Phytochemical analysis by UPLC-MS/MS, in silico molecular docking, and evaluation of the antimicrobial, antioxidant, and enzyme inhibitory effects of Thymus leucotrichus var. leucotrichus Hal.

Abstract

This study investigated the phytochemical composition and biological potential of the methanol extract of the aboveground parts of Thymus leucotrichus var. leucotrichus (METL), a high-altitude Thymus taxon used as a spice and medicinal plant. METL was evaluated for antioxidant activity (Fe3+, Cu2+, and Fe3+-TPTZ reduction; DPPH, ABTS, and DMPD radical scavenging assays), antimicrobial activity, and its inhibitory effects on α-glycosidase (α-Gly), acetylcholinesterase (AChE), carbonic anhydrase I and II (CA I and II), total phenolic and flavonoid content, phenolic compound analysis via UPLC-MS/MS, and the binding effect of the major active compound, rosmarinic acid, on enzymes. At 20 μg/mL, METL showed absorbances of 0.718, 0.856, and 1.152 in the Fe3+, Cu2+, and Fe3+-TPTZ reduction assays, respectively. The IC50 values for DPPH, ABTS, and DMPD radical scavenging were 20.39, 19.16, and 94.53 μg/mL, respectively. The IC50 values for METL’s inhibitory effects on AChE, α-Gly, and CA I and II enzymes are 3.40 μg/mL, 9.2 μg/mL, 11.50 μg/mL, and 12.9 μg/mL, respectively. The total phenolic content of METL is 186.66 mg/g GAE, and the total flavonoid content is 161.94 mg/g QE. UPLC-MS/MS analysis of METL identified rosmarinic acid (RA) at 17,961.65 μg/g, cinnamic acid at 2,189.88 μg/g, and chlorogenic acid at 281.16 μg/g as the major components. The correlation between RA and enzymes was then investigated using molecular docking analysis. The antimicrobial activity of METL was tested against a total of 12 isolates, comprising 8 standard strains and 4 hospital strains. METL exhibited a minimum inhibitory concentration (MIC) of 0.5 mg/mL against Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, colistin-resistant K. pneumoniae, Bacillus cereus, Proteus mirabilis, and Candida albicans. Overall, the findings demonstrate that METL combines marked antioxidant capacity, antimicrobial activity, and inhibitory effects on AChE, α-Gly, and CA I-II, with RA as its predominant quantified phenolic constituent.