 1403–1410. [17] D. Pal, P. Chakraborty, H.N. Ray, B.C. Pal, D. Mitra, S.N. Kabir, Reproduction 138 (2009) 453–462. [18] Y.-P. Sun, X.-Q. Li, W.-X. Zhang, H.P. Wang, Colloids Surf. A: Physicochem. Eng. Aspects 308 (2007) 60–66. [19] Y.-P. Sun, X.-Q. Li, J. Cao, W.X. Zhang, H.P. Wang, Adv. Colloid Interf. Sci. 120 (2006) 47–56. [20] K.L. Niraimathi, V. Sudha, R. Lavanya, P. Brindha, Colloids Surf. B: Biointerf. 102 (2013) 288–291. [21] http://www2.chemistry.msu.edu/faculty/reusch/VirtTxtJml/Spectrpy/ InfraRed/infrared.htm. [22] J.P. Ruparelia, A.K. Chatterjee, S.P. Duttagupta, S. Mukherji, Acta Biomater. 4 (2008) 707–716.
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Biosynthesis, characterization and antibacterial studies of silver nanoparticles using pods extract of Acacia auriculiformis Pradnya Nalawade a, Poulomi Mukherjee b, Sudhir Kapoor a,⇑ a b
Radiation & Photochemistry Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400 085, India Nuclear Agriculture & Bio-Technology Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400 085, 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
Formation of Ag nanoparticles
Effect of pH on the yield, absorption spectrum and size of Ag nanoparticles.
without using any reductant and stabilizer. Growth mechanism shows dependence on pH of the solution. The formed nanoparticles show good bactericidal activity.
a r t i c l e
i n f o
Article history: Received 29 November 2013 Received in revised form 3 March 2014 Accepted 18 March 2014 Available online 27 March 2014 Keywords: Metals Nanostuctures Optical properties Chemical synthesis Antibacterial activity
a b s t r a c t The present study reports an environmental friendly method for the synthesis of silver nanoparticles (Ag NPs) using an aqueous extract of Acacia auriculiformis that acts as reducing agent as well as capping agent. The obtained NPs were characterized by UV–vis absorption spectroscopy and showed a sharp surface plasmon absorption band at 400 nm. Fourier transform infrared spectroscopy (FTIR) showed nanoparticles were capped with plant compounds. Transmission electron microscopy (TEM) showed that the particles were spherical in nature with diameter ranging from 20 to 150 nm depending on the pH of the solution. The as-synthesized Ag NPs showed antibacterial activity against both Gram negative and Gram positive bacteria with more efficacy against Gram negative bacteria. Ó 2014 Elsevier B.V. All rights reserved.
Introduction Synthesis of metal nanoparticles (NPs) has received increased attention due to their novel optical, chemical, photo-electrochemical and electronic properties which are different from that of bulk [1]. Generally, in chemical reduction methods reducing agents are
⇑ Corresponding author. Tel.: +91 22 25590298; fax: +91 22 25505151. E-mail address: [email protected] (S. Kapoor). http://dx.doi.org/10.1016/j.saa.2014.03.032 1386-1425/Ó 2014 Elsevier B.V. All rights reserved.
added to reduce metal ions. These chemicals can be toxic and highly reactive to the environment and humans, or the procedures are too expensive to be reasonable at an industrial scale [1,2]. Therefore, efforts have been taken by various scientists for the search of inexpensive, reliable, safe, and ‘‘green’’ approach to the synthesis of stable metal NPs [3]. In this respect, an environmental acceptable solvent and an eco-friendly reducing and capping agent are essential elements for a complete ‘‘green’’ NPs synthesis [3]. Recently, the use of NPs in biomedical applications has received great interest [3]. In particular, the biomedical applications of Ag
P. Nalawade et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 129 (2014) 121–124
Materials and methods Dry pods of Acacia auriculiformis leaves were collected from the wild growing trees in Mumbai – Maharashtra, India, as and when required. The pods were washed several times with Millipore purified water to remove the dust particles. Silver nitrate (s.d. fine chem.) and sodium hydroxide (Sigma) were used as received. The experiments were performed using Millipore purified water having electrical resistivity 18.2 MX. The glasswares were washed with chromic acid/aqua regia to remove the traces of metal contaminant and rinsed with Millipore water and dried in an oven at 107 °C. Preparation of aqueous extract of plants Aqueous extract of Acacia auriculiformis was prepared by boiling about 10 g of the washed pods of Acacia auriculiformis (after removing the seeds) in 100 ml of Millipore water for 10 min. After cooling, the extract was filtered using Whatman No. 1 filter paper and kept at 4 °C until used.
