## 2 Materials and Methods
### Characterization  
The first indication for successful green synthesis of AuNPs using plant aqueous extract is provided by the color change of the solution. The purple color intensity was monitored by measuring the absorbance of the solution at a wavelength varying between 200 and 800 nm using UV-visible spectroscopy (JENWAY 6305 spectrophotometer, Jenway, Bibby Scientific UK, Staffordshire, UK). In this assay, 2 mL of synthesized purple solution was added to a quartz cuvette, and the spectrum was monitored at regular intervals of time to detect the maximum surface plasmon resonance (SPR) [40].  
The various functional groups related to specific molecules or metabolites in plant aqueous extract and their role in the reduction and stabilization of as-formed AuNPs were assessed using Fourier transform infrared (FT-IR) [41]. In this method, 10 mg of synthesized AuNP powder or 3 mL of plant aqueous extract was well-mixed with KBr, left to completely dry, and compressed to form a loaded KBr disk. The spectra were acquired by scanning in the range of 400-4000 cm\({}^{-1}\) using FT-IR spectrophotometry (JASCO FT-IR 4100 spectrometer, Jasco Co., Hachioji, Tokyo, Japan).  
The morphological characteristics (shape and size) of plant-based AuNPs were investigated by transmission electron microscopy (TEM) (JEOL JEM-1010, JEOL, Tokyo, Japan). The synthesized AuNPs were suspended in ultrapure water (Milli-Q water) and submitted to sonification to ensure effective dispersion. A few drops of the suspended solution were loaded on the TEM carbon grid and left to dry before being analyzed by TEM.  
The crystalline nature of as-formed AuNPs was characterized by X-ray diffraction analysis using a PANanalytical-X'Pert-Pro-MRD apparatus (Almelo, the Netherlands) equipped with a Cu K\({}_{\alpha}\) source (\(\lambda\) = 1.5406 A). Operating conditions were set at 40 kV voltage and 30 mA current. Scanning was performed in the range of 2\(\theta\) values of 5-80\({}^{\circ}\). The average value of crystallite size for synthesized AuNPs was calculated using the Debye-Scherrer equation applied to the experimental XRD pattern [42]:  
\[\text{Average crystallite size}=\frac{\text{K}\,\times\,\lambda}{\beta\times\cos\theta} \tag{1}\]  
where K is the Scherrer constant (i.e., 0.94), \(\beta\) is the full width of the diffraction peak at a half maximum, and \(\theta\) is the diffraction angle.  
The size distribution in the colloidal solution was assessed by dynamic light scattering (DLS, Nano-ZS, Malvern Ltd., Malvern, UK). The synthesized AuNPs were dispersed under ultrasonification in high-purity water (Milli-Q H\({}_{2}\)O) to avoid extra peaks or appearance of shadows on peaks during the scattering. The measurement was performed in triplicate at T: 24.6 \({}^{\circ}\)C, pH \(\approx\) 6.5; the refractive index was 1.33, and the loading index was 2.56. Finally, the surface charge of AuNPs was investigated by a zetasizer (Nano-ZS, Malvern, UK) using the Smoluchowski model. Electrokinetic measurements were performed at T: 25 \({}^{\circ}\)C, pH \(\approx\) 6.5 in highly pure water. Twelve zeta runs were operated, and the count rate was 55.2 kcps.