## 3 Results and Discussion
### Antibacterial and Antioxidant Properties  
Since no previous work has been reported yet on _S. ebulus_ extract-loaded ZnO NPs, some antibacterial properties in this study were compared with similar studies but using different natural extracts and commercial/chemical synthesized ZnO [62; 63; 64; 65; 66; 67; 68; 69; 70; 71; 72; 73; 74; 75; 76; 77; 78; 79; 80; 81; 82; 83]. The antibacterial activity of the biosynthesized ZnO NPs using leaf extract and the pure extract was investigated on _B. cereus_, _S. aureus_, and _E. coli_ bacteria (Figure 7a). The biosynthesized ZnO NPs exhibited antibacterial activity over all three bacteria, but was higher for Gram-negative bacteria compared to Gram-positive bacteria. Moreover, the ZnO NPs exhibited a considerable zone of inhibition for _E. coli_ and _B. cereus_ compared with _S. aureus_. Similar observations were reported for the antibacterial properties of ZnO NPs/plant extracts against _E. coli_ strains [62; 63]. The results show more activity toward Gram-negative bacteria, which can be due to the existence of a thick rigid layer that surrounds their cells. This effect has already been reported [63]. Figure 7: _Cont._The antioxidant properties of the biosynthesized NPs are presented in Figure 7b. The H\({}_{2}\)O\({}_{2}\) activity of ascorbic acid (standard), the biosynthesized ZnO NPs, and leaf extract exhibited IC\({}_{50}\) values of 36, 43, and 62 ug/mL, respectively. Enhancement in the antioxidant properties of the biosynthesized ZnO NPs in comparison with the leaf extract could be a consequence of metal or Zn ions existing in the structure. Generally, it has been reported that phenolic species existing in plant extracts have revealed high antioxidant properties, which is an important characteristic for bio-applications [76]. For comparison, strong antioxidant activity has been found in ZnO NPs isolated from extracts of _Allium sativum_, _Rosmarinus officinalis_, and _Ocimum basilicum_ for comparison [77]. Microorganisms are protected by a cell membrane covering them and through this membrane sufficient nutrient are transported [64; 65]. Figure 8 shows different mechanisms adopted by Gram-negative bacteria for attachment of ZnO NPs and their transport inside the bacteria cell. Gram-negative bacteria have a triple thin layer of peptidoglycan compared with Gram-positive bacteria and show less toleration to nanoparticle interaction with the cell membrane. According to Figure 8, a Gram-negative bacterium (such as _E. coli_) has a layer of peptidoglycan in its cell wall where porins are present in the outer layer. Porins are a family of proteins that exist in the outer peptidoglycan layer that helps passive diffusion of NPs inside the cell. ZnO NPs prefer to dissolve in the aqueous medium containing bacteria and release Zn\({}^{2+}\) ion (because of free energy) [78]. The interaction between zinc ions and the cell wall results in a change in surface tension which leads to the membrane depolarization at the point of contact. As a result, the bacterial membrane shows abnormal textures such as membrane rupture or membrane blebs. ZnO NPs, as an antibacterial agent, act with a series of mechanisms; in this way, cell membrane integrity loss (induced by disruption of the phospholipid bilayer) and oxidative stress (induced by reactive oxygen species (ROS) generation which causes cell death by inhibiting) are two important events (Figure 8) [64; 79]. ZnO and its related defects can be easily activated by UV and visible light, which leads to electron-hole pair generation [35]; the formed excitons could increase ROS concentration. ROS molecules could be superoxide, hydroxyl, singlet oxygen, and peroxide ions which are produced on the surface of NPs and possess high electronegativity that can enhance the antimicrobial activity. For example, the generated H\({}_{2}\)O\({}_{2}\) molecules can penetrate into the bacterial cell membrane inducing structural changes to membranes and hence disturbing nutrient/protein transport and causing bacteria death.