1.2
6 hrs 0.8
The antibacterial activity of the Ag NPs was tested using minimum inhibitory concentration (MIC) method. The Acacia auriculiformis extract synthesized Ag NPs sol was subjected to low temperature evaporation in a dry bath at 70 °C. The volume was reduced to varying amounts to increase the concentration of Ag NPs in the sol. Accordingly 4 different concentrations of Ag NPs were obtained which were 10.8 lg/ml, 14.4 lg/ml, 21.6 lg/ml, 31.8 lg/ ml. The antimicrobial effect of the Ag NPs sols were tested on several Gram positive and Gram negative organisms. Among the Gram
1.2 0.9 0.6 0.3 0.0 0
3 hrs
2
4
6
8
10
Time (hrs)
0.6
2 hrs 0.4
1 hr 0.2
5 min 0.0 300
400
500
600
700
Wavelength, nm Fig. 1. Time dependant UV–Vis spectrum of Ag NPs obtained by adding 1 ml of aqueous extract to 1 10 3 M AgNO3 at inherent pH 2.3. Inset: Plot of absorbance vs. time for the growth of Ag NPs.
positive organisms Bacillus cereus and Staphylococcus sps. were used. Wild type Escherichia coli BW 25113 and Klebsiella sps. were two representative Gram negative organisms. 100 ll of each culture containing approximately 106 colony forming unit (CFU) per ml was spread on Nutrient agar plates containing 1.4% agar. Wells of diameter 10 mm were bored into the solidified medium and 100 ll of each sample was added. The plates were incubated for 18 h at 37 °C and were examined for the appearance of a clear zone of inhibition as opposed to an uninhibited mat like or dense growth. The diameters of the zones were recorded and mean values for each sample for the respective bacteria are tabulated. Characterization UV–visible absorption spectra were obtained from Chemito spectrascan UV 2600 spectrophotometer. The spectra were recorded at room temperature using a quartz cuvette of path length either 1 cm or 0.2 cm. Zeta potential measurement were carried out on Zetasiezer Nanoseries (Malvern instrument). Samples for transmission electron microscopy (TEM) were prepared by putting a drop of the colloidal solution on a copper grid coated with a thin amorphous carbon film. The excess solvent was removed by using a filter paper, and letting the solvent evaporate under IR lamp. TEM characterization was carried out using a Phillips
Absorbance
2.5
Antibacterial assay
9 hrs
1.0
Biosynthesis of Ag NPs Ag NPs were prepared by adding 1 ml of aqueous extract of Acacia auriculiformis to 5 ml aqueous solution of 1.0 10 3 M AgNO3. The reaction is carried out at room temperature and at inherent pH. To study the effect of pH on the formation of NPs the pH of the solution was varied using 1.0 10 3 M NaOH.
