Nelumbo nucifera leaf extract mediated synthesis of silver nanoparticles and their antimicrobial properties against some human pathogens

In the present report, bio-reduction of silver nitrate into silver nanoparticles using the leaf extract of Nelumbo nucifera is explained. The synthesized nanoparticles exhibited surface Plasmon resonance at 410 nm. The crystalline nature of the biosynthesized silver nanoparticles was confirmed from the X-ray diffraction pattern. The functional groups responsible for bio-reduction of silver nitrate into silver were analyzed by Fourier transform infrared spectroscopy and confirmed by X-ray photoelectron spectrum. Field emission transmission electron microscope micrographs showed the formation of well-separated silver nanoparticles of size in the range of 30–40 nm. The result of dynamic light scattering also confirms the mono-dispersed silver nanoparticles with average size of 35 nm. The synthesized nanoparticles exhibited excellent antibacterial activity against the Gram-positive bacteria B. subtilis.


Introduction
Peculiar physical and chemical properties of materials in nanoscale gain the span of attention of major research groups compared to bulk of the same chemical composition. Therefore, fabrication of materials with novel applications can be made possible by controlling the morphology and size at nano scale. Among the nanomaterials, metal nanoparticles have many important applications in medicine and pharmacy. For instant, silver nanoparticles can be used in medicine to reduce infection in burn treatment, to prevent bacteria colonization on dental materials, stainless steel materials, to eliminate microorganisms on textile fabrics, and for water treatment (Panacek et al. 2006;Bo et al. 2009;Tomsic et al. 2008). Although, many chemical methods are available to reduce silver nitrate into silver nanoparticles, they expect toxic chemical and complicated procedures (Alarcon et al. 2012;Panácek et al. 2009;Prucek et al. 2011;Shanmugam et al. 2014). As an alternate, researchers are revealing their interest in the synthesis of metallic nanoparticles through green technology (Rastogi and Arunachalam 2011;Vijayakumar et al. 2011;Yilmaz et al. 2011;Guidelli et al. 2011;Kouvaris et al. 2012). Many researchers have reported the biosynthesis of metal nanoparticles by plant leaf extracts and their potential applications (Salem et al. 2014;Abdel-Aziz et al. 2014;Sulaiman et al. 2013;Prabhu et al. 2013). Nelumbo nucifera (Lotus), an aquatic plant with medicinal values, is seen in India and China. This species can be multiplied either by sexual (seeds) or asexual (rhizomes) reproduction. N. nucifera is reported to have antidiarrhoeal and antimicrobial properties. In the present work, the bio-reduction of AgNO 3 into AgNPs from the broth of N. nucifera leaf is reported. Further, the antimicrobial activity of the synthesized silver nanoparticles has also been discussed against Gram-positive and Gram-negative bacteria.

Materials
Silver nitrate (AgNO 3 ) was purchased from Merck. The glass wares used in this experimental work were acid washed. Ultrapure water was used for all dilution and sample preparation.

Preparation of the leaf extract
Fresh and healthy leaves were collected and rinsed thoroughly first with tap water followed by distilled water to remove all the dust and unwanted visible particles, cut into small pieces, and dried at room temperature for 10 days. The dried leaves were made into powder using a kitchen blender. Broth of the Nelumbo nucifera was prepared by mixing 20 g of dried leaf powder with 100 mL of water and then boiled at 80°C with constant stirring on a magnetic stirrer. The extracts were then filtered thrice through Whatman No. 1 filter paper with pore size in the range of 20-25 micron to remove particulate matter and stored in the Erlenmeyer flasks for further experiments. Throughout the experiment, sterility conditions were maintained for effectiveness and accuracy in results.

Preparation of silver nanoparticles
The 10 ml of filtered extract was mixed with 90 ml of 5 mM AgNO 3 and incubated in the dark environment for 12 h, and the control was also maintained without the addition of plant extract. The bio-reduction of AgNO 3 into AgNPs can be confirmed visually by the change in color from colorless to reddish brown ( Fig. 1).

