Evaluation of the antibiofilm activity on Mycobacterium smegmatis, redox modulating potential, and safety-related effects of Salvia aurea L. and Sphedamnocarpus pruriens (A. Juss.) Szyszyl

• Highly selective antimycobacterial biofilm activity by two medicinal plant extracts. • Demonstration of noteworthy anti-oxidant activity comparable to ascorbic acid. • Both extracts were deemed moderately cytotoxic. • Significant CYP450 inhibitory activity across all tested isoforms was noted. • First time report of CYP450 modulatory activity by these two species. With over one-quarter of the global population infected with Mycobacterium tuberculosis (Mtb), the tuberculosis (TB) epidemic and efforts aimed at its epidemiological stabilisation remains a major global health concern. Drug resistance, extended therapeutic regimens, mycobacterial biofilm formation and cytotoxic side effects as a result of the intense pharmacological load of current therapies, have prompted interest and awareness in natural products as potential adjunctive agents. This study aimed to investigate ethanolic extracts of two ethnobotanically selected Southern African medicinal plants namely Salvia aurea L. (synonymous to Salvia africana-lutea ) and Sphedamnocarpus pruriens (A. Juss.) Szyszyl., for their selective antibiofilm, antimycobacterial, anti-oxidant, cytotoxic, and cytochrome P450 (CYP450) modulatory activity relevant to TB management. Mycobacterium smegmatis was employed as a relevant, fast-growing, non-pathogenic model organism to simulate mycobacterial biofilm-forming characteristics. Antibiofilm activity was assessed using crystal violet staining across three biofilm stages to determine minimum biofilm inhibitory concentration (MBIC), while antimycobacterial minimum inhibitory concentration (MIC) values were determined using a PrestoBlue® microdilution assay. Anti-oxidant potential was measured via DPPH radical scavenging, cytotoxicity via a microtiter assay using the human kidney HEK293 cell line and CYP450 inhibition across isoforms CYP2C9, CYP2C19, and CYP3A5 using a fluorometric assay. Both extracts demonstrated selective antibiofilm activity, with MBIC values of 250 µg/mL for S. pruriens and 500 µg/mL for S. aurea with biofilm selectivity indices (BSI) of 4.68 and 5.84, respectively, suggesting targeted antibiofilm effects independent of direct antimycobacterial action. At 25 µg/mL, S. pruriens (91.61 %) and S. aurea (82.19 %) outperformed ascorbic acid (75.24 %) in terms of % radical scavenging activity. Cytotoxicity results indicated moderate toxicity, with S. pruriens demonstrating a slightly less toxic IC 50 value 101.73 µg/mL when compared to that of S. aurea (40.67 µg/mL). Notably, both extracts inhibited major CYP450 isoforms CYP2C9, CYP2C19, and CYP3A5, in certain instances by up to 80 %, raising caution for potential herb-drug interaction. This is the first report of CYP-modulatory activity for these species. Together, the results suggest that S. aurea and S . pruriens hold promise as selective biofilm-targeted agents with supportive antioxidant potential. Further research into compound isolation to determine active and toxic metabolites, pharmacokinetic safety, and in vivo validation in M. tuberculosis models is necessary to evaluate their integration into TB therapy with minimal metabolic risk.