Fagonia stabilized gold nanoparticles as antimicrobial agents

In this study, gold nanoparticles (GNPs) were synthesized using an aqueous extract of Fagonia , as a stabilizing and reducing agent, applying the green approach. The phytochemicals present in Fagonia extract are responsible for the creation of GNPs. The reaction kinetics of Fagonia stabilized GNPs (FGNPs) was observed through the optical absorption spectra and the absorption maxima occurred at 547 nm. The face-centered cubic (FCC) nature of the GNPs was analyzed by the XRD pattern and average crystallite size (D) was measured about 10 nm. TEM images showed roughly spherical shapes of FGNPs. Evidence of successful formation of FGNPs was revealed by FTIR spectra of pure Fagonia and FGNPs. Fluorescence spectroscopic analysis of FGNPs exhibited a sharp red emission at about 700 nm. TGA technique showed a weight loss of about 19.3% in FGNPs confirming the presence of ligand onto the surface of GNPs. As-synthesized GNPs were investigated for their biomedical application i.e. antimicrobial activities against E. coli and Cocci. The eco-friendly prepared GNPs could play an important role in antimicrobial applications and their visible emission property may suggest the use of such FGNPs as potential biomarkers.


Introduction
Nanotechnology is a propitious arena marketing with the design, consumption and utilization of nanoparticles (NPs) in creating advanced applications [1][2][3]. Nanoparticles have tremendous and unique optical, electromagnetic, and biological features to serve in various fields. Nanoparticles also interpret basic phenomena taking place at the nanoscale range [4]. Metal NPs come up with an obviously prevalent field of implementation [5]. Gold nanoparticles (GNPs) are amongst the utmost substantial metal nanoparticles because of their peculiar physicochemical characteristics and therefore can be implemented in chemical and biological diagnosis and treatments [6].
During the last few decades, an increasing number of current occurrences of diseases owing to microbial infections have been cured by the noble metal NPs, with GNPs having a noticeable antimicrobial performance [7]. It is well documented that silver possesses an enhanced toxicity potential than elements Au and Cu [8,9]. Recently, the cytotoxicity of silver nanoparticles (AgNPs) has been revealed to be associated with their accumulation and penetration in the mitochondrial membrane resulting in the impairment of mitochondrial function [10]. Unfortunately, the strong oxidative activity of AgNPs releases silver ions, which results in several negative effects on biological systems by inducing cytotoxicity, genotoxicity, and even cell death [11,12].
Although many researchers have reported the formation of NPs using silver metal, it is found that gold is more compatible than silver [13]. Gold NPs have chemical inertness and are optically active in biomedicines [14,15], such as in cancer treatment, biomedical imaging and sensing, and also have excellent antioxidant, antifungal, antibacterial activities [16,17]. Gold NPs are highly photostable that is why photoluminescence (PL) in GNPs is not quenched by light like in organic dyes. The PL in gold nanostructures usually results when an excited electron from the 6sp conduction band recombines with a hole in the 5d 10 band. In this perspective, Imura group measured a visible PL band around 650 nm in gold nanostructures [18]. The remarkable PL efficiency and their chemical Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI.
inertness propose the high potential of GNPs as a material for wide biomedical applications. Apart from the shape and size, the surface capping also influences the PL emission and cytotoxicity of GNPs [19].
Nowadays, green chemistry has received great attention from researchers to avoid the use of toxic stabilizing agents like sodium borohydride and sodium citrate that are increasing the health hazard and environmental poisonousness [30]. To eliminate this factor, a harmless green approach is being used which is economically safer and it follows an ecofriendly environmental procedure to construct metal NPs [31]. A green method is also an alternative approach to physical and chemical methods [32]. Among various biomaterials, plant extraction is most popular because of the extensive availability of plants [33]. Metal NPs based on plant extract as reducing and stabilizing agents are found more stable and green [34][35][36]. The role of GNPs as an environmental disinfectant and their safe synthesis are sections that remain to be explored.
In this present article, GNPs were synthesized using green strategy and Fagonia indica aqueous extract was used as a reducing and stabilizing agent together. The as-prepared NPs were characterized by UV-visible spectroscopy, x-ray diffraction (XRD) analysis, Fluorescence spectroscopy, Thermo-gravimetric analysis (TGA), Fourier-transform infrared (FTIR) spectroscopy, and transmission electron microscopy (TEM). The antimicrobial activities of Fagonia stabilized GNPs were also investigated (figure 1).

Chemicals and materials
Sigma Aldrich (99.9% pure) acquired hydrogen tetrachloroaurate trihydrate (HAuCl 4 ·3H 2 O) was used as the precursor of gold. Fagonia indica shrub (in powder form) was collected from Cholistan Institute of Desert Studies (CIDS), The Islamia University of Bahawalpur, Pakistan. The bacterial strains E. coli and Cocci were collected from Civil Hospital Bahawalpur, Pakistan. Deionized water (DI H 2 O) was used as a solvent throughout our experimental work.

