Nanomaterials often exhibit properties and behaviors drastically different from their corresponding bulk materials of the same chemical composition. Metallic nanoparticles (NPs) have generated novel materials that exhibit unique optical, catalysis, electrochemistry or biological properties making them attractive for a number of application areas [1–4]. Among all metallic NPs, silver NPs exhibit unique optical and electrical properties at the nanoscale, along with a most valuable enhanced-antibacterial activity. They have received considerable attention owing to their remarkable applications in nanomedicine, food container, anti-bacterial intentions, and material engineering . The Ag-NPs can be prepared through biological methods using microorganisms such as fungus, , bacteria , enzymes , and plant extracts [9–11]. Komal and Kashyap have recently prepared Ag NPs employing plant extracts of Actinidia deliciosa with applications in antimicrobial and detecting Hg2+ heavy metal ions . Rawat’s group also reported a green synthesis of silver NPs by Mulberry leaves extract as a reducing agent as well as a stabilizing agent . In the green synthesis of NPs, the bioreduction of silver nitrate by reducing agents in plant extracts led to the formation of Ag-NPs. Researchers reported that reducing agent in plant extracts such as carbohydrates, reducing sugars, alkaloid, terpenoids, tannin, saponins, phenolics, and flavones compounds are responsible for the reduction of silver nitrate to Ag-NPs [14,15].
As a traditional medicinal plant of the Scrophulariaceae family, Scrophularia striata (S. striata) Boiss is used as a home remedy in Iran and is known by the local name of Tashnedari among the people of Ilam province in Iran. The S. striata have been and still used by natives as a useful remedy for different diseases such as infectious diseases, allergies, wound healing, rheumatism, and chronic inflammatory disorders. Several studies on its biological activities using aqueous extracts of the S. striata showed that S. striata have promising antioxidant, antibacterial, anti-tumor, and anti-inflammatory characteristics [16–18].
While there has been a high level of attention devoted to Ag-NPs, there is still an undeniable need in finding new routes with several improved characteristics like; simplicity, utilizing environment/ user-friendly precursors, cost-effectiveness, the scaling-up potent, with the final products exhibiting promising applications. For this purpose, the present manuscript presents a success in providing a facile way to bio-synthesize Ag-NPs using an aqueous extract of S. striata with a capability of up-scaling. Then, their potential application as an antibacterial agent was examined against several Gram-positive and -negative pathogenic bacteria. Results showed an excellent antibacterial activity even better than conventional antibiotics, nominating the present Ag-NPs as a suitable case for future biological intentions.
MATERIAL AND METHODS
Preparation of plant extracts
The S. straita plants were collected from Ilam province in Iran from August to September 2016. Aerial parts of S. straita were washed with sterile distilled water, dried, and then reduced to a fine powder. After that, a fine dried powder of samples (10 g) was added to 100 mL of deionized water. Then, it was transferred to a 100 ºC water bath and remained at that temperature for 15 min. Finally, the solution was allowed to cool down to room temperature and the extracts were filtered using Whatman No. 2 filter paper.
Biosynthesis of silver NPs
For the green synthesis of Ag-NPs, 10 mL of extract was added to varying concentration of AgNO3 (Merck, 99.0-100.5 %) precursor solutions and the reaction mixture was stirred at 3000 rpm for about 30 min at the room temperature. Bio-reduction of Ag+ ions was monitored by the color change of the solution from colorless to brown along with results of UV-Vis measurements. After 24 h incubation at room temperature, the reaction solution was centrifuged at 10000 rpm for 15 min and the obtained precipitations were then washed with deionized water, followed by 3 times centrifuging. Finally, the obtained powders were placed in an oven at 60 ºC for 24 h and were used for further experiments.
