Exploring the Antimycobacterial Potential of Podocarpusflavone A from Kielmeyera membranacea: In Vitro and In Vivo Insights

<b>Background/Objectives</b>: Tuberculosis (TB) is one of the leading infectious causes of death worldwide, highlighting the importance of identifying new anti-TB agents. In previous research, our team identified antimycobacterial activity in <i>Kielmeyera membranacea</i> leaf extract; therefore, this study aims to conduct further exploration of its potential. <b>Methods</b>: Classical chromatography was applied for fractionation and spectrometric techniques were utilized for chemical characterization. For in vitro tests, samples were assessed against <i>Mycobacterium tuberculosis</i> and <i>Mycobacterium marinum</i>. The toxicity and efficacy of active samples were evaluated in vivo using different zebrafish models. Chemogenomics studies were applied to predict the isolated active compound's potential mode of action. <b>Results</b>: We performed fractionation of <i>K. membranacea</i> ethanolic extract (EE) and then its dichloromethane fraction (DCM), and the biflavonoid podocarpusflavone A (PCFA) was isolated and identified as a promising active compound. The EE and PCFA were found to be non-toxic to zebrafish larvae and were able to inhibit <i>M. tuberculosis</i> growth extracellularly. Additionally, PCFA demonstrated antimycobacterial activity within infected macrophages, especially when combined with isoniazid. In addition, the EE, DCM, and PCFA have shown the ability to inhibit <i>M. marinum's</i> growth during in vivo zebrafish larvae yolk infection. Notably, PCFA also effectively countered systemic infection established through the caudal vein, showing a similar inhibitory activity profile to rifampicin, both at 32 µM. A reduction in the transcriptional levels of pro-inflammatory cytokines confirmed the infection resolution. The protein tyrosine phosphatase B (PtpB) of <i>M</i>. <i>tuberculosis</i>, which inhibits the macrophage immune response, was predicted as a theoretical target of PCFA. This finding is in agreement with the higher activity observed for PCFA intracellularly and in vivo on zebrafish, compared with the direct action in <i>M. tuberculosis</i>. <b>Conclusions</b>: Here, we describe the discovery of PCFA as an intracellular inhibitor of <i>M. tuberculosis</i> and provide evidence of its in vivo efficacy and safety, encouraging its further development as a combination drug in novel therapeutic regimens for TB.