Document Type : Original Research Article
1 Department of Medical Nanotechnology, School of Advanced Medical Sciences and Technologies, Shiraz University of Medical Sciences, Shiraz, Iran
2 Social Security Organization,shahid beheshti hospital,shiraz,Iran
3 Department of Basic Science, Payame Noor University, Iran
4 Department of Chemistry and Nanochemistry, Faculty of Sciences & Modern Technologies
5 Department of Medical Microbiology, School of Medicine, Shahroud University of Medical Sciences, Shahroud, Iran
6 Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
7 Department of Material Engineering, Shiraz University of Technology, Shiraz, Iran
8 Department of Medical Nanotechnology, School of Medicine, Shahroud University of Medical Sciences, Shahroud, Iran
Silver nanoparticles considered the attention of researchers because of their electrical, opt-ical, thermal and biological properties [1, 2]. These properties cause that they be suitable for applications in the fields such as drug delivery, sensing, antibacterial and catalysis [3-11].
It has been reported that the excessive usage of antibiotics to eradicate the bacteria resulted in the development of resistance to various antibiotics as well as the spread of infectious diseases [12-14]. Therefore, the find of new antibacterial agents is essential for the prevention of bactericidal growth. Silver nanoparticles have significant antibacterial activity, due to having small size and the surface: volume ratios of these particles. Therefore, owing to high antibacterial properties of these nanoparticles, they could be employed for the increase of the safety in the manufacturing such as food packaging [15, 16].
These nanoparticles prepare thought several physical and chemical methods such as gamma irradiation , electrical irradiation, thermal decomposition, and sol-gel. But these methods require to long-term experiments, a large space, use of toxic chemicals, high energy and expensive[21, 22] . So, synthesis of nanoparticles using microorganisms[23-26], enzymes, and plant extracts is best choose, due to advantageous such as the short-term experiments, cost-effective, easily, available, and safe. Leaves, barks, stems, seeds, flowers and roots are parts of plant which can be used as reducing and stabilizing agents for the construction of nanomaterials[29-33]. The compounds like terpenoids, flavonoids, phenols , saponins, polysaccharide , quinones  present in the plants cause the reduction of metal ions.
Euphorbia Pseudocactus Berger (Euphorbiaceae) Euphorbia pseudocactus Berger (candelabra spurge) is a multibranched, dwarf-stemmed, candelabra-shaped, succulent herb, 60–120 cm tall. The stems often have distinctive yellow V-shaped markings. It is originating in the subtropical coast of South Africa. It grows in thorny bush-lands and savannah often forming colonies.
some species of Euphorbia are useful for the treatment of boils, cuts, and wounds. It is useful for cardiovascular complaints, asthma, cough, and spleen disorders. Certain Euphorbia species have been reported to possess cytotoxic[43-47], antimicrobial[48-52],larvicidal, insecticidal, anti-inflammatory, hepatoprotective, and antioxidant activities [54-56]. The diterpenoid ingredients, particularly those with tigliane, ingenane, and abietane skeletons, are believed to be the major bioactive and toxic agents  .So, in this paper, we study the synthesis of silver nanoparticles by using Euphorbia Pseudocactus Berger (Euphorbiaceae) extract and its antibacterial property.
Materials and method
Fresh leaves of Euphorbia Pseudocactus Berger (Euphorbiaceae) were gathered from Jam, Bushehr, Iran. Silver nitrate (AgNO3) was procured from Merck. The gathered herb was cleansed by using distilled water, and was dried at ambient temperature. Then they were powdered and stored for done experimental.
Fresh Euphorbia Pseudocactus Berger (Euphorbiaceae) extract was preferred to reduce aqueous Ag+ solution to Ag NPs. So, 5g powder plant was refluxed in 50 ml distillated water under stirred extremely for 2h. The resulting solution gradually cooled down at 25°C and then filtered using Whatman filter paper No. 1. Fresh extracts were employed for the preparation of Ag NPs.