Characterization of synthesized silver NPs
The optical property of Ag-NPs was determined by Cary 300 Bio UV-vis spectrophotometer (VARIAN) in the wavelength range of 275–675 nm. The morphological features of the bio-synthesized NPs were observed by field emission scanning electron microscopy (FE-SEM; Hitachi S-4200) under an acceleration voltage of 15 kV. Transmission electron microscopy (TEM) image was recorded using a Philips CM30 electron microscope. The chemical composition of the silver NPs was studied by an energy dispersive X-ray spectrometer (EDX) instrument (Oxford INCA II energy solid state detector). The FT-IR spectroscopy (Bruker Vertex 70 spectrometer with 0.5 cm−1 resolution) was used to obtain information about the possible interactions during NPs’ preparation. Information about phase variety and grain size of the bio-synthesized Ag-NPs was determined by X-ray diffraction pattern (Philips X'pert diffractometer by Cu-Kα radiation at 40 kV and 30 mA). The size of the crystallite was estimated by using the well-known Scherrer equation as follows;
where D is the approximate crystal size, λ is the wavelength of X-ray radiation used (1.54 Å), θ is the Braggs angle correspond to the most intense peak, and βD is the full width at half maximum of the peak in radians.
Antibacterial activity study
The antibacterial activity of the bio-synthesized silver NPs was evaluated by disk diffusion method, against Gram-negative (Escherichia coli and Salmonella typhi ATCC) and Gram-positive (Staphylococcus aureus and Bacillus cereus) bacteria. Briefly, the Muller–Hinton Agar (MHA-Merck) powder was utilized as a culture medium for bacterial growth. A single colony of each strain with a turbidity of 0.5 McFarland was swabbed uniformly on the individual plates using sterile cotton swabs. Using a micropipette, 100 µL of the Ag-NPs solution was poured into the plate, and after incubation at 37 °C for 24 h the antibacterial activity was measured based on the inhibition zone (clear zone). The diameter of the inhibition zones in the plate was measured carefully, and the mean values were recorded. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were also determined using the microdilution technique.
RESULTS AND DISCUSSION
Physical and chemical characteristics
The XRD pattern of the purified silver NPs (Fig. 1a), clearly determines the FCC structure of metallic silver. The observed diffraction peaks at 2 theta values of 38.18º, 44.2º, 64.5º, and 77.5º can be well-indexed to (111), (200), (220), and (311) miller indices-related planes (standard metallic silver XRD pattern JCPDS data 04-0783), respectively . Using the Scherrer formula, the average crystallite size of the Ag NPs was estimated to be about 15 nm. The obtained broad peaks further indicate that the present bio-synthesized samples are in the nanoscale range. Several unassigned peaks were also found in XRD pattern which can be attributed to the crystallization of bio-organic phases at the surface of NPs .
To identify the effective presence of reducing agent groups, we utilized the FT-IR spectroscopy. As seen in Fig. 1b, and noted in Table 1, the observed bonds indicate the key roles of amine, alcohol, and aldehyde groups in authority to reduce Ag+ ions to Ag0. EDX analysis of a typical synthesized silver NPs shows excellent purity of Ag-NPs (Fig. 1c). However, observation of C, and O species in the obtained spectrum, further determines the presence of different functional groups in active phytoconstituents of the plant extract, at which is in accordance with present FT-IR outcomes and other reports .
Finally, the successful formation of the Ag-NPs was monitored by employing UV-Vis spectroscopy at different concentrations of the metal ion (Fig. 1d). As can be seen, a very broad peak located around 435 nm is a characteristic peak of Ag-NPs , which undergoes a slight red-shift to 445 nm with the increase in metal ion concentration. This absorption peak is attributed to the collective oscillation of electrons known as localized surface plasmon resonance (LSPR) , resulting in the yellow-brown color of silver NPs in different media. On the other hand, based on the Si and Mandal  report, synthesis of metallic NPs is occurred through initial reduction of metal ions to metals and their nucleation, followed by an Ostwald ripening growth step. Therefore, the higher concentration of silver ions would probably lead to larger NPs.
Morphology and particle size analysis on bio-synthesized Ag-NPs were achieved by FE-SEM and TEM measurements. The typical top view FE-SEM images in Fig. 2a, b reveals smooth and spherical morphology of the Ag-NPs with the mean size of about 19.7 nm. As suggested elsewhere, the shape of Ag-NPs is significantly influenced by their optical property and spherical NPs usually show a similar absorption property . On the other hand, the TEM image of that sample (Fig. 2c) confirms the formation of relatively monodisperse and nanometer size particles with average size value of 16± 1.4 nm.
The antibacterial activity of the bio-synthesized Ag-NPs against various pathogenic bacteria (B. cereus, S. aureus, S. typhi ATCC., and E. coli) was performed by disk diffusion method, which was compared with those examined for aqueous plant extract and ciprofloxacin antibiotic. Results have been presented in Fig. 3.