Absorbance
NPs have attracted increasing interest due to its antibacterial and antimicrobial properties [3]. Consequently, Ag NPs have been synthesized by using microorganisms and plant extracts [3–12]. However, the synthesis of NPs using plant extract is potentially advantageous over microorganisms due to the ease of biohazards and the culture of the microorganisms. Synthesis of metal NPs using plant extract is very cost effective, so can be used as an economic and suitable substitute for the largescale production of metal NPs [3–12]. Biomimetic synthesis of NPs using medicinal plants [3–12] and vegetable extracts [13] have also been explored in recent years. Acacia auriculiformis belongs to the family of Fabaceae with medicinal properties [14]. In India, its wood and charcoal are widely used for fuel. It is known to contain tannins and terpenoids along with polyphenols [15]. It has pharmacological properties like antimutagenic, cytotoxic, antihelminthic, antifilarial, microbicidal, antioxidant etc. [15,16]. Recently, it has been suggested that extract of Acacia auriculiformis possesses spermicidal activity (anti-fertility) even at significantly lower concentrations [17]. In the present paper, we demonstrated a hitherto unreported green biological route for the synthesis of Ag NPs using pods extract derived from Acacia auriculiformis. The effect of pH on the formation kinetics of Ag NPs is also demonstrated. The NPs were characterized by UV–visible spectroscopy, FTIR, and TEM analysis. The NPs showed excellent antibacterial activity against both Gram negative and Gram positive bacteria.
Absorbance
122
2.0
a - pH 2.3 inherent b - pH 6.3 e c - pH 7.3 d - pH 9.5 e - pH 11 d c
1.5 b
1.0 0.5
a
0.0 300
350
400
450
500
550
Wavelength, nm Fig. 2. Effect of pH on UV–Vis spectrum of Ag NPs prepared by adding 1 ml of aqueous extract to 1 10 3 M AgNO3 solution. The spectra were recorded after 2 h. Inset: Photographs of (A) aqueous extract, (B) Ag NPs at pH 11 and (C) Ag NPs at pH 2.3 (inherent pH).
P. Nalawade et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 129 (2014) 121–124
450 2.5 440
430 1.5 420
λ max
Absorbance
2.0
1.0 410 0.5 400 0.0 2
4
6
8
10
12
pH Fig. 3. Plot of absorbance and kmax vs. pH of Ag NPs. Other conditions are same as in Fig. 2.
CM 200 electron microscope. Particle size was measured from the TEM micrographs and calculated by taking the average of at least 100 particles. Results and discussion Synthesis of Ag NPs On addition of silver nitrate solution to the aqueous pods extracts of Acacia auriculiformis at room temperature and at inherent
123
pH (2.3) the color of the extract changed from clear light brown to cream whitish to light brown and finally to dark clear brown, which indicated the formation of Ag NPs [15]. The formation kinetics of the reduction reaction was studied by recording the UV–visible absorption spectra after different time interval. In Fig. 1, the broad absorption band appears in the region of 380–700 nm is a characteristic of surface plasmon resonance of Ag NPs. The formation and growth of the particles was slow in the beginning but get accelerated once the seed gets formed. Such phenomenon generally shows sigmoidal kinetics as shown in the inset of Fig. 1. After about 21 h of Ag NPs formation brown precipitate was seen. To see the effect of extract concentration, if any, on the stability of the Ag NPs the concentration of the extract was increased by different volumes ranging from 0.5 ml to 3.5 ml. However, it was observed that at all the extract concentrations the Ag NPs precipitated. Effect of pH on the formation and stability of Ag NPs Increase in the pH of the extract caused the formation of the NPs immediately after addition of Ag+ ions. At pH 11 instant formations of Ag NPs was observed. Fig. 2 shows the effect of pH on the UV–visible absorption spectrum of Ag NPs. The color of the silver sol was also found to be changed from brown to yellow when pH of the extract was increased up to pH 11 as shown in photograph (inset Fig. 2). As pH of the solution increased above 2.3, the wavelength of absorption maximum due to Ag NPs also decreased linearly from 432 nm to 403 nm. It can be seen from Fig. 3 that along with blue shift the increase in the absorbance also occurs at definite time which indicates that at higher pH rate of reduction of
Fig. 4. TEM of Ag NPs prepared at (a) inherent pH (b) corresponding diffraction pattern and (c) pH 11. Other conditions are same as in Fig. 2.
