Comparative extracellular and intracellular anti-Mycobacterium tuberculosis activity of three tanshinones from Salvia miltiorrhiza and oral cryptotanshinone efficacy in mice

Tuberculosis (TB) remains a major global health threat, particularly due to multidrug-resistant (MDR) Mycobacterium tuberculosis (M.tb). Although recent years have seen the introduction of new drugs and shorter regimens for TB, additional chemical scaffolds with activity against drug-resistant and intracellular bacilli are still needed. Tanshinones from Salvia miltiorrhiza represent a natural product scaffold with potential anti-mycobacterial activity. We systematically compared three major tanshinones—Tanshinone I (TAN), Dihydrotanshinone I (DTI), and Cryptotanshinone (CTS)—against M.tb H37Rv and a panel of drug-resistant clinical isolates. Extracellular minimum inhibitory concentrations (MICs) were determined, and intracellular activity was evaluated in M.tb-infected THP-1 macrophages by colony-forming unit (CFU) enumeration after 48 h of treatment. CTS was further evaluated in an acute murine TB infection model via daily oral gavage (25 mg/kg/day) for 28 days starting 14 days post-infection. All three tanshinones inhibited extracellular growth of drug-sensitive and drug-resistant M.tb with MICs in the 5–20 μM range, with DTI showing the lowest extracellular MIC (5 μM). In infected THP-1 macrophages at 48 h, CTS (10 μM; 1 × MIC) reduced intracellular CFU by approximately 1.00 Log10 relative to vehicle. Under the same conditions, TAN reduced CFU by 0.14 Log10 at 10 μM (1 × MIC) and 0.52 Log10 at 20 μM (2 × MIC), whereas DTI showed minimal intracellular activity (0.17 Log10 reduction at 10 μM; 2 × MIC). In mice, oral CTS produced statistically significant reductions in bacterial burden in lungs and spleens and improved lung histopathology compared with vehicle control. Major tanshinones display measurable in vitro activity against drug-sensitive and drug-resistant M.tb, but their extracellular and intracellular profiles differ. CTS showed consistent activity across extracellular, intracellular, and in vivo assays, supporting further studies on mechanism of action, pharmacokinetic optimization, and evaluation in combination regimens.