**2. Results**

#### *2.1. Characterization of Peel Extract, Silver Nanoparticles and Formulations*

The pH and the dry matter of the peel extract obtained by maceration followed by percolation were 3.13 and 86.39 ( ±0.96) % *w/w*, and the total phenolics expressed in gallic acid and the ellagic acid were 392.0 ( ±9) and 3.64 ( ±0.03) mg/g, respectively.

The formation of silver nanoparticles was confirmed by comparing the XRD patterns and the corresponding standard patterns of cubic of silver nanoparticles (Figure 1), according to the diffraction standard (JCPDS file No. 04-0783). The reflection peak (2 2 2) is characteristic of the substrate (Si), where silver particles were deposited as a thin film. TEM images (Figure 2) showed different forms and sizes of silver nanoparticles produced by green and conventional chemical routes as well as in their respective formulations. In general, green-synthesis produced particles with a larger size than those obtained by conventional synthesis. Dynamic Laser Scanning (DLS) analyses of the formulations prepared with green or conventional silver nanoparticles demonstrated different particle sizes, being the mean values of 89 ± 21 and 19 ± 4 nm for the green and conventional formulation, respectively. The values of zeta potential of green and conventional silver nanoparticles were lower than −30 mV (−46.2 ± 6.06 mV green, and −67.5 ± 3.69 mV conventional), indicating the stability of both colloidal silver nanoparticles.

**Figure 1.** X-ray diffraction (XRD) of the green and chemical silver nanoparticles.

**Figure 2.** Images of transmission electron microscopy (TEM): (**A**) Green silver nanoparticles; (**B**) Silver nanoparticles green formulation; (**C**) Chemical silver nanoparticles; (**D**) Silver nanoparticles chemical formulation.

Almost 100% of the Ag+ ions coming from AgNO3 were reduced by the pomegranate peel extract (99.89%) and sodium citrate (99.51%). However, in the spray formulation containing chemical-silver nanoparticles, the percentage of reduction was diminished to 68.18% although the formulation maintained stable regarding Ag+ ions concentration for 28 days (Table 1). Zeta potential data confirmed the stability of the spray formulations regardless of the method used to obtain the silver nanoparticles (Table 2). The total phenolics in the spray formulations with or without silver nanoparticles were quantified at 0, 7, 14, and 28 days after having been prepared (Figure 3), and it has been significantly reduced in the green-synthesized silver nanoparticle formulation after 14 days with values ranging from 0.405 to 0.295 mg/g.


**Table 1.** Values of the silver ionic reduction and zeta potential for green and chemical silver nanoparticle formulations in different periods.

**Table 2.** Silver ion concentration (μgAg+/mL) and percentage of silver ions reduction after the reactions, AgNP percentage, and values of minimum inhibitory concentration (MIC) of silver nanoparticles and pomegranate peel extract found for *Staphylococcus aureus and Candida albicans.*


\* Control = Carboxymethylcellulose, propylene glycol, silver nitrate.

**Figure 3.** Total phenolics concentration for the silver nanoparticles green formulation and pomegranate peel extract formulation in different periods. Different capital letters denote significant difference (*p* < 0.05; one-way ANOVA followed by Tukey's multiple comparison test) among the groups.

## *2.2. Antimicrobial Activity*

The antimicrobial activity expressed as MIC values of silver nanoparticles and pomegranate peel extract (μg/mL) (Table 1) was, in general, considerably lower for the spray formulations than the active inputs regardless of the microorganisms tested. MIC values against *C. albicans* for active inputs and spray formulations were 781 and 0.18 for the peel extract, 68.75 and 16.87 for the green-, and 0.25 and 1.12 for the chemical-silver nanoparticles. While for *S. aureus*, the values were 391 and 0.37, 67.5, and 0.26, and 0.5 and 0.56 for pomegranate peel extract, green-, and chemical-silver nanoparticles in the active inputs and spray formulations, respectively. In addition, different conditions of humidity and temperature did not affect the effectiveness of the spray formulations against both microorganisms.