124
P. Nalawade et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 129 (2014) 121–124
Table 1 Zone of inhibition of bacterial growth in mm. Bacteria
Concentration of Ag NPs in lg/ml 10.8
14.4
21.6
30.8
E. coli Klebsiella sps. Bacillus cereus Staphylococcus sps.
1.67 1.35 1.37 1.54
1.78 1.43 1.3 1.38
1.67 1.37 1.37 1.49
1.84 1.52 1.37 1.47
Ag+ ions is greater than at inherent pH. The blue shift of absorption maxima of Ag NPs can be correlated to the formation of small size NPs at higher pH. It was also seen that the Ag NPs obtained at pH higher than 9.5 have shown enhanced colloidal stability compared to those obtained at lower pH. To confirm this, zeta potential (f) measurement was carried out. The zeta (f) potential is commonly used to find out the stability of colloidal systems [18,19]. The colloids are normally considered as stable when its zeta potential value is P30 mV [18]. The as-synthesized Ag NPs had f potentials 22.4, 25.1, 26.4, 28.6 and 33.2 for samples prepared at pH 2.3, pH 6.2, pH 7.3, pH 9.5 and pH 11, respectively. Representative f potential plot at pH 11 is given in Fig. S1. The Ag NPs obtained at pH 11 were stable for more than two months at room temperature and above 7 months at 4 °C. It was also seen that the particles formed at higher pH are nearly monodispersed with polygonal morphology. Representative TEM of Ag NPs obtained at inherent pH and pH 11 is shown in Fig. 4. It can be seen that at inherent pH (Fig. 4a) particles tend to aggregate and form aggregates of average diameter of 150 nm, however, at higher pH 11 (Fig. 4c) particles are nearly monodispersed with an average particle size of 20 nm. Role of A. auriculiformis pods extract in formation, stability and growth of NPs The Acacia auriculiformis is known to have tannins and terpenoids along with the polyphenols. It is known that polyphenols and tannins act as reductants and stabilizers for Ag NPs [13,20]. Therefore, the FTIR spectrum of dried biomass of Acacia auriculiformis pods extracts before and after bioreduction of silver ions was recorded (Fig. S2). It is seen that the stretching peak intensity due to AOAH (of polyphenol) at 3333 cm 1 and AOAR (ester) at 1021 cm 1 is drastically reduced after bioreduction with silver [21]. In addition, a new band at 1540 cm 1, which is assigned to C@O of amide II band, appeared in the FTIR spectrum of Ag NPs. This clearly indicates the role played by hydroxyl groups of polyphenols and ester groups of terpenoids in the reduction of Ag+ ions. Screening of antibacterial activity by agar well diffusion method Of the four bacteria studied the Acacia auriculiformis pods extract stabilized Ag NPs were most effective against E. coli as evident from the higher values of zone of inhibition while lowest values were obtained for Bacillus cereus (Fig. S3). Increasing the Ag NPs concentration did not result in significant increase of the zone of inhibition in most cases while there were a few exceptions to this trend as can be seen from the Table 1. The absence of drastic increase in the zone of inhibition with increase in concentration of Ag NPs could be due to lesser diffusibility of the concentrated sol in the agar gel. The Ag NPs sol was effective against Gram positive
