UV–Vis spectral analysis
Figure 1 shows the leaves of the Pedalium murex plant. Figure 2 shows the AgNO3 solution before and after adding the leaf extract. The UV–visible absorption spectra of the Ag nanoparticles with different concentrations of Pedalium murex leaf extract of 1, 2, 3, 4 and 5 ml are recorded and shown in Fig. 3. The formation of the AgNPs during the reduction process is indicated by change in the color of the reaction solution from colorless to dark brown which can be visually observed (Fig. 2). Metal nanoparticles have free electrons, which yield a surface plasmon resonance (SPR) absorption band, due to the mutual vibration of electrons of metal nanoparticles in resonance with light wave. The appearances of the peaks show the characteristics of surface plasmon resonance of silver nanoparticles.
The absorption spectra exhibit a gradual decrease of the absorbance, accompanied by a shift in the wavelength from 430 to 424 nm. A decrease in the FWHM value is also observed from Fig. 3. The increases in the intensity of the plasmon bands indicate the decrease in the band width (Zhang et al. 2006). The UV–Vis spectrum shows the important role of AgNO3 and the presence of ingredients in the leaves for the formation of silver nanoparticles. The increase in the concentration of the leaf extract will also increase the absorbance intensity. It is also observed that the surface plasmon peak that occurs at 424 nm is slowly shifted toward lower wavelength at high concentrations. This shift may be due to blue shift and depends on the particle size and shape (Kelly et al. 2003; Lee and El-sayed 2006). According to Njagi et al. (2011), this band corresponds to the absorption by colloidal silver nanoparticles in the region (400–450 nm) due to the excitation of surface plasmon vibration. So the high-concentration sample is used for further analysis. A high concentration of Pedalium murex leaf extract increases the number of biocompounds required to reduce Ag+ to Ag0.
Fluorescence
The PL of the synthesized bio-inspired AgNPs by Pedalium murex leaf extract is also studied via fluorescence emission spectroscopy. Photoluminescence (PL) spectrum is one of the methods to estimate the optical property of silver nanoparticles as photonic materials. The colloidal silver nanoparticles are dispersed in water and the PL emission spectra are recorded for the excitation wavelength at 420 nm. A broad emission is obtained at 478 nm (Fig. 4). The intensity of fluorescence emission peak is gradually increased up to 478 nm, after which it is slowly decreased up to 650 nm. Earlier, a characteristic fluorescence peak of AgNPs in the water phase at 465 nm was reported (Jiang et al. 2005). Vigneshwaran et al. (2006) reported an emission peak of AgNPs produced from soluble starch at 553 nm. When compared with the value of AgNPs in the water phase (465 nm), the present peak is redshifted.
FTIR analysis of AgNPs
FTIR measurements were carried out to identify the possible biomolecules in the Pedalium murex extract. FTIR spectra of dried aqueous extract and synthesised AgNPs are shown in Fig. 5. The phytochemical analysis of Pedalium murex reveals the presence of flavonoids, alkaloids, steroids, rosins, saponins and proteins (Rajashekar et al. 2012; Patel et al. 2011). In leaf extract, the peaks are observed at 445, 617, 1075, 1287, 1421, 1602, 3157 and 3785 cm−1,respectively. After reaction with AgNO3, the peaks are shifted to a higher wave number side, such as 456, 614, 1074, 1382, 1592, 3158 and 3881 cm−1. The peak at 445 cm−1 of the extract is shifted toward a higher wave number side at 456 cm−1 due to the O–Si–O network and ring opening vibration. The band observed at 617 cm−1 is shifted to the lower side at 614 cm−1, which corresponds to C–Cl stretching in the alkyl group. The strong intense peaks at 1382 cm−1 correspond to C–N stretch vibrations, as well as to the amide I bands of proteins in the leaf extract (Gurunathan et al. 2015). The strong bands at 1074 cm−1 are due to ether linkages and suggest the presence of flavanones adsorbed on the surface of metal nanoparticles (Shankar et al. 2004). The phenolic groups participating in ion replacement response are placed in the 1315–1037 and 1456–1600 cm−1 regions for the plant extract (Jeeva et al. 2014b). The very strong band at 1592 cm−1 is due to C=C stretching in the aromatic ring, confirming the presence of the aromatic group (Reddy et al. 2014). The silver nanoparticles of O–H stretching in carboxylic acids vibration is shifted from 3785 to 3881 cm−1. The immediate reduction and capping of silver ion into silver nanoparticles in the present analysis might be due to flavanoids and proteins. The flavonoids present in the leaf extract are powerful reducing agents which may be suggestive of the formation of AgNPs by reduction of silver nitrate. The flavonoid compounds in the water extract of M. pendans might be actively involved and responsible for the reduction of Ag+ to Ag0 (Zuas et al. 2014). The involvement of water-soluble flavonoid in the reduction of metal ions using plant extracts is also evidenced from another study (Prabhu et al. 2010).
XRD studies
The nanoparticles synthesised in this method are characterized using powder XRD to confirm the particles as silver and to know the structural information. Figure 6 shows the XRD pattern of silver nanoparticles.
The pattern clearly shows the main peaks at (2θ) 38.19, 44.37, 64.56 and 77.47 corresponding to the (111), (200), (220) and (311) planes, respectively. By comparing JCPDS (file no: 89-3722), the typical pattern of green-synthesized AgNPs is found to possess an fcc structure. The average crystalline size of the silver nanoparticles was estimated using (Eq. 1), the Debye–Scherrer’s equation (Ajitha et al. 2014):
$$D = 0.9\lambda /\beta \, \cos \theta .$$
(1)
By determining the width of (111) Bragg’s reflection, the estimated average size of the particle is 14 nm.
