## 4 Discussion
; thus, they induce apoptosis by the internalization of the hydrogen peroxide radicals or the loss of the cellular integrity following the interaction between ZnO NPs and the cell wall [68]. Biologically fabricated ZnO nanoparticles gain two benefits that result from the biological functionalization: high biocompatibility and decreased microbial drug resistance [69]. Based on the estimated marginal means (Figure 3a), after loading the LPME, the antimicrobial action of the ZnO was improved, which was the result of the biofunctionalization by the _L. pubescens_ methanol extract, which contains compounds with antimicrobial properties (Section 3.1). We observed the interaction effects between the microorganisms, solvent type, and ZnO nanoparticles (Table 2 and Figure 3a,b) (i.e., how the examined strains of bacteria and fungi responded to the antimicrobial activities of the nanoparticles in various solvents). We observed that the ZI was higher in DMSO than in water (Figure 2a), which was probably attributable to the higher solubility of the LP-ZnO NPs in DMSO than in water. Interestingly, the antibacterial activity of the 50 mg LP-ZnO NPs in water against _S. aureus_ and _P. aeruginosa_ was significantly better than that of the 100 mg LP-ZnO NPs (Figure 2a). This could be the result of the good biofunctionalization and smaller particle size of the synthesized 50 mg LP-ZnO NPs, which was ascertained by the aforementioned characterization findings (Section 3.2). The 50 and 100 mg LP-ZnO NPs both showed stronger antifungal efficacy in water than in DMSO (Figure 2a-c), which might be because the ZnO nanoparticles dispersed in water could promote ROS, and especially H2O2 radicals, which are capable of internalizing the cell and causing apoptosis. In this regard, [PERSON] et al. [70] examined ZnO NPs as an antifungal agent against _Candida albicans_, and they proposed that the generation of ROS might be the factor that led to the fungal apoptosis in the aqueous medium. The antimicrobial activity of ZnO alone in DMSO, but not in water, was enhanced by functionalizing the nanoparticles with _L. pubescens_ shoot extract (Figure 2a-c). The ZnO surface may physically adsorb the bioactive compounds found in LPME, thus reducing and capping the ZnO to produce functionalized LP-ZnO NPs. [PERSON] et al. [71] concluded that, after the crystal loses a photoexcited electron to O2, organic compounds, such as phenolate and alcoholate anions adsorbed onto the zinc oxide surface, are oxidized either by the transfer of the electrons to the photopositive zinc oxide surface, or through the absorption of hydrogen by the peroxide radical. The broadening of the inhibition zone for diverse bacteria reflects the species-sensitive nature of the cytotoxic impact of a particular metal oxide nanoparticle [72]. Despite the lower ZIs of the ZnO nanoparticles in water versus DMSO, they were still significantly higher in the ZnO nanoparticles than in the control drugs, augmentin/voriconasole, in water. In this study, the alterations noticed in the inhibitory zones after the bacterial and fungi cultures were exposed to the biosynthesized LP-ZnO-NP treatments could be due to the enhancement of the ZnO by the LPME bioactive compounds adsorbed onto its surface. The LPME, as a functionalizing material, demonstrated antimicrobial potential, such as indole, 3-(4-nitrophenylamino), which has anticancer and antibacterial properties against _S. aureus_[39; 40]; nonanoic acid, methyl ester, which is an antifungal agent [41]; 3-methyl-4-isopropylphenol, which has synergistic antibacterial effects against _Streptococcus mutans_[42]; tridecanoic acid, methyl ester, which has antienteric efficacy against enteropathogenic bacteria, such as _Enterococcus faecalis_ (Gram-positive bacterium) and _Salmonella enterica_ (Gram-negative bacterium) [45]. The cell cytotoxicity required direct particle-cell interaction, and the toxicity of the particles was unrelated to the concentration of the soluble ZnO under the cell growth conditions.