Characterization techniques
Characterization of nanoparticles is important to understand and control nanoparticles synthesis and application. The formation of AgNPs was confirmed by sampling the reaction mixture at regular intervals and the absorption maximum was scanned by UV-Visible spectra, in a range of wavelength between 300 and 700 nm using LAMDA 25 PERKIN ELMER spectrometer. The X-ray diffraction (XRD) patterns of the silver nanoparticles were recorded using X'PERT PRO diffractometer with Cu-Ka radiation (k = 1.5406 Å ). The crystallite size was estimated using the Scherrer's equation. The metal state of AgNPs was examined using Thermo Fisher Scientific Inc., K-Alpha, USA, X-ray photoelectron spectroscopy (XPS) system. The presence of functional groups in leaf extract and synthesized AgNPs was identified by SHIMADZU-8400 FT-IR Spectrometer using KBr pellet technique. DLS measurements were carried out with a DLS particle size analyzer [ZETA Seizers Nanoseries (Malvern Instruments Nano ZS)] to estimate the average size distribution of the prepared particles. Field emission transmission electron microscopy (FE-TEM) analysis was performed (JSM 2100F JEOL) to study the morphology and size of the Ag nanoparticles.

Antibacterial activity
Silver is known for its antibacterial properties and has been used for years in the medical field for antimicrobial applications and even has shown to prevent HIV binding to host cells (Nino-Martinez et al. 2008;Alt et al. 2004;Russel and Hugo 1994;Lee et al. 2007;Jeong et al. 2005). In addition, silver has been used in water and air filters to eliminate microorganisms (Chou et al. 2005;Jin et al. 2007). In the present work, the synthesized Ag nanoparticles in aqueous were tested against bacterial pathogens by agar disc diffraction method (Karting et al. 1991). The pathogens namely Escherichia coli, Bacillus subtilis, Salmonella typhi, and Vibrio cholerae were obtained from Raja Sir Muthaiya Medical College, Annamalai University. About 150 CFU/ml of inoculums was swabbed onto MHagar plates uniformly and allowed to dry in a sterile environment. Sterile disc of 6 mm (HIMEDIA) was loaded with 30 ll of test solution (AgNO 3 and Ag nanoparticles). Ampicillin (10 mg in 1 ml) was used as positive control. The plates were incubated at 25°C for 3 days to measure the zone of inhibition. The mean was calculated by performing the experiments in triplicates.

UV-Visible absorption spectra
Bio-reduction of silver nitrate into silver nanoparticles in the presence of N. nucifera extract was confirmed from UV-Vis spectral measurements. AgNPs have free electrons, which have the probability to give rise to a surface Plasmon resonance (SPR) absorption band (Binupriya et al. 2010), due to the combined vibration of electrons of metal nanoparticles in resonance with the light wave. The UV-Vis absorption results of AgNPs with different time of incubation are shown in Fig. 2. As shown in figure, the broad spectrum of AgNPs with absorption maximum at 410 nm was seen due to the surface plasmon resonance. It is also noted that the increase in incubation time increases the reduction process and intense absorption has been predicted at 120 h of incubation. The presence of amides and methoxy groups in the extract of N. nucifera may play an important role in reduction and stability of AgNPs. Figure 3 depicts the X-ray diffraction pattern of silver nanoparticles prepared using Nelumbo nucifera leaves extract. The diffraction pattern has four main diffraction features corresponding (111), (200), (220), and (311) planes and all the four peaks can be indexed to standard cubic phase of silver (JCPDS card No: 04-0783) (Shanmugam et al. 2014). No reflection planes related to nitrate ions and other impurities were detected in the diffraction pattern, revealing the high purity of the synthesized product. Obviously, it is seen that the obtained reflections are sharp and high in intensity, indicating that the prepared silver nanoparticles are well crystalline in nature. Further, the lattice constant of the prepared cubic silver is found to be a = 4.085Å , which is consistent with the JCPDS value (a = 4.086Å ). The average crystalline size estimated from Debye-Scherrer's formula is around 25 nm. The overall spectrum indicates the presence of strong C 1 s, O 1 s, N 1 s, and Ag 3d core levels (Fig. 4a). The presence of carbon at 284.8 eV corresponds to the carbon contribution from the phenyl rings of the protein (Jeona and Kang 2008). The N 1 s peak at 399.8 eV could be assigned to neutral amine groups present in the protein moiety (Sharma et al. 2004). The O 1 s core level assigned at 532.1 eV can be ascribed to the carboxylate groups  (Jeona and Kang 2008;Carroll et al. 2011). From the spectrum of Ag 3d (Fig. 4b), the binding energies of Ag 3d 5/2 and Ag 3d 3/2 are found to be at 368 and 374 eV, respectively (Sumesh et al. 2011;Lai et al. 2010). Therefore, it is concluded that the proteins were responsible for the bio-reduction of AgNO 3 into Ag nanoparticles.

