Evaluation of Green Synthesis of Ag Nanoparticles Using Eruca sativa and Spinacia oleracea Leaf Extracts and Their Antimicrobial Activity

1. Background

Nano-biotechnology is considered as one of the mainly vigorous field of research in new material science. Recently, biosynthetic methods employing both biological microorganisms such as bacteria (1) and fungus (2) or plants extract (2, 3), have developed speedily as a trouble-free and feasible choice to obtain nanomaterials alternative to more complex chemical synthetic procedures. The particular distinctiveness like size, allocation and shape give the nanoparticles different properties from the bulk material (4).

Current nanotechnology developments have led to nanomedicine, a new field which includes many diagnostic and therapeutic applications involving nanomaterials and nanodevices (5). Synthesis of nanoparticles using plant extract supplies progression more than chemical and physical method as it is cost helpful, environment safety, simply scaled up for great range production and in this process there is no requirement to use high pressure, power, temperature and poisonous chemicals (6).

The green production of AgNPs requires three main processes, which have to be checked based on green chemistry phenology, include (1) the assortment of solvent medium, (2) the choice of environmentally kind reducing agent, and (3) the choice of safe substances for the steadiness of AgNPs.

Bio reduction of silver and gold ions to yield metal nanoparticles using plant extract (3, 7), Geranium leaf broth (2), Neem leaf broth (8) lemongrass extract (9), Tamarind leaf extract (10) and Aloe vera plant extracts (3) have been reported. Shankar et al. (8) reported on the synthesis of clean spiky nanoparticles of silver and gold by the reduction of Ag⁺ and Au³⁺ ions by Neem (Azadirachta indica) leaf broth. Most of the reported green synthesis methods using plants took more than 1 hour for the formation of colloidal silver (11, 12).

Nanobiotechnology has enhanced the production of minor AgNPs with little toxic effect to human and more effectiveness alongside bacteria (13-16). Furthermore, nanoparticles are alternative to antibiotics viewing better action against multidrug opposing bacteria and consequently, plant derived nanoparticles proved better to other methods (17-19). The method of the AgNPs antibacterial action is efficiently explained in conditions of their interaction with cell membranes of bacteria by troubling its permeability and respiratory role (20, 21).

2. Objectives

In this study, we used aqueous plant extract of Eruca sativa and Spinacia oleracea leaves for the creation of silver nanoparticles and study their antiseptic activity against Streptococcus pneumoniae and Pseudomonas aeruginosa.

3. Materials and Methods

3.1. Plant Material and Synthesis of the Extract

Fresh leaves of (Eruca sativa and Spinacia oleracea) were used to make the extract. 25 g of fresh green leaves were systematically washed with distilled water followed by double distilled water to take away the dust particles and other pollutants. Then the plant substance was chopped into fine slices and taken in a clean 250 mL Erlenmeyer conical flask and 100 mL of germ-free double distilled water was added and incubated on a sand bath at 60 ºC for 30 min to facilitate the formation of aqueous leaf extract. The extract was then filtered using Whitman No. 1 filter paper. The plant extract is used for the synthesis of AgNPs and the extract can be stored at 4 ºC for further use.

3.2. Preparation of 1 mM Silver Nitrate Solution

For the preparation of 1 mM Silver nitrate (AgNO₃) 0.02 g of AgNO₃ was added to 100 mL of double distilled water. The solution was mixed thoroughly and stored in an amber colored bottle in order to prevent auto oxidation of silver.

3.3. Synthesis of Silver Nano Particles

For the production of 5% plant mediated AgNPs; 5 mL of plant extract was added to 95 mL of 1 mM silver nitrate solution and incubated on a sand bath at 60 ºC for 30 min after that the color change was observed. This indicates the preliminary confirmation for the formation of AgNPs. The brown color formation indicates that the AgNPs were synthesized from the herbs extract and they were centrifuged at 5000 rpm (Hettich EBA20S Portable Centrifuge) for 10 min in order to obtain the pellet which is used for further study.

3.4. Transmission Electron Microscope (TEM)

TEM measurements and photographs were carried out on a JEOL-TEM 1200 EX II Transmission Electron Microscope in the Faculty of science in Alexandria University (Alexandria, Egypt). The sample was dried in order to take the photograph.

