European Journal of Medicinal Chemistry 73 (2014) 135e140
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Biofabrication of silver nanoparticles using Andrographis paniculata Venkata S. Kotakadi a, *, Susmila Aparna Gaddam b, Y. Subba Rao a, T.N.V.K.V. Prasad d, A. Varada Reddy c, D.V.R. Sai Gopal a, b a
DST-PURSE Centre, Sri Venkateswara University, Tirupati 517502, Andhra Pradesh, India Department of Virology, Sri Venkateswara University, Tirupati 517502, Andhra Pradesh, India c Department of Chemistry, Sri Venkateswara University, Tirupati 517502, Andhra Pradesh, India d Agricultural Research Station, AN.G.R.A University, Tirupati 517502, Andhra Pradesh, India b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 November 2012 Received in revised form 14 September 2013 Accepted 5 December 2013 Available online 16 December 2013
New and novel strategies are of recent interest in the development of silver nanoparticles. The plant extracts are eco-friendly, economical and cost effective for synthesis of nanoparticles. In this paper, we represent biofabrication of silver nanoparticles (AgNPs) using Andrographis paniculata and the synthesized AgNPs was monitored by ultra-violet visible spectroscopy (UVeVis). The morphology and crystalline nature of AgNPs were determined from scanning electron microscopy (SEM) with Energy dispersive X-ray (EDX), X-ray diffraction patterns (XRD), Fourier transform-infrared spectroscopy (FT-IR). The size and the stability were detected by using Nanoparticle analyzer. The average size of the AgNPs was found to be 54 2 nm and the Zeta potential was found to be 50.7 mV. The synthesized AgNPs have very good antifungal activity. Ó 2013 Elsevier Masson SAS. All rights reserved.
Keywords: Biofabrication EDX FTIR Nanoparticle analyzer Zeta potential Antifungal activity
1. Introduction “There is plenty of room at the Bottom”, a lecture of Richard Feynman at the annual meeting of American Physical Society at the California Institute of technology in the year 1959, was the beginning of a new Science of Nanotechnology. Nanomaterials have a long list of applicability in improving the human life and its environment due to its optical and magnetic properties [1,2]. New and novel strategies are a recent interest in the development of gold and silver nanoparticles which have potential applications in different fields like Biology, Chemistry, Medicine and material Science [3e5]. Nanoparticles of very small size are essential for biological labeling for SER Studies [6]. Presently, silver nanoparticles have received greater attention due to their antimicrobial properties [7]. The size, shape and structure of metal nanoparticles, can be controlled and are very important to have strong correlation between different optical, electrical and catalytic properties. There are different physical and chemical methods available for synthesis of nanoparticles which are not eco/environmentally friendly [8e12]. Priosynthesis of nanoparticles is now an emerging area of
* Corresponding author. E-mail address: [email protected] (V.S. Kotakadi). 0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.ejmech.2013.12.004
nanoscience research by utilizing the rich source of plant, microorganisms. There are several reports on the use of plant, bacteria, fungi, yeast and honey for synthesis of silver and gold nanoparticles [13e22]. Plants like Aloe vera, Azadirachta indica, Camellia Sinensis, Carica papaya, Capsicum annum, Coriandrum sativeem, Diopyros kaki, Emblica officianalis, Natural rubber, Tamarindus indica, Hibiscus rosa sinensis, ocimum sanctum, Musa paradisiaca, Rosa rugosa and Vinca rosea have been reported to form silver, gold, palladium and platinum metal nanoparticles. All the above reports of synthesis of nanoparticles have a wide range of novelty regarding the size, shape, synthesis conditions and stability. So by considering diversity of plants, rapid synthesis of nanoparticles has to be explored, because only a little progress has been made on green synthesis of nanoparticles using plant extracts. The present study contains a description of our efforts to synthesize silver nano-particles by green synthesis. Here we represent rapid and simple biosynthesis of silver nanoparticles by reducing the metal (AgNo3), using the leaf extract Nelavemu (Andrographis paniculata). The leaves of A. paniculata are widely used in Indian folk-medicine for treatment of different illnesses, especially as an anti-diabetic and as a crude drug, as suggested by tribal and nontribal herbalists. The entire plant is used as medicine. According to Ayurveda, the plant is bitter, acrid, cooling, laxative, vulnerary, antipyretic, anti-periodic, anti-inflammatory, expectorant, cures
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hyperdispia, burning sensation, wounds, ulcers, malaria, diarrhea etc. It also has been proven to be a hepato-protective drug [23,24]. 2. Experimental details 2.1. Materials and methods 2.1.1. Synthesis of silver nanoparticles Nanoparticles of Ag were prepared by using Nelavemu (A. paniculata) plant extract. Healthy plant leaves was taken and washed thoroughly and were shade dried. The dried leaves were made into a fine powder. 1 g of the fine powder was dissolved in 100 ml of sterile distilled water, heated at 90 C for 10 min and filtered firstly through sterile muslin cloth and then through Whatmann filter paper. The filtrate was used for the preparation of silver nano-particles. This filtrate solution was treated as source extract and was utilized in subsequent procedures. To the 10 ml diluted filtrate, 20 ml of 0.025(M) AgNO3 was added and the sample was left at room temperature, until the color of solution changes from light yellow to brown color (in the web version) (Fig. 1b). The solution containing AgNPs was confirmed by the dark brown color. The rate of nanoparticle synthesis was very high when compared with microorganisms’ methods. Because the process can be suitably scaled up for large scale synthesis of NPs when compared with microorganisms [17,25]. In the present process for production of silver nanoparticles with Nelavemu (A. paniculata), plant leaf extract in aqueous solution was made without any external, man-made chemicals. 2.1.2. Characterization The bio-reduction of pure Ag þ ions done with the leaf samples of Nelavemu (A. paniculata) was monitored by periodic sampling of the 1 ml aliquots and the optical absorbance of silver nanoparticles suspended in distilled water was recorded on UVeVis spectrophotometer (Shimadzu 2400 UVeVis double beam model) in 200e 800 nm wavelength range. The reaction solutions were carried out at room temperature on spectrophotometer at a resolution of 1 nm.
