Involvement of oxidative stress in protocatechuic acid‐mediated bacterial lethality

The involvement of oxidative stress in protocatechuic acid‐mediated bacterial lethality was investigated. Minimum inhibitory concentrations (MIC) and minimum bactericidal concentration (MBC) of protocatechuic acid against Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus are 600 and 700 μg/ml, 600 and 800 μg/ml, and 600 and 800 μg/ml, respectively. The optical densities and colony‐forming units of protocatechuic acid‐treated bacteria decreased in time‐dependent manner. Protocatechuic acid (4× MIC) significantly increased the superoxide anion content of E. coli, P. aeruginosa, and S. aureus compared to dimethyl sulfoxide (DMSO). Superoxide dismutase, catalase, and NAD+/NADH in protocatechuic acid‐treated E. coli, P. aeruginosa, and S. aureus increased significantly when compared to DMSO. Conversely, level of reduced glutathione decreased in protocatechuic acid‐treated E. coli, P. aeruginosa, and S. aureus, while glutathione disulfide increased when compared to DMSO. Furthermore, malondialdehyde and fragmented DNA increased significantly following exposure to protocatechuic acid. Protocatechuic acid inhibited the activity of complexes I and II. From the above findings, protocatechuic acid enhanced the generation of reactive oxygen species (superoxide anion radical and hydroxyl radical) in E. coli, P. aeruginosa, and S. aureus, possibly by autoxidation, fenton chemistry, and inhibiting electron transport chain resulting in lipid peroxidation and DNA fragmentation and consequentially bacterial cell death. Protocatechuic acid enhanced the generation of superoxide anion and hydroxyl radicals. This increased superoxide anion and hydroxyl radical levels perturbs the redox status of bacteria leading to lipid peroxidation, protein oxidation, DNA fragmentation, ATP depletion, and cell death.