3.5. Energy-Dispersive X-Ray Spectrometer (EDX) Analysis

AgNPs were cut-off by centrifuging 20 mL of solution in water including AgNPs for 10 min at 15,000 rpm (Hettich EBA20S Portable Centrifuge). The pellets were obtained and dehydrated in oven at 50 ^ºC to get rid of water. The AgNPs obtained in the powder and was used for EDX investigation. To perform EDX investigation, the leaf extract AgNPs were dehydrated and fall covered on to carbon layer. EDX investigation was then done using electron microscope (SEM) set with EDX.

EDX can be used to confirm the composition and distribution of the nanoparticles through spectrum and elemental mapping by using an EDX spectrometer incorporated into a scanning electron microscopy (SEM) system.

3.6. UV-Visual Observation

UV-Vis absorption spectra were measured using LKB spectrophotometer.

3.7. Microorganisms

The evaluation of antibacterial action was done using various strains. The subsequent microorganisms were used: Streptococcus pneumoniae (Thermo Fisher Scientific, AS Polyvalent 2 R671260, Waltham; USA) and Pseudomonas aeruginosa (Thermo Fisher Scientific, Set R670372, Waltham; USA). The microbial cultures were maintained by the Department of Botany and Microbiology, College of Science, King Saud University, Riyadh, Saudi Arabia.

3.8. Antibacterial Activity Study

Antiseptic action of the synthesized AgNPs was performed using the agar well diffusion examine process (22). In this technique, disinfected Mueller-Hinton Agar plates were arranged. Pathogenic bacteria used in the current research were widening above the agar plates by sterile cotton wipe down. The plates were allowed to dry and a sterile well-cutter of diameter 5.0 mm was used to bore wells in the agar plates. Subsequently, a 50 µL of the synthesized nanoparticle suspension (mass concentration = 0.02 µg.µL^-1) was introduced into wells of the inoculated Mueller-Hinton Agar plates. Another two concentrations 10 and 25 µL of the synthesized Ag-nanoparticles were used and introduced into wells of the Agar. The plates containing the bacterial and AgNPs were stand for 1 hour to allow diffusion to take place and then incubated at 37 ºC for 24 hours, and then observed for indication of zones of inhibition, which show as a clear region around the wells (23). The length of such inhibition zone was calculated using a metre ruler, and the significant value for each type of bacteria was documented and spoken in millimeters.

4. Results

When plant leaf extract was uniformed in aqueous solution of 1 mM AgNO3, the reduction of Ag was measured by UV-Vis spectrum of the process. The reduction of AgNO₃ into AgNPs during contact to plant extracts is followed by a slow raise in color progress from clear yellow to brown (Figure 1). The absorption spectra of AgNPs formed, shows the creation of AgNPs with almost 100 % plant reduction of Ag ions as supported by qualitative testing of supernatant after the decontamination of silver nanoparticles by heat (Figure 1).

SEM examination was approved out to recognize the topology, surface morphology and the dimension of the AgNPs, which viewed the production of higher density poly dispersed round AgNPs of different sizes that varied between 4.97-14.4 nm for Eruca sativa and from 2.31-16.07 nm for Spinacia oleracea. The majority of the AgNPs collected and only some of them were spread, as examined under SEM (Figure 2A and B). From the images, the nanoparticles are appearing to be aggregated and the surface of the aggregates is rough. Scanning electron micrograph (SEM) was equipped with energy dispersive spectroscopy (EDX). The presence of silver was confirmed from the Ag peak obtained from the EDX spectrum as shown in Figure 2C and D.

Examination of AgNPs by Energy dispersive X-ray (EDX) spectrometer established the existence of elemental indication of the Ag and homogenous distribution of AgNPs (Figure 2C and D). The pointed sign peak of Ag powerfully established the reduction of AgNO₃ to AgNPs. The upright axis expresses the number of X-ray counts while the parallel axis shows energy in KeV. Detection lines for the main release energy for Ag were clarified and these communicate with peaks in the spectrum, thus giving affirmation that Ag has been properly recognized and present in the solution. The appearance of some other minute signals may be due to the thin film made on the glass slide taken for the EDX.