Fig. 2. UVeVis absorption spectra of AgNPs synthesized from Nelavemu leaves extract with 2 103 M silver nitrate.
The formation and quality of compounds were checked by XRD technique. The X-ray diffraction (XRD) pattern measurements of drop-coated films AgNPs on aluminum foil substrate were recorded in a wide range of Bragg angles 2q at a scanning rate of 2 min1, carried out on a spectroscopy using Seifert Rayflex 300TT X-ray diffractometer with CuK (l ¼ 1.542 A) radiation that was operated at a voltage of 40 kV and a current of 30 mA with CuK (l = 1.542 A) radiation (1.5405 A). Scanning Electron Microscopy (SEM) and EDX was performed by Oxford Inca Penta FeTX3 EDS instrument attached to Carl Zeiss EVO MA 15 Scanning Electron Microscope (200 kV) machine with a line resolution 2.32 (in A). These images were taken by drop coating AgNPs on an aluminum foil. Energy Dispersive Absorption Spectroscopy photograph of AgNPs were carried out by the SEM equipment, as mentioned above. Particle size and zeta potential measurement experiments were carried out by using a Nanopartica (HORIBA). The FT-IR analysis was carried by using Alpha T model, FTIR Spectrophotometer, Bruker Company, the synthesized AgNPs were carefully prepared by centrifuging at 9000 rpm for 20 min and the pellet is washed thoroughly with sterile distilled water thrice to remove the unbound plant extract residues. The isolated AgNPs are now used for IR analysis. 2.1.3. Antifungal activity Sterile Potato dextrose agar medium was prepared and poured into sterile petri plates and allowed to solidified, after solidification fungal cultures were swabbed on these plates. The sterile discs were dipped in AgNPs solution and placed in the agar plate and kept for incubation for 7 days. After 7 days the discs dipped in plant extracts and 5 mm AgNO3 solution were also placed in the agar plates along with AgNPs as control to compare the results. 3. Results and discussions 3.1. UVeVis spectral analysis
Fig. 1. (a) Aqueous extract of Nelavemu and (b) AgNPs solution after addition of extract to AgNO3 solution.
The plant extracts are eco-friendly, economical and cost effective for synthesis of nanoparticles. The present work deals with the synthesis of AgNPs using leaf extract of Nelavemu (A. paniculata). The synthesis of AgNPs with leaf extract was successful, confirmed by the fact that nanometallic Ag exhibits a well-defined absorption peak at 433 nm and consequent color changes were observed from without color to reddish brown as shown in Fig. 1 and the
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absorption spectrum of the AgNPs was shown in Fig. 2. Due to surface plasmon resonance (SPR), a strong absorption of electromagnetic waves is exhibited by metal nanoparticles in the visible range. The SPR at 433 nm was broad and the concentration of different groups and molecules responsible for capping and stabilizing of nanoparticle reductions. Similar results have already been reported in the case of the stabilization effect of biological extracts on the formation of metal nanoparticles [20,26,27]. Further characterization of these AgNPs reveals that the crystals consist of small, reasonably monodispersed spheres in the 48 nm to 124 nm size range and the average of size of the particles is 53.4 nm 5 nm.
plant extracts are surrounded by a thin layer of some capping organic material from the plant leaf broth, so they are stable in solution up to 4 weeks after synthesis [29]. This is another advantage of nanoparticles synthesized using plant extracts over those using chemical methods. Silver (90.49%) was the major constituent element compared to carbon (2.259%) and oxygen (5.8%) (Fig. 4). The spectrum around 3 keV indicates a characteristic strong signal for nanosized particles of silver [30,31]. There are no peaks for silver compounds were observed. This determines that the silver compound has been reduced completely to AgNPs as determined by the spectrum.