Preparation of fagonia extract
Fagonia indica (a medicinal plant) powder is used for extract formation. A 2 g of Fagonia indica (commonly known as Dhaman) powder was added to 100 ml of DI H 2 O. This solution was heated at about 100°C using a hotplate magnetic stirrer for 30 min. The extract was well filtered to remove any residues. The filtrate was stored at 4°C in a glass bottle for further use in the experiments. This freshly prepared Fagonia plant extract was used for the biological reduction and stabilization of GNPs.

Biosynthesis of gold nanoparticles
For the formation of GNPs, 5 ml (0.01 M) aqueous solution of HAuCl 4 ·3H 2 O and 3 ml Fagonia extract were mixed in a glass vial and stirred for 30 min at room temperature. The mixture solution turned to purplishbrown color from pale yellow, indicating the formation of colloidal GNPs. For the separation of GNPs from colloidal solution, the prepared GNPs solution was washed 3-4 times with DI H 2 O while centrifuging at a speed of 6000 rpm at room temperature for 15-20 min, to eliminate the water-soluble biological molecules and secondary metabolites. The concentrated solution of GNPs was dried at room temperature for 48 h and finally crushed to get fine powder for further characterization.

Instrumentation
To confirm the formation and stability of Fagonia stabilized gold nanoparticles (FGNPs), the UV-Vis absorption spectroscopic analysis was done by using Aquarius 7400 model Cecil spectrophotometer having a scale from 290 to 1100 nm. The XRD technique was used to investigate the structural analysis of FGNPs and applied using a Bruker D8 advance x-ray diffractometer in the 2θ range of 20°-80°while operating at 40 keV and 35 mA. A JEOL JEM-1010 transmission electron microscopy (TEM) operated at 100 kV was used to analyze the shape and size of FGNPs. For the purpose, a drop of the solution containing the FGNPs sample was put on an amorphous carboncoated copper grid and kept for 10 min at room temperature in order to stand the film. Any additional solution was removed by means of blotting paper and the grid was dried for the next few minutes. In order to determine the size distributions of FGNPs appearing in TEM images, the ImageJ analyzer was utilized. The Cary Eclipse MY18060003 photoluminescence spectrometer was utilized to measure the fluorescence of pure Fagonia extract and FGNPs in the wavelength range 350-800 nm while the sample was excited at 350 nm wavelength. Thermal gravimetric analysis (TGA) being a very useful technique to determine initial and final destroy temperatures of the NPs and amount of weight loss of the NPs, was run using Indium TGA/DTA 6000 under a Nitrogen gas environment at the flow rate of 50 ml/min. To perform the experiment, a 1.32 mg powder sample was put into a combustion cell and the temperature of the sample was gradually raised from 30°C to 450°C at a constant heating rate of 10°C/min. After the completion of experimentation, a solid piece of gold was residue at the bottom of the combustion cell. To confirm the attachment of Fagonia coating onto the surface of GNPs, a Tensor: 27 (Bruker) FTIR spectrometer was operated, where a few mg of the dried sample was mixed with KBr powder to form a pellet for the analysis. The IR spectra of pure Fagonia powder and FGNPs were examined in the wavelength range 400 to 4000 cm −1 .

Antibacterial performance of fagonia stabilized GNPs
Nutrient agar medium has been utilized to prepare Petri plates for the growth of microorganisms. To sterilize the nutrient agar medium, 20 g of the medium was added in 50 ml DI H 2 O in a conical flask and kept it in an autoclave at 121°C. Subsequently, the medium was poured in sterilized Petri plates placed in the incubator to avoid any contaminations. The bacterial culture obtained from Civil Hospital Bahawalpur was already growing in the incubator at 37°C for 24 h. After 24 h, this bacterial culture was spread on Petri plates followed by the sample disks and the control disks placed on those Petri plates.