It was indicated that the maximum antibacterial activity was observed against B. cereus followed by S. aureus, S. typhi ATCC, and E. coli with a zone inhibition diameters of 33, 30, 28, and 25 mm, respectively (Table 2). The results indicated that MIC and MBC test have the same trend so that E. coli with lower zone inhibition diameters have higher MIC and MBC values (Table 3). Interestingly, the plant extract did not show any antibacterial activity against different pathogenic bacteria. In all experiments, ciprofloxacin antibiotic was used as a control for better evaluating of the antibacterial activity of present bio-synthesized silver NPs. There were significant differences (p<0.05) between plant extract, ciprofloxacin, and Ag-NPs in all bacteria so that the antibacterial activity of bio-synthesized Ag-NPs against both Gram-positive and Gram-negative bacteria was remarkably greater than that of ciprofloxacin antibiotic and those reported by others (see Table 2).
In general, the as-prepared Ag NPs were found to be more effective on Gram-positive bacteria than Gram-negative ones, which can be attributed to the structural and compositional differences of the cell wall. The mechanism of antibacterial activity of Ag-NPs is still not well known. However, regarding the high degree of scientific attention, several mechanisms have been proposed for the bactericidal effects of Ag-NPs.
1. On one hand, the electrostatic interaction between the positive surface charge of the Ag-NPs and negatively charged cell membrane of bacteria, and on the other hand, the small size of Ag-NPs with high surface energy, cause effective adhesion of Ag-NPs onto the cell membrane. This adhesion leads to subsequent damages on cell wall and membrane such as disruption of the cell wall, alteration of membrane structure and permeability, the formation of pit and leakage of cellular content, and also disturbance in transport activity .
2. After absorption of Ag-NPs to the bacterial membrane, the Ag-NPs can transport across the cell membrane and penetrate into the bacterial cell. Inside the bacterial cell, the Ag-NPs can affect the cell’s macromolecules such as DNA, Proteins, and lipids, which results in negative effects on vital cellular functions, including protein synthesis , sugar metabolism , and cell division .
3. Formation of reactive oxygen species (ROS) and free radicals by the Ag-NPs. The effects of an increase in ROS level in the bacterial cells can be denaturation of protein, hyper-oxidation of lipids and DNA . Therefore, high levels of ROS in a bacterial cell can lead to cell death.
4. Ag-NPs can effect on the signal transduction pathway in the bacterial cell. These signal transduction pathways influence different cellular activities including recombination, DNA replication, cell cycle, and bacterial metabolism. Ag-NPs with modulation of these signaling pathways would inhibit bacterial growth .
Although, the general antibacterial activity of bio-synthesized NPs depends on different parameters such as plant species, plant organ type, precursors and methods used, and concentration of nanomaterials in antibacterial activity assay, however, the results obtained in the present work are remarkably better than the best results reported by other groups. This shows great potential for our cost-effective and scalable bio-synthesized Ag-NPs using S. striata Boiss plant extract, for future biological applications.
A novel, facile and green approach was suggested for bio-synthesis of high-quality Ag-NPs using a very safe, biocompatible and accessible S. striata Boiss plant extract. The synthesized Ag-NPs were relatively spherical in shape exhibiting FCC cubic structure with the average size of ~16 nm and plasmonic-related absorption peak at about 435 nm. The presence of different functional groups in active phytoconstituents of the plant extract has been shown by FT-IR spectroscopy. The antibacterial activity of the as-prepared Ag-NPs was also investigated using the disk diffusion method. Results revealed a strong antimicrobial activity against different Gram-positive and -negative microorganisms (zone of inhibition of 25, 28, 30, and 33 mm for E. coli, S. typhi ATCC, B. cereus, and S. aureus, respectively). It demonstrated better bactericidal property against Gram-positive bacteria compared to Gram-negative bacteria. On the other hand, the bactericidal activities of NPs were better than those of Ciprofloxacin antibiotic and even than other reports. Therefore, the present scalable bio-synthesized Ag-NPs using S. straita plants can be considered as excellent candidates for a wide range of biological intentions.
The authors gratefully acknowledge from Ilam university science research laboratories complex for providing laboratory facilities.
CONFLICT OF INTEREST
The authors declare that there is no conflict of interests regarding the publication of this manuscript.