Functional group analysis
The functional groups of N. nucifera responsible for the bio-reduction of AgNO 3 into Ag nanoparticles can be explained from FT-IR analysis. The IR spectra of N. nucifera extract and Ag nanoparticles stabilized by the extract are shown in Fig. 5. For the leaf extract, FT-IR shows the major absorption peaks around 3361,2922,2360,1647,1540,1242,1058, and 667 cm -1 . The broad absorption band observed around 3361 cm -1 is the characteristic band of hydrogen-bonded NH groups. The absorption band positioned at 2922 cm -1 could be assigned to the C-H stretching vibration of methyl, methylene, and methoxy groups (Feng et al. 2009). The appearance of peak at around 1647 cm -1 indicates the C=O stretching of amide I bands of peptide linkage (Jeona and Kang 2008). The peaks at 1540 and 1242 cm -1 are the stretching vibration of N-H groups and the bending vibration of C-N groups, amide II and III bands, in the proteins (Carroll et al. 2011). The broad absorption positioned at 1058 cm -1 reveals the presence of aliphatic amides (Bouropoulos et al. 2001). The absorption band positioned at 667 cm -1 is due to the plane bending vibration of N-H groups in the proteins (Shikuo et al. 2007). A similar kind of IR bands was observed for Ag nanoparticles with minor shift in these absorption bands. The minor shift may be due to the interaction of leaf extract with AgNPs which changed the original transmittance level of the extract. From the FT-IR spectra, it is concluded that the proteins are responsible for stabilizing the silver nanoparticles synthesized by the leaf extract of N. nucifera.

Morphology and particle size
The morphology and size of the synthesized AgNPs were analyzed by FE-TEM. The FE-TEM image of AgNPs synthesized after 120 h of incubation is shown in Fig. 6. The figure exhibited the morphology of the particles being spherical with sizes in the range of 30-40 nm. To confirm the size of the Ag NPs, DLS measurement was recorded. Figure 7 shows the DLS histogram of the AgNPs obtained after 120 h of incubation. The average size of the particles as predicted from the histogram is 35 nm. The obtained size is almost consistent with the result of FE-TEM.

Antimicrobial activity
The antimicrobial susceptibility of the synthesized Ag nanoparticles was investigated against the Gram-positive bacteria B.subtilis and gram-negative E.coli, S.typhi, and V.cholerae through disc diffusion method. Figure 8 illustrates the antibacterial activity of AgNPs and the positive control (Ampicillin) against the pathogens subjected for analysis. The diameter of each zone of inhibition was measured and the results are given in Table 1. The results indicate that the synthesized silver nanoparticles showed a considerable antimicrobial activity for all the pathogens studied; however, the highest zone of inhibition was recorded for B. subtilis (16 mm). The difference in antimicrobial activity between Gram-positive and Gram-negative could be due to their difference in membrane structure. The Gram-positive bacteria have a thick peptidoglycan layer, whereas, peptidoglycan layer in the Gram-negative bacteria is thinner but surrounded by a lipid layer outside (Gogoi et al. 2015). In the antimicrobial activity, initially AgNPs attach to the surface of the bacterial cell membrane and then penetrate into the bacteria. After penetration, they inactivate the enzymes of the microbes, generating hydrogen peroxide and causing bacterial cell death. The antibacterial properties of these green-synthesized AgNPs suggest their usage in medical devices as an antimicrobial coater.

Conclusion
In conclusion, a novel synthesis of silver nanoparticles through bio-reduction of silver nitrate using aqueous leaf extract of N. nucifera was demonstrated. The formation of AgNPs was confirmed by the presence of surface plasma resonance at 410 nm. The crystalline nature of the synthesized product was confirmed by the diffraction results. FT-IR and XPS results suggest that proteins present in the extract were largely responsible for the bio-reduction of the silver nitrate into silver nanoparticles. The morphology of the particles analyzed by FE-TEM showed the presence of spherical particles with sizes in the range of 30-40 nm. The DLS histogram also suggests the mono-dispersed size distribution of the particles. The antimicrobial activity of the AgNPs exhibits potential inhibitory activity against the Gram-positive bacteria B. subtilis, despite their activity being moderate for all other pathogens studied. Hence, the N. nucifera leaf extract-mediated synthesis of AgNPs can be suggested as good therapeutic agents against human pathogens and also for the successful development of drug delivery in future.