3.2. SEM-EDX Analysis of AgNPs
3.3. Particle size determination
The morphology and size of the Nanoparticles in the solution is determined by SEM images. The AgNPs are spherical in shape and are polydispersed in natured with varied sizes. The SEM micrograph of synthesized AgNPs is shown in Fig. 3a and b. Energy dispersive X-ray analysis (EDX) reveals strong signal in the silver region and confirms the formation of silver nanoparticles which may have originated from the biomolecules bound to the surface of the silver nanoparticles (Fig. 4). EDX profile shows strong silver signal along with weak oxygen, chloride and carbon peak, which may originate from the biomolecules that are bound to the surface of the AgNPs, indicates the reduction of silver ions to elemental silver. Metallic silver nanocrystals generally show typical optical absorption peak, approximately at 3 keV due to surface plasmon resonance [28]. There is also a weak signal for oxygen in the EDX data. It has been reported that nanoparticles synthesized using
Particle size of the synthesized AgNPs was determined by Laser diffraction intensity, revealed that particles obtained were polydisperse mixture with the size ranging from 48 to 124 nm (Fig. 5). The average diameter of the particles was found to be 53.2 nm. 3.4. Zeta potential measurement The zeta potential of the synthesized AgNPs was found to be 50.7 mV in water as dispersant. On the other hand, the electrostatic repulsive forces between the nanoparticles when they are negatively charged possibly protect them from forming an association. This prevents the particles from agglomeration in the medium leading to long term stability [32]. The high value confirms the repulsion among the particles and thereby increases in stability AgNPs and the formulation. The AgNPs in the present study were negatively charged with a zeta potential of 34.3 mV which proves evident that the particles were dispersed in the medium, proving the verdict that they are stable (Fig. 6). 3.5. XRD analysis of silver nanoparticles The structure of biofabricated AgNPs was analyzed by XRD measurement. AgNPs show diffraction peaks characteristic of metallic face-centered cube as shown in Fig. 7. This is a characteristic XRD pattern of the AgNPs synthesized by green synthesis. The Bragg reflections at 2q ¼ 38.40, can be indexed to the (111) orientations confirmed the presence of AgNPs. These results clearly indicated that the nanoparticles are composed of highly crystalline Ag. It can be noted that diffraction peaks corresponding to potential silver oxides (AgO or Ag2O) cannot be observed. Dubey et al. [33] reported that the size of silver nano-crystallites as estimated from the full width at half maximum of the (111) peak of silver using the Scherrer formula was 20e60 nm. Moreover, two small insignificant impurity peaks were observed at 60 and 70 , which may be attributed to other organic substances in culture supernatant. The XRD pattern clearly illustrates that the silver NPs formed in this present synthesis were crystalline in nature. We also observed a few intense additional and yet unassigned peaks were also noticed in the vicinity of characteristic peaks of silver and these peaks might have resulted from some bioorganic compounds/proteins in the nanoparticle during the synthesis [17]. The average grain size of the silver nanoparticles formed in the bioreduction process can be determined using DebyeeScherrer’s equation by determining the width of (111) Bragg reflection [34] and was found to be 59 nm. The XRD pattern thus clearly illustrates that the silver nanoparticles formed in this present synthesis are crystalline in nature. 3.6. IR spectral analysis
Fig. 3. SEM micrograph of synthesized AgNPs.
The main constituent of A. paniculata leaves extract is Andrographolide [35]. FTIR showed that the leaves extract contains oxygen
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Fig. 4. EDX spectrum of synthesized silver nanoparticles.
Hence, it may be conclude that Andrographolide are responsible for capping and efficient stabilization. Similar type of results were also observed in earlier reports, The peak at 2855 cm1 may correspond to ketones with C H stretches correlating to a hepatoprotective diterpenoid lactone of the plant, andrographolide [36]. The peak at 3465 and 1385 cm1 can be correlated to H2O adsorption [37]. The peak at 1636 cm1 can be attributed to carbonyl stretch of amides and thereby could be related to proteins that may be encapsulated. The peak at 668.25 cm1 could be due to the OeH bends related to phenolic constituents of the plant. 3.7. Antifungal activity Fig. 5. Particle size distribution curve for AgNPs.
atoms in functional groups (OeH, C]C, C]O, CeH, CeO). The IR spectrum of the silver nanoparticles is shown in Fig. 8. From the spectrum reveals that carbonyl group, hydroxyl group ketones and (CeH 2855 cm1 C]O, 1636 cm1, OeH, 3465 cm1 OeH bending of phenols 668.25 cm1) are involved in the reduction of Agþ to Ag.
Fig. 6. Zeta potential of synthesized AgNPs.
Further the AgNPs synthesis by green route was found highly toxic against 2 fungal species at a concentration of 10 mg Ag nanoparticles, revealed higher antifungal activity against Aspergillus niger and Penicillium sp. Fig. 9. The inhibitory activities in culture media of the Ag nanoparticles reported in Table 1 were comparable with reference drug viz. Fluconazole Fu10(SD114, Himedia). The antimicrobial activity of silver has been recognized by clinicians for over 100 years. But since past few decades the modes of action of silver nanoparticles synthesized by green
Fig. 7. X-ray diffraction pattern of dried powder of synthesized AgNPs.
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Fig. 8. IR spectrum of the AgNPs.
synthesis have been widely studied as an antimicrobial agent [38,39]. The silver nanoparticles also exhibited the antifungal activity against both fungal sp A. niger and Penicillium sp. and formed the zone of inhibition of diameters 12 mm and 14 mm respectively and the control drug Fluconazole Fu10(SD114, Himedia) showed 10 mm and 11 mm respectively (Fig. 9 and Table 1). Similar types of results were also observed in different fungal pathogen [39]. The most important application of silver and silver nanoparticles is in medical industry such as tropical ointments to prevent infection against burn and open wounds [40]. And also states that addition of AgNPs to Fluconazole can hence significant antifungal activity further more, so in future the synthesized AgNPs can be useful biomedical application to cure fungal diseases [41]. Silver NPs stabilized by polymers and surfactants on incorporation with antifungal drugs exhibited reasonably high antifungal activity as a result of their enhanced aggregate stability. Further, it can be concluded that AgNPs formulations can be used as effective agents against fungal pathogens. However, exhaustive experimental trials on animals are needed before using AgNPs as potential antimicrobial agents.
Table 1 Antifungal activity of the silver nanoparticles. Fungal species
Leaf extract
Silver nitrate solution
Nelavemu (Andrographis paniculata). Inhibition zones (mm)
Aspergillus niger Penicillium sp.
No inhibition zone No inhibition zone
No inhibition zone No inhibition zone
Control
AgNPs
10
12
11
14
4. Conclusion In the present study biofabrication of AgNPs was performed by easy and reproductable way for the synthesis. The total process take place with any other chemicals so it is considered “green” route of synthesis thus permeating the synthesized nanoparticles can be used in sensitive areas such as biomedicine. The average size of the silver nanoparticles was calculated as 54 nm. The bio-reduced silver nanoparticles are characterized using UVeVis, SEM, EDX, XRD,
Fig. 9. Antifungal activity of the AgNPs: 1. Plant extract 2. Silver nitrate solution 3. AgNPs 4. Control.
