Green Synthesis of Silver Nanoparticles using Strobilanthes flaccidifolius Nees. Leaf Extract and its Antibacterial Activity

The leaf extract of Strobilanthes flaccidifolius Nees. was used for the synthesis of silver nanoparticles through a green technique of synthesis. The nanoparticles was characterized by UV-VIS spectroscopy which proves the formation silver nanoparticles. FTIR (Fourier Transmission infra red spectroscopy) study was carried out to assess the biomolecule as indigo precursors, Energy dispersion X-ray analysis(EDX) data further proves it. EPR (Electron paramagnetic resonance technique) shows the free radical in silver neutral state and XRD(X-ray diffraction technique) also repots silver neutral formation.The morphology and the shape of the silver nanoparticles were determined by Scanning electron microscopy(SEM) and Tunneling electron microscopy (TEM).The nanoparticles adopted spherical morphology and the size ranging from 6nm to 54.11nm and average size was determined as 12.15± 5.3nm.The nanoparticles had antimicrobial activity.


Plant material and preparation of extract
The freshly leaves weighing 5g were collected in separate beakers. Then it was thoroughly rinsed with distilled water. The sample was heated in 200 mL of solution of 50% ethanol in water on a steam bath till appearance of brown coloration. This brown colored extract was cooled to room temperature and filtered using Whatman filter paper (no.42). This extract was taken as the stock solution.

Synthesis
Volume of stock solution (40 mL) was made to double by addition of distilled water. The solution was treated with AgNO3 solution (20 mL) and warmed on steam bath for approximately 10min till reddish brown colour precipitate was observed, allowed to cool at room temperature. The silver nanoparticles were collected by centrifugation process for 10 min at 150000 rpm, washed with distilled water and dried at 30°C in a closed oven [34].

Characterization of nanoparticles 2.3.1. FTIR analysis
FTIR data was recorded on a Shimadzu FTIR instruments. The sample was made into KBr pellet and the spectra were recorded. The FTIR data of both the raw extract as well as the silver nanoparticles formed was recorded.

UV-VIS analysis
For UV spectra the raw extract was diluted with 50% ethanol and 0.03 M AgNO3 in the ratio of 4:1. The solution was heated on a water bath for 5min. It was allowed to stand and the absorption was recorded in a Shimadzu UV-VIS spectrophotometer. Further four fold dilution was performed before recording the spectra. For comparison and confirmation of formation of silver nanoparticles, the spectrum of the raw extract was also recorded along with the solution of the nanoparticles.

XRD analysis
The XRD of the sample was loaded on a PAN analytical XPERT-PRO instrument. The spectra were recorded from 20 to 80, 2 theta with step size [°2Th.] of 0.0500. Anode Material was Cu, K-Alpha1 [Å]:1.54060. Generator settings were at 30 mA, 40 kV with scan step time [s] was 2.0000, and divergence slit size was 0.9570 and receiving slit size [mm]: was 0.2000.The Bragg-Brentano focusing geometry was used. The pattern was compared with the patterns of International Centre of Diffraction Data (ICDD) database nearest matching pattern was found with silver with XPERT HIGHSCORE. Mar c h 11, 2 0 1 4

SEM analysis
SEM images were recorded on FEI-QUANTA-250 electron microscope. The compound was adsorbed on a carbon sheet and loaded on the microscope. The sizes of particles were measured with the software IMAGE J and the average was calculated.

EPR analysis
Electron paramagnetic resonance (EPR) was recorded for free radical analysis on JEOL JES-FA200 ESR spectrometer with X-band microwave unit.

EDX analysis
The EDX analysis was performed on an EDAX Energy Dispersion X-ray spectrometer. The identification of the elemental constituents and estimation of the quantities were done without standard.

TEM analysis
For TEM analysis the compound was dispersed in ethylene glycol and measured with JEOL JEM-2100. The sizes of particles were measured with the software IMAGE J.

Antimicrobial assay
The antimicrobial activity was assessed by agar well diffusion method using 20ml of sterile Nutrient agar (NA) (Hi-Media) for testing the bacterial against Proteus mirabilis, Klebsiella pneumoniae, Escherichia coli, Salmonella paratyphi and Pseudomonas aeruginosa [35]. The sample was diluted in 5mg/ml in DMSO. The dilutions of the sample concentration were deposited 20μl on the inoculated well and left for 10 min at room temperature for the extract diffusion. Negative control was AgNO3 solution. Ciprofloxacin (Hi-Media) for bacteria were served as positive control. The plates were inoculated with bacteria were incubated at 37ºC for 24 hr. The experiment was repeated four times and the average results were recorded. The antimicrobial activity was determined by measuring the diameter of the inhibition zone around the well. The susceptibility of microbial was determined by minimum inhibitory concentration determination method [36]. The minimum inhibitory concentrations (MICs) of the sample were determined by serial dilution against the microorganisms. The minimum concentrations at which no visible growth were observed were defined as the MICs, which were expressed in mg/ml. The antimicrobial tests were calculated as a mean of three replicates and the SD was calculated using the software SPSS, version 10 (SPSS, Richmond, USA).

Synthesis
Synthesis was performed with the introduction of AgNO3 into the raw extract.

