In-Vitro Antimicrobial, Antibiofilm, Antihemolytic, and Anti-Inflammatory Activities of Ozonated Ziziphus spina-christi Oil Supported by GC–MS Analysis

Abstract

Ozonation has emerged as an effective approach to enhance the biological efficacy of plant-derived oils through chemical modification. In this study, crude and ozonated Ziziphus spina-christi oils were chemically characterized by gas chromatography–mass spectrometric (GC-MS) analysis and evaluated in vitro for their biological activities. GC–MS profiling revealed that the crude oil consisted predominantly of oxygenated monoterpenes and aromatic esters, particularly eucalyptol and methyl salicylate, along with lower proportions of fatty acids and their derivatives. Following ozonation, a marked increase in oxygenated compounds was observed, including epoxidized fatty alcohol esters and alkyl ether alcohols. Ozonated oil showed pointedly enhanced antibacterial activity, minimum inhibitory (MIC) and minimum bactericidal concentrations (MBC), with inhibition zones of 37.0 ± 0.6, 31.0 ± 0.1, 25.0 ± 0.8, and 29.0 ± 0.8 mm against Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa and Salmonella typhi, respectively. Ozonated oil inhibited biofilm formation by more than 80% at sub-MBC levels and reduced bacteria-induced hemolysis by up to 98.1% at 75% MIC. Time-kill kinetics confirmed rapid bactericidal effects within 180 min. Moreover, ozonated oil exhibited superior anti-inflammatory activity with an IC₅₀ of 2.40 ± 0.09 µg/mL. These findings highlight ozonated Z. spina-christi oil as a promising multifunctional bioactive agent.