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zeta potential and particle size measurements techniques. The synthesized AgNPs have very good antifungal activity. Acknowledgment The authors are grateful to DST-PURSE, Department of Science and Technology, New Delhi for providing Research Fellowships, to work under PURSE Programme at Sri Venkateswara University, Tirupati. References [1] H. Bar, D.K. Bhui, G.P. Sahoo, P. Sarkar, S.P. De, A. Misra, Colloids Surf. A 339 (2009) 134e139. [2] T. Tuutijarvi, J. Lu, M. Sillanpaa, G. Chen, J. Hazard. Mater. 166 (2009) 1415e 1420. [3] D. Xia, X. Luob, Q. Ninga, K. Yaod, Z. Liud, J. Nanjing Med. Univ. 21 (2007) 207e212. [4] D.R. Bhumkar, H.M. Joshi, M. Sastry, V.B. Pokharkar, Pharm. Res. 24 (2007) 1415. [5] C.A. Lin, T.Y. Yang, C.H. Lee, S.H. Huang, R.A. Sperling, M. Zanella, J.K. Li, J.L. Shen, H.H. Wang, H.I. Yeh, W.J. Parak, W.H. Chang, ACS Nano 3 (2009) 395e401. [6] G.W. Jeong, Y.W. Lee, M. Kim, S.W. Han, J. Colloid Interface Sci. 329 (2009) 97e102. [7] I. Sondi, B. Salopek-Sondi, J. Colloid Interface Sci. 275 (2004) 177e182. [8] C. Petti, P. Lixon, M.P. Pilent, J. Phys. Chem. 97 (1993) 12974e12983. [9] S.A. Vorobyova, A.I. Lesnikovich, N.S. Sobal, Colloids Surf. A 1525 (1999) 375e379. [10] G. Sandmann, H. Dietz, W. Pileth, J. Electroanal. Chem. 491 (2000) 78e79. [11] S. Keki, J. Torok, G. Deak, J. Colloid Interface Sci. 229 (2000) 628e634. [12] C.H. Bae, S.H. Nam, S.M. Park, Appl. Surf. Sci. 197 (2002) 628e637. [13] J.L. Gardea-Torresdey, K.J. Tiemann, G. Gamez, K. Dokken, S. Tehuacanero, M. Jose- Yacaman, J. Nanopart. Res. (1999) 397e404. [14] A. Ahmad, P. Mukherjee, S. Senapati, D. Mandal, M.I. Khan, R. kumar, M. Sastry, Colloids Surf. B 28 (2003) 313e318. [15] S.S. Shankar, A. Ahmad, M. Sastry, Biotechnol. Prog. 19 (2003) 1627e1631. [16] M. Kowshik, S. Ashtaputre, S. Kharrazi, W. Vogel, J. Urban, S.K. Kulkarni, K.M. Paknikar, Nanotechnology 14 (2003) 95e100. [17] S.S. Shankar, A. Rai, A. Ahmad, M. Sastry, J. Colloid Interface Sci. 275 (2004) 496e502. [18] B. Ankamwar, M. Chaudhary, M. Sastry, Synth. React. Inorg. Met.-Org. NanoMet. Chem. 35 (2005) 19e26.
[19] N.C. Sharma, S.V. Sahi, S. Nath, J.G. Parsons, J.L. Gardea-Torresdey, T. Pal, Environ. Sci. Technol. 41 (2007) 5137e5142. [20] A.R. Shahverdi, S. Minaeian, H.R. Shahverdi, H. Jamalifar, A.-A. Nohi, Process. Biochem. 42 (2007) 919e923. [21] M.I. Husseiny, M. Abd EI-Aziz, Y. Badir, M.A. Mahmoud, Spectrochim. Acta A 67 (2007) 1003e1006. [22] V.S. Kotakadi, Y.S. Rao, G.S. Aparna, T.N.V.K.V. Prasad, A.V. Reddy, D.V.R. Sai Gopal, Colloids Surf. B Biointerfaces 105 (2013) 194e198. [23] www.hort.purdue.edu. [24] www.ayushveda.com/herb/andrographis-paniculata. [25] D. Mandal, M.E. Bolander, D. Mukhopadhyay, G. Sarkar, P. Mukherjee, Appl. Microbiol. Biotechnol. 69 (2006) 485e492. [26] N. Vigneshwaran, N.M. Ashtaputre, P.V. Varadarajan, R.P. Nachane, K.M. ParAlikar, R.H. Balasubramanya, Mater. Lett. 61 (2007) 1413e1418. [27] Muhammad Amin, Farooq Anwar, Muhammad Ramzan Saeed Ashraf Janjua, Muhammad Awais Iqbal, Umer Rashid, Int. J. Mol. Sci. 13 (2012) 9923e9941. [28] P. Magudapatty, P. Gangopadhyayrans, B.K. Panigrahi, K.G.M. Nair, S. Dhara, Phys. B 299 (2001) 142e146. [29] J.Y. Song, B.S. Kim, Bioprocess. Biosyst. Eng. 32 (2008) 79e84. [30] M. Sathishkumar, K. Sneha, S.W. Won, C.W. Cho, S. Kim, Y.S. Yun, Colloids Surf. B 73 (2009) 332. [31] K. Vijayaraghavan, S.P. Kamala Nalini, N. Udaya Prakash, D. Madhankumar, Colloids Surf. B Biointerfaces 94 (2012) 114e117. [32] A.K. Suresh, M.J. Doktycz, W. Wang, J.W. Moon, B. Gu, H.M. Meyer III, D.K. Hensley, D.P. Allison, T.J. Phelps, D.A. Pelletier, Acta Biomater. 7 (2011) 4253. [33] M. Dubey, S. Bhadauria, B.S. Kushwah, Green synthesis of nanosilver particles from extract of Eucalyptus hybrida (safeda) leaf, Dig. J. Nanomater. Biostruct. 4 (2009) 537e543. [34] H. Borchert, E.V. Shevchenko, A. Robert, I. Mekis, A. Kornowski, G. Grubel, Langmuir 21 (2005) 1931e1936. [35] S.S. Handa, A. Sharma, Indian J. Med. Res. 92 (1990) 284. [36] Udhayaraj Suriyakalaa, Jacob Joe Antony, Subramanian Suganya, Durairaj Siva, Raman Sukirtha, Soundarrajan Kamalakkannan, P.B. Tirupathi Pichiah, Shanmugam Achiraman, Colloids Surf. B Biointerfaces 102 (2013) 189e194. [37] L.J. Jha, S.M. Best, J.C. Knowles, I. Rehman, J.D. Santos, W. Bonfield, J. Mater. Sci. Mater. Med. 8 (1997) 185. [38] Sukumaran Prabhu, Eldho K. Poulose, Int. Nano Lett. 2 (2012) 32. [39] K.J. Kim, W.S. Sung, B.K. Suh, S.K. Moon, J.S. Choi, J.G. Kim, Biometals 22 (2009) 235e242. [40] M. Ip, S.L. Lui, V.K.M. Poon, I. Lung, A. Burd, J. Med. Microbiol. 55 (2006) 59e63. [41] Manoj Singh, Manish Kumar, R. Kalaivani, S. Manikandan, A.K. Kumaraguru, Bioprocess. Biosyst. Eng. 36 (2013) 407e415.
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Biofabrication of silver nanoparticles using Andrographis paniculata Venkata S. Kotakadi a, *, Susmila Aparna Gaddam b, Y. Subba Rao a, T.N.V.K.V. Prasad d, A. Varada Reddy c, D.V.R. Sai Gopal a, b a