UV-VIS analysis
When the raw extract was treated with 0.03 M AgNO3 in the ratio of 4:1 and treating as described in the method and recording the spectra at different intervals of time a band started appearing at 450 nm after keeping for 10min which corresponded to the surface Plasmon resonance band of noble metal silver. After 30min band started flattening as illustrated in figure1 [3 7]. The IR bands at 1708 cm -1 and 1606 cm -1 were characteristic of carbonyl group whereas the band at 1589 cm -1 was characteristic of NH group respectively [39]. Band at 1508 cm -1 disappeared in the case of nanoparticles. B a n d a t 7 6 1 cm -1 is because of NH wagging. These structural changes indicated that the reduction and stabilization of silver nanoparticles proceed via the coordination between N of the indigo precursors and silver ions. The FTIR studies had confirmed the fact that the hydroxyl and N form indigo precursors had the stronger ability to bind metal indicating that the glycosides could possibly form a layer covering the metal nanoparticles (i.e. capping of silver nanoparticles) to prevent agglomeration and thereby stabilized the medium.

X-ray diffraction analysis
The XRD pattern of the Ag nanoparticles prepared using S. flaccidifolius extract showed a number of strong Bragg reflections at 38.2, 44.3, 64.4 and 77.3 ( Figure 3) which corresponded to the (1 1 1), (2 0 0), (2 2 0) and (3 1 1) facets of the face centered cubic crystal structure, respectively [40]. No diffraction peaks corresponding to the precursor (AgNO 3 ) and/or bi-products (such as silver oxide) were observed, which confirmed that only metallic Ag w a s formed by S. flaccidifolius extract reaction.

EDX identification and estimation
The scanning SEM-EDX mapping images of the nanoparticles synthesized at different temperatures showed the presence of estimated elements (silver, carbon, oxygen, Nitrogen and Silver) ( Figure 5). It also confirmed the formation of silver nanoparticles with the capping that reported earlier of indigo precursors and the formation of silver nanoparticles [42].

EPR analysis
The EPR signal showed the presence of lone pair indicating of the silver in neutral d 9 (Ag0) state which was absent in the d 8 (Ag+) state ( Figure 6).This further proves the formation of silver nanoparticles.

Transmission electron microscopy
Spherical shaped particles were showed by TEM of the size ranging from 6nm to 54.11nm (Figure 7a). The average size was 12.15± 5.3nm in TEM image (Figure 7c). The SAED (Selected Area Electron Diffraction) pattern showed the miller planes of silver metal and additional peaks of the organic capping (Figure 7b) that proved the crystalline nature of the compound. The inter planar distance from the adjacent lattice fringes in HR-TEM image of one particle was 2.36Ǻ which corresponded to face centre cubic of Ag (Figure 7d

Antimicrobial activity
Antimicrobial effects of synthesized silver nanoparticles using S. flaccidifolius were tested. against human pathogens gram negative bacteria. The result showed inhibitory action when compared to silver nanoparticles at high level of zone of inhibition against P. aeruginosa. Moderate level of inhibitory activity was observed for P. mirabilis, K. pneumonia, E. coli and S. paratyphi. P. aeruginosa (MIC =0.00122070312mg/ml) was found to be the most susceptible bacterial pathogen (Table 1). Crude silver blend shows potent activity than the purified silver nanoparticles, implies the bioactivity of combined plant and silver nanoparticles conjugate [43]. Exact antimicrobial effect of silver nanoparticles was still unclear, suggesting three different possible mechanism of action, involving first silver ions attach to the bacterial cell membrane and caused plasmolysis (cytoplasm of bacteria separated from bacterial cell wall), inhibited the bacterial cell membrane synthesis [44]. Secondly, AgNPs could strongly interact with sulphur-phosphorus containing compounds present inside (DNA, proteins) and outside (membrane proteins) of bacterial cell, affecting respiratory chain reaction, cell division and finally lead to cell death [45]. Finally, AgNPs released silver ions that will penetrate into the cell wall, causing condensation of DNA damage and also by affecting the protein synthesis [46]. Overall results showed the involvement of the phenomena of synthesized particles leading to the damage to pathogens or killing the pathogens. Raut et al. [47] investigated the antibacterial activity of photosynthesized silver nanoparticles against E. coli, P. aeroginosa and K. pneumoniae. Similarly, Kim et al. [48] reported antimicrobial activity of silver nanoparticles against E. coli and S. aureus. Kotakadi et al. [49] showed antibacterial activity of silver nanoparticles against E. coli. Sathishkumar et al. [50] inhibited antimicrobial activity of phyto-synthesis of silver nanoscale particles against E. coli, P. aeroginosa and K. Pneumonia.

CONCLUSION
The silver nanoparticles were synthesized with green technique using the extract of S. flaccidifolius as the reductant and the stabiliser. The formation of AgNPs was confirmed by IR, UV, EPR, EDX and XRD technique and size was characterized by SEM and HRTEM. The antimicrobial activity of the silver nanoparticles shows much potent inhibitory activity against clinically isolated pathogens. Hence the plant mediated synthesized nanoparticles can be used as good therapeutic agent against human pathogens and also for the successful development of drug delivery in near future.