Characterization and Antibacterial Activity of Silver Nanoparticles Biosynthesized Using Leaves Extract of Artemisia sieberi and Calotropis procera

The prevalence of antibiotic-resistant bacteria has increased recently leading to the need for novel, natural antibacterial agents such as plant-synthesized silver nanoparticles. Such synthesis is safe, cheap, rapid, non-toxic and environmentally friendly. In this study, characterization of biosynthesized silver nanoparticles from extracts of A. sieberi and C. procera was carried out using transmission electron microscopy, fourier transform infrared and energy dispersive x-ray analysis. Spherical nanoparticles with an average size was ~10 nm for A. sieberi and ~14 nm for C. procera were synthesised; synthesis was most effective using A. sieberi. Antibacterial activity of silver nanoparticles was carried out using the agar-diffusion method and by determination of the minimum inhibitory concentration. Biosynthesized silver nanoparticles showed antibacterial activity against Staphylococcus aureus, MRSA, Salmonella typhimurium and Escherichia coli, with silver nanoparticles extracts from A. sieberi being the most antibacterial.


1.Introduction
In recent years, the emergence of antibiotic-resistant bacteria has become a serious public health concern [1,2]. Antimicrobial agents are important in reducing the prevalence of multidrug resistant (MDR) strains in pathogenic bacteria [3]. Many medicinal plants are known to be a sources of natural antimicrobial compounds and can provide alternatives to antibiotics [4]. The synthesis of nanoparticles, particularly silver nanoparticles (AgNPs), has increased due to their varied application as antimicrobial agents and in, for example, biomedicine, optics and catalysis [5][6][7][8][9].

Materials
AgNPs were synthesized using A. sieberi and C. procera obtained from Riyadh, Saudi Arabia as described in our previous study [43].

Method 2.2.1. Characterization of AgNPs
Characterization of AgNPs synthesized using A. sieberi and C. procer, including UV-visible spectroscopy and scanning electron microscopy was conducted as described in our previous study [43]. Transmission electron microscopy (TEM) Analysis was used to observe the morphology and size of silver nanoparticles using a TEM, JEOL microscope JEM-1011). Fourier transform infrared (FTIR) Analysis was used to determine functional groups of plants extracts and synthesized silver nanoparticles using a Perkin-Elmer 1000 FTIR instrument (Waltham, MA, USA). Energy dispersive Xray (EDX) analysis was done by using Jeol SEM model JSM 6360A (Japan) in order determine the elemental composition of nanoparticles and the morphology.

Agar-diffusion method
The Agar-diffusion Method was used to determine the in vitro antibacterial activity of AgNPs. Concentrations 1.6 and 4 mg/disk were tested against bacteria, which were inoculated onto Muller Hinton Agar (Scharlau Microbiology, Spain) using a swab. Disks containing the selected AgNP concentration were then transferred to the surface of the inoculated media and incubated at 37°C for 24 h; any zones were then measured (mm).

Minimum inhibitory concentration (MIC)
The MIC of AgNPs, was determined using the broth media dilution method using Muller Hinton broth inoculated with 100 μL of the bacterial cultures and incubated for 24 h.at 37°C.

Characterization of AgNPs
The biogenic synthesis of AgNPs was achieved as describe previously [43] and confirmed by UVvis spectroscopy and scanning electron microscopy. Here, we also include characterization of the A.sieberi and C.procera extracts with FTIR, EDX and TEM.

FTIR spectra
FTIR spectra were conducted to characterize the synthesized AgNPs and to determine the presence of functional groups responsible to the biocomponents in extracts of A. sieberi and C. procera used in the preparation of AgNPs. FTIR spectra of A. sieberi extract and AgNPs produced by A. sieberi exhibited more functional bonds in the plant extract compared to synthesized nanoparticles (Figure 1a,b). A peak was observed at 3410 cm -1 corresponding to the hydroxyl (O-H), while peaks at 2849 to 2917 cm −1 correspond to C-H. The band found at 1629 cm -1 is allocated to C=O of the amide groups and the bands at 1033 cm -1 corresponding to C-O. On the other hand, FTIR spectrum of AgNPs by A. sieberi was observed the disappearance of all the bonds except the bond at 1602 cm -1 corresponding to C=O of the amide groups. (Figure 2 a,b) exhibits the spectrum of C. procera plants and synthesized AgNPs. All the peaks shown are comparably similar to the A.sieberi plant peaks. Similarly, in several previous studies, these peaks were recorded for different plant extracts used in biogenic AgNP synthesis. [10,12,37,40,41].

Figure 1. FTIR spectra of A. sieberi extract (A) and
AgNPs produced using A. sieberi (B) Figure 2A. FTIR spectra of C. procera extract Figure 2B. FTIR spectra of AgNPs produced using C. procera

EDX analysis
EDX was used to observe elemental composition of nanoparticles of AgNPs synthesized using A. sieberi and C. procera extract (Figure 3a,b). The results showed strong signals at ~3 keV in silver (Ag) zone for AgNPs synthesized using A. sieberi and C. procera extracts. This signal at ~3 keV for AgNPs has been previously widely reorted [44][45][46]10,12]. The appearance of signal at ~3 keV was as evidence of the presences of Ag elements in the AgNPs.

TEM analysis
Transmission electron microscopy (TEM) images (Figure 4a,b) indicating the size and morphology of the synthesized nanoparticles. TEM results show the spherical and monodisperse of AgNPs produced by A. sieberi and C. procera extracts. The average diameter of AgNPs was almost 10 nm for A. sieberi and 14 nm for AgNPs produced using the C. procera extract. The diameter of AgNPs produced here using A. sieberi are smaller compared to previous studies, which gave a size of 25 nm for leaf extract of A. vulgaris [37], 17 nm for A.monosperma [40] 22 nm for A.turcomanica leaf extract [41] and 20-90 nm for A. annua L. extract [39]. On the other hand, diameters of AgNPs produced by C. procera extracts were similar to results for C.procera reported by Mohamed et al. [47] and latex of C. gigantea L [48]. https://doi.org /10.37358/Rev.Chim.1949

Antibacterial activity
The Agar-diffusion Method was used to determine the antibacterial activity of AgNPs against some pathogenic bacteria (Table 1). In general, all bacteria were inhibited by AgNPs produced by A. sieberi and C. procera extracts and Gram-positive bacteria showed the most marked effect. Bacterial inhibition was directly related to AgNPs concentration where inhibition zones increase with increasing AgNPs concentration (1.6 to 4 mg). AgNPs synthesized by A. sieberi showed greater antibacterial activity than those synthesized using C. procera. Inhibition zones values (mm) of Staphylococcus aureus and MRSA ranged from 9 to 14 mm for AgNPs synthesized using A. sieberi from 8 to 10 mm for AgNPs synthesized by C. procera. Inhibition zones values (mm) of Salmonella typhimurium and Escherichia coli ranged from 7 to 13 mm for AgNPs synthesized using A. sieberi and 7 to 9 mm for AgNPs synthesized using C. procera. MIC values are shown in Table 2. The results confirmed that bacterial inhibition for AgNPs synthesized using A. sieberi was more marked than those synthesized using C. procera [10,12,13,37,38]. Table 1. Inhibition zone (mm) of AgNPs against some pathogenic bacteria at concentrations of 1.6 and 4 mg Table 2. MIC values of AgNPs against pathogenic bacteria at (mg/L)