introductionThe increasing resistance to anti-TB drugs has become a global issue, highlighting an urgent need to investigate new drug candidates for combating Mycobacterium tuberculosis (M.tb.). Asiatic Acid (AA), a major phytoconstituent of Centella asiatica, exhibits anti-microbial and anti-biofilm activities against several microbes. In the present investigation, AA was explored for prospective anti-biofilm activity against mycobacteria.

methodsWe first used a computational approach to probe the in silico inhibitory potential of AA against selected target proteins of the FAS-II pathway, involved in mycolic acid biosynthesis, which contributes to mycobacterial cell wall and biofilm formation. Further, in vitro anti-mycobacterial assays were performed to determine the inhibitory concentration and biofilm inhibition against M. smegmatis, which was quantified by crystal violet staining and validated by SEM and CLSM.

resultsThe structure-based inhibitory potential of AA was evaluated against β-Ketoacyl ACP Synthase (KasA) and Enoyl acyl carrier protein (InhA) by molecular docking analysis, showing good binding affinities, with binding energies of -9.53 kcal/mol and -10.53 kcal/mol, and inhibition constants of 3.04 μM and 80.20 nM, respectively. Further, the in vitro anti-mycobacterial assays confirmed the MIC as 20 μg/ml against M. smegmatis. A conspicuous reduction in mycobacterial biofilm formation was observed upon exposure to AA at the MIC, as quantified by crystal violet staining and validated by SEM and CLSM. The EC50 value, the concentration showing 50% inhibition of biofilm formation, was observed as 40 μg/ml against M. smegmatis. DISCUSSION: This study shows advancement in understanding the natural compound AA as a potent anti-tubercular compound by elucidating its dual mechanism of action, including antimycobacterial and anti- biofilm activity, and by targeting enzymes of the FASII pathway, i.e., InhA and KasA. The present in vitro investigation suggests that asiatic acid may serve as a good anti- TB compound, with the possibility of potentiation of anti- mycobacterial effects via additional biofilm-inhibition activity.

conclusionThese findings are expected to pave the way for the design and development of novel anti-TB medications and strategies that enhance treatment efficacy and minimize resistance development in this persistent pathogen. Further investigations are required to determine the mode of action and validate its candidacy as a promising anti-biofilm agent in the current clinical setting.