Synthesis of Ag NPs
Typically, 20 ml fresh filtered extract was interacted with 5 ml aqueous 0.01 mM AgNO3 under reflux by magnetic stirred. After interaction for 30 min, color of suspension was converted from light-yellow to brown which this change illustrated formation of Ag NPs. The brown Ag suspension was cooled at room temperature, and it was identified by using several techniques (Fig. 2).
Characterization of Ag NPs
UV-Vis spectroscopy (UV-Vis) was performed through Varian Cary 50 UV–vis spectrophotometer. The X-ray diffraction (XRD) of samples was done on Holland-Philips X-ray powder diffractometer using Cu Ka radiation (= 0.1542 nm). TEM of synthesized nanoparticles was performed using Transmission Electron Microscopy model of CM30 3000Kv. FT-IR spectra was recorded through Bruker VERTEX 80 v model.
Microorganisms and growth conditions
The antimicrobial activity of the prepared Ag NPs has tested through standard micro dilution technique-reveals the minimum inhibitory concentration (MIC), which MIC value is the lowest concentration of Ag NPs to inhibit bacterial growth up to 99%. To investigate of antibacterial effect of prepared Ag NPs was used several type of Gram-negative and Gram-positive bacteria. Gram-negative bacteria involved Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27852) and Gram-positive bacteria including Staphylococcus aureus (ATCC 25923) and Enterococcus faecalis (ATCC 27852). Cultivating bacteria was carried out on Mueller-Hinton agar (MHA) at 37 ˚C for 18-24 h.
Antibacterial activity of synthesized nanoparticles was studied on standard species of bacteria including both gram-positive and gram-negative bacteria of S. aureus, E. coli, E. faecalis, P. aeruginosa by micro dilution broth method (M27-A3) documented by CLSI. In brief, for designation of antibacterial activity, serial dilutions of synthesized nanoparticles (0.5-128 μg/mL) were prepared in a 96-well micro-titer plate using Müller-Hinton broth (MHB, EMD Millipore) medium (pH=7 ± 0.1; 25°C) based on the M07-A10 protocol. Stock inoculums ready using transferring some pure colonies in 5 mL sterile DW and adjust the turbidity of the inoculums to 0.5McFarland standard at 530 nm wavelengths which are equal to 1–1.5 × 108 cells/mL for bacteria. Working suspension was prepared by production a 1/100 dilution with Müller-Hinton broth to add wells. The one column of 96-well microplate was filled with 200 µL of MHB media culture as positive control. The other column was filled with 200 µL of bacterial suspension as negative control. The two other columns were filled with 100 µL inoculum and 100 µL of nanoparticles dilution sequentially. The growth of bacteria treated with nanoparticles was compared with those grown in the control group. The values of MIC were defined by the lowest concentrations of the reduction in the bacterial growth in comparison with the growth in the control group. The experiments were performed in triplicate.
In this work, both molecular dynamics (MD) and monte carlo (MC) simulations were done by Materials Studio v22.214.171.124 to simulate the XRD and UV-vis, respectively. To geometry optimization, universal force field, the atom based van der Waals, and Ewald Electrostatic were performed. The simulated structures of Ag is face centered cubic (FCC) with lattice parameters of 4.08oA as shown in Fig. 3. UV-vis and XRD simulation can be evaluated using VAMP and diffraction module, respectively. Also, XRD pattern of Ag simulated investigate based on freely equilibrated conﬁgurations by Cu and Kα radiation (λ= 1.54 Å).
RESULTS AND DISCUSSION
UV-Vis Spectroscopy a quantitative technique applied to measure the optical absorption spectra of metal nanoparticles. Absorption spectrum shows that maximum plasmon resonance absorption of simulation result and biosynthesized Ag NPs is in region of 447 and 426 nm, respectively, which confirmed formation of of Euphorbia Pseudocactus Berger (Euphorbiaceae) (Fig. 4).
