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Pharmaceutics 2020, 12, 1044 11 of 19 4.2. Free Radical Generation Biological molecules such as lipids, proteins and nucleic acids are adversely affected by free radicals. This causes alteration of the normal redox status and lead to increased oxidative stress [99]. Although, oxidative stress is a normal cellular process that occurs in several phases of cellular signaling however, extreme oxidative stress can be detrimental. Literature has showed that MNPs can trigger cellular oxidative stress [100–102]. These free radicals are either in the form of reactive oxygen species (ROS) or reactive nitrogen species (RNS). They result from either endogenic sources (endoplasmic reticulum mitochondria, peroxisomes, etc.) or exogenic sources (heavy metals, pollution, transition metals and specific drugs) [99]. When cells are exposed to stress, they show defensive responses via enzymatic or non-enzymatic mechanisms [102,103]. Damage to the DNA, cell wall, proteins and lipids usually occurs when the defense mechanism is overpowered by oxidative stress. Free radicals such as singlet oxygen, hydrogen peroxide (H2O2) and hydroxyl radical (-OH) are released when the defense mechanism is weakened by the oxidative stress. All these can lead to lipid oxidation, which inhibits or kills bacteria growth. Cell membranes can easily be disrupted by both endogenic and exogenic ROS [104,105]. Chakraborty et al. [106] evaluated the antimicrobial effects of Thevetia peruviana mediated AgNPs on E. coli. The as-synthesized AgNPs showed an effective inhibitory effect against E. coli with an inhibition zone of 20 mm. This suggests that the antibacterial potency of the AgNPs might be related to the membrane structure of the bacteria. Electron spin spectroscopy (ESR) was used to investigate if the free radical production from AgNPs formed at pH 7 after 48 h of reaction time is related to the antimicrobial activity. The results showed that the growth inhibition was due to the formation of free radical species from the surface of AgNPs, which altered the permeability of the outer membrane and inactivated the respiratory function of the bacteria. Soo-Hwan et al. [107] showed that the mechanism of bactericidal effect of AgNPs against S. aureus and E. coli was by the production of ROS due to increased membrane permeability and the inactivation of lactate dehydrogenase, which eventually led to protein breaks. More protein leakage occurred in the membrane of E. coli compared with that S. aureus. This observed difference was possibly attributed to the thickness of the peptidoglycan layer of S. aureus [107]. Gomaa [108] corroborated these results in the study of the bactericidal mechanism of AgNPs with respect to S. aureus and E. coli. The growth curve was measured, followed by an estimation of the protein and reducing sugar leakage. Furthermore, lethal ROS and respiratory chain dehydrogenase activity were evaluated. The study showed that 50 mg/mL AgNPs completely inhibited the growth of bacterial cells and damaged the bacterial membrane permeability, depressing the activity of some membranous enzymes, which eventually led to bacteria cell death. In this study, Dye 20, 70-dichlorofluorescein diacetate (DCFH-DA) was used to measure the ROS. It was observed that after 6 h incubation of the E. coli and S. aureus with AgNPs, there was a significant increase in ROS production however, this was not observed in the control groups. Significantly, AgNPs are stress inducers for bacteria. Qayyum et al. [109] expanded their study to Gram negative (K. pneumoniae, P. aeruginosa and E. coli) and Gram positive (S. mutans and S. aureus) strains. The results showed that green AgNPs produced ROS after 4 h of incubation with the bacterial cells. It was observed that increased contact time of the AgNPs with the bacterial cells led to increased production of the ROS. Additionally, the quantity of ROS increased several times compared to that of the control group for both Gram positive and Gram negative bacteria. However, more ROS production was observed in the treated Gram negative E. coli bacterial cells compared to treated Gram positive S. mutans bacterial cells [110]. ROS formed as a result of bacterial interactions with AgNPs that causes damage to the bacterial cell membrane, protein structure and intracellular systems. In studying the conditions and mechanism of antibacterial activity of silver nanoparticles (AgNPs) against E. coli O157:H7 (CMCC44828), it was also established that the presence of oxygen generated more ROS, which led to increased antimicrobial activity [111]. The mechanism of antibacterial activity of AgNPs against multidrug resistant P. aeruginosa was studied by using H2DCF-DA staining and fluorescence microscopy [112]. It was observed that there was anPDF Image | Bactericidal Antibacterial Mechanism of Plant Nanoparticles
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