Beyond Traditional Use: Mechanistic Insights into the Medicinal Potential of Thymus tiflisiensis and Modulatory Enhancement Effects of Gallic Acid

Background: Thymus tiflisiensis is used in Georgian traditional medicine, but its pharmacological properties remain poorly characterized. This study evaluated the antioxidant, protein denaturation inhibitory, and antibacterial activities of leaf and root extracts and examined the modulatory effects of gallic acid (GA) on these activities. Methods: Methanolic extracts of leaves and roots were analyzed for total phenolic and flavonoid contents, rosmarinic acid (RA), ascorbic acid, and glutathione. Antioxidant activity was evaluated using the DPPH assay, protein denaturation inhibitory activity using a bovine serum albumin denaturation assay, and antibacterial activity against Streptococcus agalactiae and Escherichia coli using broth microdilution. The combined effects of the plant extracts and GA were evaluated across multiple concentration matrices. Molecular docking was used to predict the interactions of RA and GA with the bacterial enzymes MurF and LpxC. Results: The methanolic leaf extract exhibited greater antioxidant activity (IC50 = 34.8 µg/mL) than the root extract (IC50 = 81.9 µg/mL), accompanied by higher total phenolic (113.6 vs. 56.9 mg GAE/g DW) and flavonoid (63.8 vs. 19.0 mg RE/g DW) contents and higher ASC/DHA and GSH/GSSG ratios. Conversely, the root extract exhibited greater protein denaturation inhibitory activity (IC50 = 51.3 vs. 159.7 µg/mL for the leaf extract) and moderate antibacterial activity against S. agalactiae (MIC = 6.0 mg/mL) and contained higher levels of RA. The addition of 5 µM GA enhanced the antioxidant activity of both leaf and root extracts. In protein denaturation assays, 10 µM GA produced a minor enhancement at low extract concentrations, whereas 100 µM GA reduced the inhibitory effects. At 1 mM, GA enhanced the antibacterial activity of both extracts against S. agalactiae and that of the root extract against E. coli. Molecular docking predicted more favorable interactions of RA with MurF and LpxC than those of GA. Conclusions: T. tiflisiensis root extracts exhibited greater protein denaturation inhibitory and antibacterial activities, whereas leaf extracts showed greater antioxidant activity. GA modulated these activities in a concentration- and tissue-dependent manner, with enhancement observed at specific concentrations and attenuation at higher concentrations in the protein denaturation assay. Exploratory molecular docking predicted favorable interactions of RA with the bacterial enzymes MurF and LpxC. These findings support further investigation of the bioactive properties of T. tiflisiensis and the mechanisms underlying their modulation by GA.