*4.9. In Vivo Antibacterial Activity*

#### 4.9.1. Animals

We obtained 30 white albino male rats (190–210 g and 8 weeks old) from the animal house at the Faculty of Veterinary Medicine, Cairo University. All rats were kept in an environment with a constant temperature of 25 ± 2 ◦C and a pathogen-free atmosphere with a 12-h light/dark cycle. They were permitted free access to a standard pellet diet in addition to filtered water. The rats were given one week for acclimatization before being utilized in the research. The in vivo experiment performed in the current study followed the standards of the use of the laboratory animals authorized by the Faculty of Pharmacy Research Ethical Committee (Tanta University, Al Gharbiyah, Egypt) with approval number TP/RE/11-21-P-001.

#### 4.9.2. Wound Model

The 30 rats were distributed into three groups, randomly, each with 10 rats. The groups were as follows: the control group (0.9% normal saline), Betadine™ ointment (Mundi pharma as standard drug) group, and AgNP group. The rats were anesthetized using diethyl ether; then a small area was carefully shaved on their backs. After that, full-thickness excisional wounds were created and infected with *S. aureus* bacteria (10<sup>6</sup> CFU/mL). For 7 days, the groups were administered the drugs topically on the surface of the wound [63].

#### 4.9.3. Macroscopic Wound Healing

The day of the wound creation was considered as day 0, and the wound healing process was observed for 7 days starting from day 0. The wound images were taken on days 0, 3, and 7 using a digital camera. Wound areas (cm<sup>2</sup> ) were calculated on days 0, 3, and 7 for evaluation of the healing efficacy using a ruler beside the wounds [64]. The percentage of the wound healing was calculated using the following equation [65]:

$$\text{Percentage of women} = \frac{\text{wound area at day 0} - \text{wound area at nth day}}{\text{wound area at day 0}} \times 100$$

where *n* represents day 3 and day 7.

In addition, on days 3 and 7 post-wounding, wound tissues were excised and homogenized in PBS (10 mL), 10-fold serially diluted in MHB, and plated onto mannitol salt agar plates for CFU quantification after overnight incubation at 37 ◦C [66,67].

#### 4.9.4. Histological Examination

The entire wound was isolated for histological examination at the end of the experiment, with a margin of about 5 mm of the surrounding intact skin. The skin sections were fixed using 10% formalin solution (pH 7.4) overnight. Then, they were processed using a series of alcohol and xylene grades. Following that, the tissues were inserted in paraffin wax at 65 ◦C. The blocks of tissues were cut into sections of 5 µm thickness, stained with hematoxylin and eosin (H&E), and finally viewed using a light microscope [63].

#### *4.10. Statistical Analysis*

All tests were accomplished in triplicate and the obtained results are presented as mean <sup>±</sup> standard deviation (SD). Prism 8® software (GraphPad, Inc., San Diego, CA, USA) was utilized to assess the statistical significance at *p* < 0.05.

#### **5. Conclusions**

According to our results, GTLE includes phenolic compounds. These substances have the ability to form and stabilize AgNPs. In conclusion, the green-synthesized AgNPs by GTLE exhibited good antibacterial activity both in vitro and in vivo against *S. aureus* clinical isolates. They significantly decreased (*p* < 0.05) the membrane integrity of 45.8% of the isolates. In addition, they reduced the membrane potential of 35.42% of isolates

by flow cytometry. AgNPs also resulted in morphological and ultrastructural changes in the tested isolates, as revealed by SEM. Furthermore, they resulted in a significant reduction in efflux activity in 41.67% of the tested isolates. For more in-depth study of the impact of AgNPs on efflux activity, qRT-PCR was utilized to examine the relative gene expression of the efflux pump genes (*nor*A, *nor*B, and *nor*C). The in vivo examination was performed on wounds infected with *S. aureus* bacteria in rats. The group treated with AgNPs was characterized by epidermis regeneration and reduction in the infiltration of the inflammatory cells. Therefore, the green-synthesized AgNPs by GTLE could be a future alternative to chemical compounds and antimicrobials that are currently used to induce healing of wounds, especially infected ones.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/ph15020194/s1, Table S1. The sequences of the primers used in qRT-PCR.

**Author Contributions:** Conceptualization: W.A.N. and E.E.; Data curation, N.G.M.A. and I.A.H.; Formal analysis, N.G.M.A.; Funding acquisition, N.G.M.A.; Investigation, E.E., W.A.N., F.A.M. and O.M.A.-F.; Methodology, E.E., W.A.N., F.A.M., I.A.H. and O.M.A.-F.; Resources, F.A.M. and O.M.A.- F.; Writing—original draft, E.E., W.A.N., F.A.M. and O.M.A.-F.; Writing—review and editing, E.E., W.A.N., F.A.M., I.A.H. and O.M.A.-F. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was funded by Princess Nourah bint Abdulrahman University Researchers Supporting Project number (PNURSP2022R204), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia.

**Institutional Review Board Statement:** This study followed the standards of the use of the laboratory animals authorized by the Faculty of Pharmacy Research Ethical Committee (Tanta University, Egypt) with approval number TP/RE/11-21-P-001.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data is contained within the article and supplementary materials.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