DST-PURSE Centre, Sri Venkateswara University, Tirupati 517502, Andhra Pradesh, India Department of Virology, Sri Venkateswara University, Tirupati 517502, Andhra Pradesh, India c Department of Chemistry, Sri Venkateswara University, Tirupati 517502, Andhra Pradesh, India d Agricultural Research Station, AN.G.R.A University, Tirupati 517502, Andhra Pradesh, India b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 November 2012 Received in revised form 14 September 2013 Accepted 5 December 2013 Available online 16 December 2013
New and novel strategies are of recent interest in the development of silver nanoparticles. The plant extracts are eco-friendly, economical and cost effective for synthesis of nanoparticles. In this paper, we represent biofabrication of silver nanoparticles (AgNPs) using Andrographis paniculata and the synthesized AgNPs was monitored by ultra-violet visible spectroscopy (UVeVis). The morphology and crystalline nature of AgNPs were determined from scanning electron microscopy (SEM) with Energy dispersive X-ray (EDX), X-ray diffraction patterns (XRD), Fourier transform-infrared spectroscopy (FT-IR). The size and the stability were detected by using Nanoparticle analyzer. The average size of the AgNPs was found to be 54 2 nm and the Zeta potential was found to be 50.7 mV. The synthesized AgNPs have very good antifungal activity. Ó 2013 Elsevier Masson SAS. All rights reserved.
Keywords: Biofabrication EDX FTIR Nanoparticle analyzer Zeta potential Antifungal activity
1. Introduction “There is plenty of room at the Bottom”, a lecture of Richard Feynman at the annual meeting of American Physical Society at the California Institute of technology in the year 1959, was the beginning of a new Science of Nanotechnology. Nanomaterials have a long list of applicability in improving the human life and its environment due to its optical and magnetic properties [1,2]. New and novel strategies are a recent interest in the development of gold and silver nanoparticles which have potential applications in different fields like Biology, Chemistry, Medicine and material Science [3e5]. Nanoparticles of very small size are essential for biological labeling for SER Studies [6]. Presently, silver nanoparticles have received greater attention due to their antimicrobial properties [7]. The size, shape and structure of metal nanoparticles, can be controlled and are very important to have strong correlation between different optical, electrical and catalytic properties. There are different physical and chemical methods available for synthesis of nanoparticles which are not eco/environmentally friendly [8e12]. Priosynthesis of nanoparticles is now an emerging area of
* Corresponding author. E-mail address: [email protected] (V.S. Kotakadi). 0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.ejmech.2013.12.004
nanoscience research by utilizing the rich source of plant, microorganisms. There are several reports on the use of plant, bacteria, fungi, yeast and honey for synthesis of silver and gold nanoparticles [13e22]. Plants like Aloe vera, Azadirachta indica, Camellia Sinensis, Carica papaya, Capsicum annum, Coriandrum sativeem, Diopyros kaki, Emblica officianalis, Natural rubber, Tamarindus indica, Hibiscus rosa sinensis, ocimum sanctum, Musa paradisiaca, Rosa rugosa and Vinca rosea have been reported to form silver, gold, palladium and platinum metal nanoparticles. All the above reports of synthesis of nanoparticles have a wide range of novelty regarding the size, shape, synthesis conditions and stability. So by considering diversity of plants, rapid synthesis of nanoparticles has to be explored, because only a little progress has been made on green synthesis of nanoparticles using plant extracts. The present study contains a description of our efforts to synthesize silver nano-particles by green synthesis. Here we represent rapid and simple biosynthesis of silver nanoparticles by reducing the metal (AgNo3), using the leaf extract Nelavemu (Andrographis paniculata). The leaves of A. paniculata are widely used in Indian folk-medicine for treatment of different illnesses, especially as an anti-diabetic and as a crude drug, as suggested by tribal and nontribal herbalists. The entire plant is used as medicine. According to Ayurveda, the plant is bitter, acrid, cooling, laxative, vulnerary, antipyretic, anti-periodic, anti-inflammatory, expectorant, cures
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V.S. Kotakadi et al. / European Journal of Medicinal Chemistry 73 (2014) 135e140
