Exploring the Antibacterial, Anti-Inflammatory, and Antioxidant Properties of the Natural Food Supplement “Protegol” as a Supportive Strategy in Respiratory Tract Infections

<b>Background/Objectives</b>: Respiratory tract infections (RTIs) remain a leading cause of morbidity worldwide and are frequently associated with the emergence of multidrug-resistant pathogens. In this context, natural compounds represent a valuable source of novel antimicrobial and immunomodulatory agents. The present study aimed to evaluate the antibacterial, anti-inflammatory, and antioxidant activities of <i>Protegol</i>, a natural food supplement enriched in bioactive phytochemicals including hydroalcoholic extracts of propolis and hedge mustard (<i>Sisymbrium officinale</i> (L.) Scop.) aerial parts, together with honey, against clinically relevant bacterial strains and in cellular models of inflammation and oxidative stress. Furthermore, the ability of the multi-herbal formulation to alter the permeability of the bacterial cell wall was assessed. <b>Methods</b>: The antibacterial properties of <i>Protegol</i> were evaluated by determining its minimum inhibitory (MIC) and minimum bactericidal concentrations (MBC) against a panel of Gram-positive and Gram-negative bacteria, using the broth microdilution method. Cell wall permeability was investigated through the propidium iodide (PI) uptake assay. The anti-inflammatory potential was investigated in LPS-stimulated RAW 264.7 macrophages by measuring nitric oxide (NO) production with the Griess assay. The antioxidant activity was evaluated in BALB/3T3 fibroblasts exposed to hydrogen peroxide, using the DCFH-DA assay. <b>Results</b>: <i>Protegol</i> exhibited a broad-spectrum antibacterial effect, with MIC values ranging from 1.5 to 6.2 mg/mL and MBC values between 3.1 and 12.4 mg/mL. The strongest activity was observed against <i>Staphylococcus aureus</i> and <i>Streptococcus pyogenes</i>, including clinical isolates, while moderate efficacy was detected against resistant <i>Klebsiella pneumoniae</i> strains. PI uptake assays confirmed a dose-dependent disruption of bacterial membrane integrity, supporting a direct effect of <i>Protegol</i> on cell wall permeability. In macrophages, <i>Protegol</i> significantly and dose-dependently reduced NO release, lowering production to 44% at the highest concentration tested. In BALB/3T3 cells, <i>Protegol</i> markedly decreased ROS accumulation to 24% at the same concentration. <b>Conclusions</b>: Overall, the findings support the potential of <i>Protegol</i> as a natural adjuvant to the conventional therapies for respiratory tract health by counteracting bacterial pathogens, reducing inflammation, and mitigating oxidative stress, thereby supporting host defense mechanisms in the context of respiratory tract infections.