Results and discussion
To check the kinetics of FGNPs, the optical spectra of reaction solution were observed at various reaction timings. Reaction kinetics is an important feature in understanding the growth progression of GNPs. In the present work, the absorbance of samples was investigated after every 30 min run of the reaction, and an average peak position occurred at λ max =547 nm. Pure Fagonia extract showed no absorbance at all as shown by an inset in figure 2. When Fagonia extract was added to gold salt and stirred for 30 min a prominent absorption peak was observed, as shown by the pink curve in figure 2. The reaction kinetics of GNPs revealed a very small hyperchromic shift in absorption peak at first (after stirring for 60 min) and then shifted toward longer wavelength with a further increase in growth progression. Additional reaction timing resulted in no countable increase in peak position but caused a little lowering and broadening of the peak position. No more noticeable bathochromic shift in the peak position was observed after 120 min run of the reaction. The actual solution colors are visible by an inset in figure 2.
For structural analysis of FGNPs by XRD technique, figure 3   respectively, based on FCC nature of Gold structure [4,37]. There were no more considerable peaks observed in the XRD pattern, giving confirmation of the purity of the sample. The average crystallite size (D) was measured about 10 nm by using Debye-Scherrer formula and the average inter-planer spacing measured between (111) peaks was about 0.17685 nm. Figure 3(B) showing the SAED pattern, ratifies that the formed nanoparticles are crystalline in nature.
The TEM image in figure 3(C) shows that FGNPs are of roughly spherical shape, including few nanotriangles of a rather large size. It is observed that the prepared FGNPs are well dispersed in the solution. The size of nanoparticles while excluding nanotriangles ranges between 21.8 nm to 60 nm with an average particle size ∼41 nm, which appears bigger as compared to the size calculated in XRD analysis by Debye-Scherrer formula. Figure 3(D) shows the histogram representing the size distribution of nanoparticles.
Apart from the mechanism discussed earlier, green ligands are also considered responsible for contributing to PL emission from GNPs [38] that is associated with metal-ligand radiative transferences, strongly suggesting the influence of nanoparticle surface on its PL emission [19]. Fluorescence spectroscopic analysis was performed for pure Fagonia extract and FGNPs aqueous solution ( figure 4). Pure Fagonia extract showed one broadened emission peak appearing at about 465 nm with an emission band ranging in 387-582 nm. For FGNPs, a sharp red emission peak appears at 700 nm with an emission band in the range of 688-710 nm. This sharp and very clear emission peak suggests that as-synthesized NPs are provided with minimum surface defects that lead to  A comparable antimicrobial performance of as-prepared FGNPs with different concentrations and control groups (Tigecycline and Levofloxacin) against E. coli and Cocci bacterial strains has been inspected. These microorganisms cause various infections in people. The present work shows that antimicrobial performance of as-prepared GNPs is very significant. In this work, after 24 h of incubation, inhibition zones (ZOI) of GNPs disks are measured against different strains of bacteria. ZOI is a region where the growth of further bacteria ends and no bacterial effect is observed in this region. Against E. coli strain, sample A (5 μg) almost showed no activity or zero ZOI, Sample B (10 μg) and sample C (15 μg) showed a considerable ZOI. While sample D (20 μg) showed a significant result with remarkable ZOI, as shown in figure 7. The control element (Tigecycline) also showed better ZOI but less than the ZOI shown by as-prepared GNPs. An analogous trend is observed for another strain of bacteria that is Cocci; the as-prepared GNPs are more effective as compared to the control element that is Levofloxacin, as clear by table 2.
It is well known that the attachment to and transportation into the cell membrane is essential for the NPs to inhibit the growth of bacteria. Following this, reactive oxygen species (ROS) is an important tool to understand  Thus, ROS responsible for the antimicrobial activity of FGNPs is supported by the fact that oxygen significantly affects antimicrobial activity. The oxidation of FGNPs into Au + ions assist their diffusion into the  bio-organism or set oxidative stress to produce ROS that change the permeability of cell membrane, causing DNA, proteins and lipids damage and finally result in cytotoxicity in the cell body of prokaryotes. From this study, we conclude that as-prepared GNPs with high concentration are more effective and less toxic as compared to commercially available control medicines, as shown in figures 7(a) and (b). This experiment was executed to know about the antimicrobial performance of FGNPs in comparison to antibiotics, which was taken as a control group. Thus, FGNPs were found to be effective against different clinical segregates of bacteria (figure 8).

Conclusion
In present work, GNPs were synthesized via biosynthesis method using Fagonia extract as a stabilizer and reducing agent both. Fagonia stabilized GNPs (FGNPs) showed an average absorption at 547 nm while Fagonia extract showed no visible absorption at all. Investigation revealed that the absorption peak showed a small hyper-chromic shift in absorbance with the increase in growth progression. The peaks position also shifted toward higher wavelength showing an overall bathochromic shift. The average crystallite size (D) was measured 10.44 nm by using Debye-Scherrer formula in x-ray diffraction. The TEM images showed that the NPs were roughly spherical in shape except a few triangular-shaped and the sizes of NPs were within 22-60 nm with an average size of 35.8 nm. FTIR spectroscopy of FGNPs indicated the presence of C-X bond (chloride) and C=C (alkenes group) bond. Optical property of both Fagonia extract and FGNPs has been analyzed by using photoluminescence (PL) spectroscopy and exhibited a sharp and defect-free emission peak at around 700 nm, suggesting these NPs very significant as biomarkers for in vivo. Also, two strains of bacteria were used for the assessment of the antimicrobial behavior of FGNPs. Through this assessment, we can say that GNPs have very significant antimicrobial properties.  Studies, IUB) for providing Fagonia indica herbs and Materials Chemistry Laboratory of IUB for providing successful PL spectroscopic results.

Data availability statement
All data that support the findings of this study are included within the article (and any supplementary files).