bacteria as well like Staphylococcus sps., strains which are potential human pathogens. Potentially pathogenic Gram negative Klebsiella sps. was also inhibited by Ag NPs synthesized and stabilized by Acacia auriculiformis pods extract. It is well known that the antibacterial action of Ag NPs by way of cleaving of the cell wall is a process more effective in Gram negative bacterial wall as opposed to thick peptidoglycan layer of Gram positive bacteria. However according to Ruparelia et al. the cell wall and membrane composition and structure is not the sole determinant of NPs mediated toxicity to bacteria [22]. Summarily, the sol is effective against laboratory strains (E. coli) environmental isolates (Bacillus cereus) as well as isolates from pathological laboratories (Staphylococcus sps. and Klebsiella sps.), exhibiting well the versatility of this preparation against many different organisms. Conclusions We have developed a simple eco-friendly and convenient green method for the synthesis of nearly monodispersed nanoparticles of silver using pods extract of medicinal plant Acacia auriculiformis as both reducing and capping agent. Pods extract of Acacia auriculiformis is found suitable for the green synthesis of Ag NPs. Stable and nearly spherical Ag NPs of particle size 20 nm are obtained at high pH. FTIR and UV–visible spectroscopy confirmed that the constituents of extract reduce the Ag+ ions. The as synthesized Ag NPs were found to have antibacterial properties. 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.2014.03.032. References [1] M.-C. Daniel, D. Astruc, Chem. Rev. 104 (2004) 293–346. [2] M. Kowshik, S. Ashtaputre, S. Kharrazi, W. Vogel, J. Urban, S.K. Kulkarni, K.M. Paknikar, Nanotechnology 14 (2003) 95–100. [3] J. Xie, J.Y. Lee, D.I.C. Wang, Y.P. Ting, ACS Nano 1 (2007) 429–439. [4] R. Iravani, Green Chem. 13 (2011) 2638–2650. [5] S.P. Chandran, M. Chaudhary, R. Pasrichia, A. Ahmad, M. Sastry, Biotechnol. Prog. 22 (2006) 577–583. [6] S.S. Shankar, A. Rai, A. Ahmad, M. Sastry, J. Colloid Interface Sci. 275 (2004) 496–502. [7] S.S. Shankar, A. Rai, A. Ahmad, M. Sastry, Chem. Mater. 17 (2005) 566–572. [8] V. Armendariz, I. Herrera, J.R. Peralta-Videa, M. Jose-Yacaman, H. Troiani, P. Santiago, J.L. Gardea-Torresdey, J. Nanopart. Res. 6 (2004) 377–382. [9] B. Ankamwar, C. Damle, A. Ahmad, M. Sastry, J. Nanosci. Nanotechnol 5 (2005) 1665–1671. [10] A. Rai, A. Singh, A. Ahmad, M. Sastry, Langmuir 22 (2006) 736–741. [11] A. Kanchana, I. Agarwal, S. Sunkar, J. Nellore, K. Namasivayam, Dig. J. Nanomater. Bios. 6 (2011) 1741–1750. [12] A.K. Jha, K. Prasad, Dig. J. Nanomater. Bios. 6 (2011) 1717–1723. [13] J. Jacob, T. Mukherjee, S. Kapoor, Mater. Sci. Eng. C 32 (2012) 1827–1834. [14] en.wikipedia.org/wiki/Acacia_auriculiformis. [15] R. Singh, S. Singh, S. Kumar, S. Arora, Food Chem. 103 (2007) 505–511. [16] R. Singh, S. Singh, S. Kumar, S. Arora, Food Chem. 103 (2007) 1403–1410. [17] D. Pal, P. Chakraborty, H.N. Ray, B.C. Pal, D. Mitra, S.N. Kabir, Reproduction 138 (2009) 453–462. [18] Y.-P. Sun, X.-Q. Li, W.-X. Zhang, H.P. Wang, Colloids Surf. A: Physicochem. Eng. Aspects 308 (2007) 60–66. [19] Y.-P. Sun, X.-Q. Li, J. Cao, W.X. Zhang, H.P. Wang, Adv. Colloid Interf. Sci. 120 (2006) 47–56. [20] K.L. Niraimathi, V. Sudha, R. Lavanya, P. Brindha, Colloids Surf. B: Biointerf. 102 (2013) 288–291. [21] http://www2.chemistry.msu.edu/faculty/reusch/VirtTxtJml/Spectrpy/ InfraRed/infrared.htm. [22] J.P. Ruparelia, A.K. Chatterjee, S.P. Duttagupta, S. Mukherji, Acta Biomater. 4 (2008) 707–716.