hyperdispia, burning sensation, wounds, ulcers, malaria, diarrhea etc. It also has been proven to be a hepato-protective drug [23,24]. 2. Experimental details 2.1. Materials and methods 2.1.1. Synthesis of silver nanoparticles Nanoparticles of Ag were prepared by using Nelavemu (A. paniculata) plant extract. Healthy plant leaves was taken and washed thoroughly and were shade dried. The dried leaves were made into a fine powder. 1 g of the fine powder was dissolved in 100 ml of sterile distilled water, heated at 90 C for 10 min and filtered firstly through sterile muslin cloth and then through Whatmann filter paper. The filtrate was used for the preparation of silver nano-particles. This filtrate solution was treated as source extract and was utilized in subsequent procedures. To the 10 ml diluted filtrate, 20 ml of 0.025(M) AgNO3 was added and the sample was left at room temperature, until the color of solution changes from light yellow to brown color (in the web version) (Fig. 1b). The solution containing AgNPs was confirmed by the dark brown color. The rate of nanoparticle synthesis was very high when compared with microorganisms’ methods. Because the process can be suitably scaled up for large scale synthesis of NPs when compared with microorganisms [17,25]. In the present process for production of silver nanoparticles with Nelavemu (A. paniculata), plant leaf extract in aqueous solution was made without any external, man-made chemicals. 2.1.2. Characterization The bio-reduction of pure Ag þ ions done with the leaf samples of Nelavemu (A. paniculata) was monitored by periodic sampling of the 1 ml aliquots and the optical absorbance of silver nanoparticles suspended in distilled water was recorded on UVeVis spectrophotometer (Shimadzu 2400 UVeVis double beam model) in 200e 800 nm wavelength range. The reaction solutions were carried out at room temperature on spectrophotometer at a resolution of 1 nm.
Fig. 2. UVeVis absorption spectra of AgNPs synthesized from Nelavemu leaves extract with 2 103 M silver nitrate.
The formation and quality of compounds were checked by XRD technique. The X-ray diffraction (XRD) pattern measurements of drop-coated films AgNPs on aluminum foil substrate were recorded in a wide range of Bragg angles 2q at a scanning rate of 2 min1, carried out on a spectroscopy using Seifert Rayflex 300TT X-ray diffractometer with CuK (l ¼ 1.542 A) radiation that was operated at a voltage of 40 kV and a current of 30 mA with CuK (l = 1.542 A) radiation (1.5405 A). Scanning Electron Microscopy (SEM) and EDX was performed by Oxford Inca Penta FeTX3 EDS instrument attached to Carl Zeiss EVO MA 15 Scanning Electron Microscope (200 kV) machine with a line resolution 2.32 (in A). These images were taken by drop coating AgNPs on an aluminum foil. Energy Dispersive Absorption Spectroscopy photograph of AgNPs were carried out by the SEM equipment, as mentioned above. Particle size and zeta potential measurement experiments were carried out by using a Nanopartica (HORIBA). The FT-IR analysis was carried by using Alpha T model, FTIR Spectrophotometer, Bruker Company, the synthesized AgNPs were carefully prepared by centrifuging at 9000 rpm for 20 min and the pellet is washed thoroughly with sterile distilled water thrice to remove the unbound plant extract residues. The isolated AgNPs are now used for IR analysis. 2.1.3. Antifungal activity Sterile Potato dextrose agar medium was prepared and poured into sterile petri plates and allowed to solidified, after solidification fungal cultures were swabbed on these plates. The sterile discs were dipped in AgNPs solution and placed in the agar plate and kept for incubation for 7 days. After 7 days the discs dipped in plant extracts and 5 mm AgNO3 solution were also placed in the agar plates along with AgNPs as control to compare the results. 3. Results and discussions 3.1. UVeVis spectral analysis
Fig. 1. (a) Aqueous extract of Nelavemu and (b) AgNPs solution after addition of extract to AgNO3 solution.
The plant extracts are eco-friendly, economical and cost effective for synthesis of nanoparticles. The present work deals with the synthesis of AgNPs using leaf extract of Nelavemu (A. paniculata). The synthesis of AgNPs with leaf extract was successful, confirmed by the fact that nanometallic Ag exhibits a well-defined absorption peak at 433 nm and consequent color changes were observed from without color to reddish brown as shown in Fig. 1 and the
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absorption spectrum of the AgNPs was shown in Fig. 2. Due to surface plasmon resonance (SPR), a strong absorption of electromagnetic waves is exhibited by metal nanoparticles in the visible range. The SPR at 433 nm was broad and the concentration of different groups and molecules responsible for capping and stabilizing of nanoparticle reductions. Similar results have already been reported in the case of the stabilization effect of biological extracts on the formation of metal nanoparticles [20,26,27]. Further characterization of these AgNPs reveals that the crystals consist of small, reasonably monodispersed spheres in the 48 nm to 124 nm size range and the average of size of the particles is 53.4 nm 5 nm.
plant extracts are surrounded by a thin layer of some capping organic material from the plant leaf broth, so they are stable in solution up to 4 weeks after synthesis [29]. This is another advantage of nanoparticles synthesized using plant extracts over those using chemical methods. Silver (90.49%) was the major constituent element compared to carbon (2.259%) and oxygen (5.8%) (Fig. 4). The spectrum around 3 keV indicates a characteristic strong signal for nanosized particles of silver [30,31]. There are no peaks for silver compounds were observed. This determines that the silver compound has been reduced completely to AgNPs as determined by the spectrum.