The X-ray diffraction patterns (XRD) of the formed AgNPs formed by the leaf extract of both Eruca sativa and Spinacia oleracea were additionally established by the distinguished peaks examined in the XRD image (Figure 3). The XRD pattern showed four intense peaks (27.94 ^º, 32.36 ^º, 46.36 ^º and 56.28 ^º) in the full spectrum of 2Ө value between from 20^º to 70^º.

Transmission electron micrographs give the closed view of spherical Ag nanoparticle and indicating that they are also spherical (Figure 4). AgNPs were not well estranged from each other in the nanotriangles in the extent range 45-60 nm are covered with minor particles due to the occurrence of small crystal and hexagonal particles of about 10-25 nm in width on the triangular face.

The antibacterial activity for silver nanoparticles was done with gram positive bacterial strains like Streptococcus pneumonia and gram negative bacterial strains such as Pseudomonas aeruginosa. The inhibition zone caused by 50 µL of synthesized silver nanoparticles in Eruca sativa leaves extract was reached to about 2.5 cm for gram positive strains Streptococcus pneumonia and 2.8 cm for Eruca sativa and 3.2 cm for Spinacia oleracea. On the other side a less effect of Ag NPS on negative strains Pseudomonas aeruginosa was observed Figure 5. Lower antibacterial activity was observed for both 10 and 25 µL for gram positive and gram negative bacteria for both plants (Data not shown).

Acknowledgments

The authors extend their appreciation to the Deanship of Scientific Research at King Saud University for funding this work through research group no. RGP-VPP 297.

Authors’ Contribution:

All authors have participated in the manuscript preparation.

Financial Disclosure:

There is no conflict of interest.

Funding/Support:

The study is self-funded.

5. Discussion

It is well-known that silver nanoparticles exhibit yellowish brown color in aqueous solution (19). The out looking of the organized samples shows that considerable alter of the color of Eruca sativa and Spinacia oleracea leaves extracts. This examination was extra established by with UV-Vis spectrophotometer as shown in Figure 1. According to our results, it could be confirmed that Eruca sativa and Spinacia oleracea leaves extracts were originate to display the reducing potential in terms of production rate and change to silver nanoparticles.

A little absorption band at 435 nm starts showing in the absorption spectra of the produced sample (0.1 mL sample). This band grew and blue changed from 435 to 495 nm with increasing time. This band related to the absorption by AgNPs in the visible area (380-450 nm) due to the occurrence of exterior Plasmon sensations (24). The raise of the peaks strength shows that the absorption of AgNPs was promoted (16). The symmetric and thin absorption peak indicates the fine size allocation of the AgNPs. The peak change of greatest absorption wavelength clarifies that the dimension of AgNPs decreases with increasing time. Our consideration here that, with prolonged time, the rate of nuclei impulsive rises and a high number of nuclei are created throughout the nucleation rupture. Thus, the amount of ending particles rises, and the variance of particle dimension therefore lowers. This examination obviously indicates the occurrence of reduction of AgNPs using Eruca sativa and Spinacia oleracea leaves extracts.

The characterization of AgNPs by EDX outline evidenced physically powerful signals for Ag atoms as shown in Figure 2. The EDX pattern clearly shows that the Ag nanoparticles are crystalline in nature, which is caused by the reduction of silver ions using Eruca sativa and Spinacia oleracea leaf extracts.

The X-ray diffraction (XRD) has confirmed to be an important study means to show the creation of AgNPs, forming the crystal formation of the as-prepared AgNPs and to estimate the crystalline particle dimension.

The antibacterial activity of biologically synthesized silver nanoparticles from leaf extracts of both Eruca sativa and Spinacia oleracea plants was evaluated against S. pneumonia and S. aeruginosa, showing more effective bactericidal activity in opposition to Gram-negative bacteria than gram-positive one. It could be suggested that AgNPs showed effective antibacterial properties owing to their exceptionally big exterior region, which provides superior contact with microorganisms and its interactions with bacteria are and localized on the membrane of the organism. Our results are consistent with Shrivastava et al., (25) who reported that the silver nanoparticles have an antimicrobial effect on S. aureus and E. coli. Similarly, Kim et al. (26) proved bactericidal activities of AgNPs in opposition to E. coli and S. aureus. They suggested that the cause was quantity needy and was more prominent in opposition to Gram-negative organisms than Gram-positive ones.