Urease Inhibitory Activity of Tilia rubra subsp. caucasica V.Engl.: Activity-Guided Fractionation of a Folk Medicine

Bacterial ureases play a key role in the pathogenesis of various diseases, including gastritis, peptic ulcers, urolithiasis, and gastric cancer. Concerns regarding the toxicity and stability of urease inhibitors have prompted the search for safer alternatives from natural sources. Therefore, this study aimed to identify the bioactive compounds responsible for the urease inhibitory activity of Tilia rubra subsp. caucasica V.Engl. [synonym of Tilia dasystyla subsp. caucasica (V.Engl.) Pigott] using activity-guided fractionation. The plant material was subjected to liquid–liquid partitioning followed by chromatographic techniques, with in vitro urease inhibitory activity against jack bean urease evaluated at each step. The structures of the isolated compounds were elucidated by LC-HRMS and NMR spectroscopy. The 80% ethanol extract exhibited notable in vitro urease inhibitory activity (47.14 ± 2.59%). The ethyl acetate subextract was the most active fraction (61.38 ± 0.64% inhibition; IC50 = 71.35 ± 3.48 µg/mL), yielding several compounds, including guaijaverin, trans-tiliroside, isoquercitrin, protocatechuic acid, and a new dimeric flavonoid glycoside (methylenebisastragalin). Guaijaverin (71.86 ± 3.55% inhibition; IC50 = 119.63 ± 3.64 μM) and isoquercitrin (48.04 ± 2.28%) showed the strongest urease inhibitory activity at 100 μg/mL. HPLC quantification showed that guaijaverin and isoquercitrin contribute to the activity of the ethylacetate subextract; however, the compounds responsible for the observed activity could not be fully identified. Molecular docking studies revealed XP GScores of −8.64 and −8.47 kcal/mol for these compounds, respectively. These findings indicate that T. rubra subsp. caucasica is a promising natural source of urease inhibitors, warranting further experimental validation, particularly against bacterial ureases, including that of Helicobacter pylori, to evaluate its therapeutic potential.