In addition, two unassigned peaks appeared at 32.25° and 46.21°. These peaks were weaker than those of silver. This may be due to the bioorganic compounds occurring on the surface of the AgNPs. Unpredicted crystalline structures (32.25° and 46.21°) are also present and might be due to the organic compounds in the leaf extract (Suvith and Philip 2014; Duraisamy et al. 2013). A similar result was observed by Kumar and Yadav (2009) and Jeeva et al. (2014b), who identified crystalline peaks (32.28°, 46.28°, 54.83°, 67.47° and 76.69°) which were also obvious in a lot of works in which the XRD pattern included the relevant 2° range. Appearances of these peaks are due to the presence of phytochemical compounds in the leaf extracts. The stronger planes indicate silver as a major constituent in the biosynthesis.
The average crystalline size, lattice parameter, cell volume and microstrain are shown in Table 1. The calculated lattice constant is in good agreement with the reported value and the sample exhibits smaller cell volumes. Earlier workers reported similar results for Ag nanoparticles (Gopinath et al. 2012; Basavegowda et al. 2014; Bindhu and Umadevi 2013).
Table 1 The variation of crystalline size, lattice parameter, cell volume and microstrain value of biosynthesized nanoparticles
FESEM analysis
The FESEM images of the silver nanoparticles are shown in Fig. 7. The surface morphology of silver nanoparticles showed even shape and spherical nature. In the present study, the histogram of the particle size ranges from 20 to 50 nm. Similar results were also reported for phyto-synthesised silver nanoparticles (Sathishkumar et al. 2012). This result strongly confirms that Pedalium murex leaf extracts might act as a reducing and capping agent in the production of silver nanoparticles.
EDAX study
Figure 8 shows the energy dispersive spectrum of the synthesized nanoparticles, which suggests the presence of silver as the ingredient element. Metallic silver nanoparticles generally show a typically strong signal peak at 3 keV, due to surface plasmon resonance (Magudapatty et al. 2001; Kaviya et al. 2011; Das et al. 2013). Figure 8 shows the quantitative information of biosynthesized AgNPs. The presence of elements such as Ag, O, C, K, Cl, Ca and Na are shown in the inset of Fig. 8.
This is one of the advantages of nanoparticles synthesized using plant extracts over those synthesised using chemical methods. In the present investigation, the synthesized silver nanoparticles show strong absorption in the range 2.5–4 keV. Similar results were reported earlier and the formation of silver nanoparticles was in the range 2–4 keV using Artemisia nilagirica leaf and Artocarpus heterophyllus seed extracts by Jagtap and Bapat (2013) and Vijaykumar et al. (2013).
TEM analysis of AgNPs
The shape and size of the resultant particles were elucidated with the help of TEM (Fig. 9). Aliquots of Ag nanoparticle solution were placed on a carbon-coated copper grid and allowed to dry under ambient conditions and TEM image were recorded. The TEM micrographs suggest that the sizes of the particles were around 50 nm. The particles were of spherical shape. The size measured by TEM analysis was lower than that measured by DLS analysis.
DLS and zeta potential
The DLS size distribution image of biosynthesized silver nanoparticles is shown in Fig. 10a. It is observed that the size distribution of AgNPs ranges from 10 to 150 nm. The calculated average particle size distribution of AgNPs is 73.14 nm. The broad spectrum of DLS analyzer confirms that the particle size is decreased when compared with the sharp SPR peak (424 nm) obtained in the UV–Vis spectra. In earlier reports, the average diameters of the particles were 53.2 nm. The zeta potential of the biosynthesized AgNPs was found as a sharp peak at −7.66 mV (Fig. 10b). It is suggested that the surface of the nanoparticles is negatively charged and dispersed in the medium. The negative value confirms the repulsion among the particles and proves that they are very stable.
Antimicrobial activity
In the present investigation, the antibacterial effect of prepared silver nanoparticles is studied on different types of bacteria such as E. coli, K. pneumoniae, P. aeruginosa (Gram negative) M. flavus, B. subtilis, B. pumilus and S. aureus (Gram positive). The antibacterial activities of three different concentrations of AgNPs with seven microorganisms were studied. The zone of incubation around AgNPs individual bacterial culture is shown in Fig. 11. The numerical value of the inhibition zone and the control antibiotic ofloxacin are given in Table 2. Bankar et al. (2010) reported the antibacterial activity of AgNPs using E. coli, E. aerogenes, Klebsiella sp. and Shigella spp. In the present study, the synthesised AgNPs had the highest antibacterial activity against E. coli and B. subtilis, respectively. Lesser antibacterial activity of AgNPs is observed against K. pneumoniae, M. flavus, P. aeruginosa, B. pumilus and S. aureus, while increasing (5, 10, 15 µl/ml) the concentration of Ag nanoparticles. These bacterial group incubations around the wall are due to the release of diffusible inhibitory compounds from silver nanoparticles. These biosynthesized nanoparticles are widely used in cancer therapy, wound healing, antimicrobial activity, water paints, cotton fabrics and textiles, etc. The green synthesis of AgNPs has also paved a better methodological approach in the medical field.
Table 2 Antibacterial activity of the silver nanoparticles