3.2. SEM-EDX Analysis of AgNPs
3.3. Particle size determination
The morphology and size of the Nanoparticles in the solution is determined by SEM images. The AgNPs are spherical in shape and are polydispersed in natured with varied sizes. The SEM micrograph of synthesized AgNPs is shown in Fig. 3a and b. Energy dispersive X-ray analysis (EDX) reveals strong signal in the silver region and confirms the formation of silver nanoparticles which may have originated from the biomolecules bound to the surface of the silver nanoparticles (Fig. 4). EDX profile shows strong silver signal along with weak oxygen, chloride and carbon peak, which may originate from the biomolecules that are bound to the surface of the AgNPs, indicates the reduction of silver ions to elemental silver. Metallic silver nanocrystals generally show typical optical absorption peak, approximately at 3 keV due to surface plasmon resonance [28]. There is also a weak signal for oxygen in the EDX data. It has been reported that nanoparticles synthesized using
Particle size of the synthesized AgNPs was determined by Laser diffraction intensity, revealed that particles obtained were polydisperse mixture with the size ranging from 48 to 124 nm (Fig. 5). The average diameter of the particles was found to be 53.2 nm. 3.4. Zeta potential measurement The zeta potential of the synthesized AgNPs was found to be 50.7 mV in water as dispersant. On the other hand, the electrostatic repulsive forces between the nanoparticles when they are negatively charged possibly protect them from forming an association. This prevents the particles from agglomeration in the medium leading to long term stability [32]. The high value confirms the repulsion among the particles and thereby increases in stability AgNPs and the formulation. The AgNPs in the present study were negatively charged with a zeta potential of 34.3 mV which proves evident that the particles were dispersed in the medium, proving the verdict that they are stable (Fig. 6). 3.5. XRD analysis of silver nanoparticles The structure of biofabricated AgNPs was analyzed by XRD measurement. AgNPs show diffraction peaks characteristic of metallic face-centered cube as shown in Fig. 7. This is a characteristic XRD pattern of the AgNPs synthesized by green synthesis. The Bragg reflections at 2q ¼ 38.40, can be indexed to the (111) orientations confirmed the presence of AgNPs. These results clearly indicated that the nanoparticles are composed of highly crystalline Ag. It can be noted that diffraction peaks corresponding to potential silver oxides (AgO or Ag2O) cannot be observed. Dubey et al. [33] reported that the size of silver nano-crystallites as estimated from the full width at half maximum of the (111) peak of silver using the Scherrer formula was 20e60 nm. Moreover, two small insignificant impurity peaks were observed at 60 and 70 , which may be attributed to other organic substances in culture supernatant. The XRD pattern clearly illustrates that the silver NPs formed in this present synthesis were crystalline in nature. We also observed a few intense additional and yet unassigned peaks were also noticed in the vicinity of characteristic peaks of silver and these peaks might have resulted from some bioorganic compounds/proteins in the nanoparticle during the synthesis [17]. The average grain size of the silver nanoparticles formed in the bioreduction process can be determined using DebyeeScherrer’s equation by determining the width of (111) Bragg reflection [34] and was found to be 59 nm. The XRD pattern thus clearly illustrates that the silver nanoparticles formed in this present synthesis are crystalline in nature. 3.6. IR spectral analysis
Fig. 3. SEM micrograph of synthesized AgNPs.
The main constituent of A. paniculata leaves extract is Andrographolide [35]. FTIR showed that the leaves extract contains oxygen
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Fig. 4. EDX spectrum of synthesized silver nanoparticles.
Hence, it may be conclude that Andrographolide are responsible for capping and efficient stabilization. Similar type of results were also observed in earlier reports, The peak at 2855 cm1 may correspond to ketones with C H stretches correlating to a hepatoprotective diterpenoid lactone of the plant, andrographolide [36]. The peak at 3465 and 1385 cm1 can be correlated to H2O adsorption [37]. The peak at 1636 cm1 can be attributed to carbonyl stretch of amides and thereby could be related to proteins that may be encapsulated. The peak at 668.25 cm1 could be due to the OeH bends related to phenolic constituents of the plant. 3.7. Antifungal activity Fig. 5. Particle size distribution curve for AgNPs.
atoms in functional groups (OeH, C]C, C]O, CeH, CeO). The IR spectrum of the silver nanoparticles is shown in Fig. 8. From the spectrum reveals that carbonyl group, hydroxyl group ketones and (CeH 2855 cm1 C]O, 1636 cm1, OeH, 3465 cm1 OeH bending of phenols 668.25 cm1) are involved in the reduction of Agþ to Ag.
Fig. 6. Zeta potential of synthesized AgNPs.