X-ray diffraction pattern (XRD) of biosynthesized Ag NPs using Euphorbia Pseudocactus Berger (Euphorbiaceae) presents in Fig. 5a. All of the peaks with Miller indices of (111), (200), (220) and (311) can be indexed to the face centered cubic (FCC) structure for biosynthesized nanoparticles (JCPDS, 04–0783). The crystalline size of Ag NPs evaluated using Scherrer’s formula (D=0.9*λ/βcosθ; where λ is X-ray wavelength, β is full width at half the maximum (FWHM) and θ is Braggs’ angle) in about 42 nm . Some extra peaks in XRD pattern related to bioorganic phases residue and impurities such as AgCl on surface of nanoparticles. Simulated XRD pattern in molecular dynamics simulation for Ag is present in Fig. 5b. The peaks at 2θ=38.1°, 44.3°, 64.4°, and 77.4° are close to experimental measurement.
Transmission Electron Microscopy (TEM) is a scientific instrument that applies a beam of highly energetic electrons to evaluate the morphology, particle size, and size distribution of nanoparticles. TEM micrograph of biosynthesized Ag NPs using Euphorbia Pseudocactus Berger (Euphorbiaceae) extract shown in Fig. 6a. It clearly indicates that the biosynthesized Ag NPs are spherical shape and well dispersed. The histograms of particle distribution present in Fig. 6b. The biosynthesized Ag NPs have size range from 5.48-60 nm with average diameter of about 9.16 -20.31 nm.
FT-IR spectral analysis of biosynthesized Ag NPs shows in Fig. 7. The main goal of FT-IR analysis is to detect the organic functional groups present in plant extract. The spectrum shows stretching vibrations as 3500 cm-1 (N-H, amide), 3472 -3413 cm-1 (O-H, alcohol and phenol), 3231cm-1 (O-H, carboxylic acid), 2923-2358 cm-1 (C-H, methyl, methylene, and methoxy), 1640 cm-1 and 1618 cm-1 (C=O, carboxylic acid), 1383cm-1(C-H, alkanes), 1111 cm-1 (C-OH, disaccharides), and 620 cm-1 (aromatic ring, carbohydrates).
Thus, FT-IR spectral showed that compounds and functional groups such as: amide, alcohol, phenol, carboxylic acid, methyl, methylene, methoxy, carboxylic acid, alkanes, disaccharides, aromatic ring and carbohydrates play significant roles in the, reduction, stabilization, and formation of these silver nanoparticles.
In fact, these functional groups diminish the stability of silver ions and subsequently their production yields.
Antibacterial effect of biosynthesized Ag NPs survey on gram positive and gram negative bacteria. The MIC results of biosynthesized Ag NPs on E. faecalis and S. aureus obtained 8 and 4 μg/mL and P. aeruginosa and E. coli obtained 16 and 4 μg/mL (Table 4 ). Synthesized nanoparticles have a significant antibacterial effect compared to the extract. The MBC test of Ag NPs showed that there was no result observed for testing all bacteria.
The antibacterial effect of biosynthesized Ag NPs is related to cell wall structure in gram-negative and gram-positive bacteria . The sulfur and phosphor atoms present in cell well of bacteria. The silver tends to interact these atoms, so silver can kill bacteria by reacting with the cell wall of bacteria. Gram positive bacteria contain rigid polysaccharide in itself cell well, which makes it difficult for silver to penetrate the walls of these bacteria. Hence, inhibitory activity of Ag NPs is stronger in gram-negative than gram-positive bacteria .
Silver nanoparticles were synthesized using Euphorbia Pseudocactus Berger (Euphorbiaceae) extract. existing Biomolecules in the plant extract act as fast bioreduction of silver ions during the formation nanoparticles. The average size of biosynthesized Ag NPs was determined 5.48-60 nm with an average diameter of about 9.16 -20.31 nm. Antibacterial results show good effect of biosynthesized nanoparticles on gram positive and gram negative bacteria. Moreover, the simulation results for XRD and UV-vis are in good agreement with experimental measurements. So, biosynthesized nanoparticles can be utilized as an antibacterial agent in medical and industrial devices and tools.
The authors kindly thank to Shiraz University of Medical Sciences for financial support of the study [grant No. 97–01-74–17873].
CONFLICT OF INTEREST
The authors declare there is no any conflict of interest.