Further the AgNPs synthesis by green route was found highly toxic against 2 fungal species at a concentration of 10 mg Ag nanoparticles, revealed higher antifungal activity against Aspergillus niger and Penicillium sp. Fig. 9. The inhibitory activities in culture media of the Ag nanoparticles reported in Table 1 were comparable with reference drug viz. Fluconazole Fu10(SD114, Himedia). The antimicrobial activity of silver has been recognized by clinicians for over 100 years. But since past few decades the modes of action of silver nanoparticles synthesized by green
Fig. 7. X-ray diffraction pattern of dried powder of synthesized AgNPs.
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Fig. 8. IR spectrum of the AgNPs.
synthesis have been widely studied as an antimicrobial agent [38,39]. The silver nanoparticles also exhibited the antifungal activity against both fungal sp A. niger and Penicillium sp. and formed the zone of inhibition of diameters 12 mm and 14 mm respectively and the control drug Fluconazole Fu10(SD114, Himedia) showed 10 mm and 11 mm respectively (Fig. 9 and Table 1). Similar types of results were also observed in different fungal pathogen [39]. The most important application of silver and silver nanoparticles is in medical industry such as tropical ointments to prevent infection against burn and open wounds [40]. And also states that addition of AgNPs to Fluconazole can hence significant antifungal activity further more, so in future the synthesized AgNPs can be useful biomedical application to cure fungal diseases [41]. Silver NPs stabilized by polymers and surfactants on incorporation with antifungal drugs exhibited reasonably high antifungal activity as a result of their enhanced aggregate stability. Further, it can be concluded that AgNPs formulations can be used as effective agents against fungal pathogens. However, exhaustive experimental trials on animals are needed before using AgNPs as potential antimicrobial agents.
Table 1 Antifungal activity of the silver nanoparticles. Fungal species
Leaf extract
Silver nitrate solution
Nelavemu (Andrographis paniculata). Inhibition zones (mm)
Aspergillus niger Penicillium sp.
No inhibition zone No inhibition zone
No inhibition zone No inhibition zone
Control
AgNPs
10
12
11
14
4. Conclusion In the present study biofabrication of AgNPs was performed by easy and reproductable way for the synthesis. The total process take place with any other chemicals so it is considered “green” route of synthesis thus permeating the synthesized nanoparticles can be used in sensitive areas such as biomedicine. The average size of the silver nanoparticles was calculated as 54 nm. The bio-reduced silver nanoparticles are characterized using UVeVis, SEM, EDX, XRD,
Fig. 9. Antifungal activity of the AgNPs: 1. Plant extract 2. Silver nitrate solution 3. AgNPs 4. Control.
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zeta potential and particle size measurements techniques. The synthesized AgNPs have very good antifungal activity. Acknowledgment The authors are grateful to DST-PURSE, Department of Science and Technology, New Delhi for providing Research Fellowships, to work under PURSE Programme at Sri Venkateswara University, Tirupati. References [1] H. Bar, D.K. Bhui, G.P. Sahoo, P. Sarkar, S.P. De, A. Misra, Colloids Surf. A 339 (2009) 134e139. [2] T. Tuutijarvi, J. Lu, M. Sillanpaa, G. Chen, J. Hazard. Mater. 166 (2009) 1415e 1420. [3] D. Xia, X. Luob, Q. Ninga, K. Yaod, Z. Liud, J. Nanjing Med. Univ. 21 (2007) 207e212. [4] D.R. Bhumkar, H.M. Joshi, M. Sastry, V.B. Pokharkar, Pharm. Res. 24 (2007) 1415. [5] C.A. Lin, T.Y. Yang, C.H. Lee, S.H. Huang, R.A. Sperling, M. Zanella, J.K. Li, J.L. Shen, H.H. Wang, H.I. Yeh, W.J. Parak, W.H. Chang, ACS Nano 3 (2009) 395e401. [6] G.W. Jeong, Y.W. Lee, M. Kim, S.W. Han, J. Colloid Interface Sci. 329 (2009) 97e102. [7] I. Sondi, B. Salopek-Sondi, J. Colloid Interface Sci. 275 (2004) 177e182. [8] C. Petti, P. Lixon, M.P. Pilent, J. Phys. Chem. 97 (1993) 12974e12983. [9] S.A. Vorobyova, A.I. Lesnikovich, N.S. Sobal, Colloids Surf. A 1525 (1999) 375e379. [10] G. Sandmann, H. Dietz, W. Pileth, J. Electroanal. Chem. 491 (2000) 78e79. [11] S. Keki, J. Torok, G. Deak, J. Colloid Interface Sci. 229 (2000) 628e634. [12] C.H. Bae, S.H. Nam, S.M. Park, Appl. Surf. Sci. 197 (2002) 628e637. [13] J.L. Gardea-Torresdey, K.J. Tiemann, G. Gamez, K. Dokken, S. Tehuacanero, M. Jose- Yacaman, J. Nanopart. Res. (1999) 397e404. [14] A. Ahmad, P. Mukherjee, S. Senapati, D. Mandal, M.I. Khan, R. kumar, M. Sastry, Colloids Surf. B 28 (2003) 313e318. [15] S.S. Shankar, A. Ahmad, M. Sastry, Biotechnol. Prog. 19 (2003) 1627e1631. [16] M. Kowshik, S. Ashtaputre, S. Kharrazi, W. Vogel, J. Urban, S.K. Kulkarni, K.M. Paknikar, Nanotechnology 14 (2003) 95e100. [17] S.S. Shankar, A. Rai, A. Ahmad, M. Sastry, J. Colloid Interface Sci. 275 (2004) 496e502. [18] B. Ankamwar, M. Chaudhary, M. Sastry, Synth. React. Inorg. Met.-Org. NanoMet. Chem. 35 (2005) 19e26.
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