UPLC-MS/MS (254 nm) chromatogram, UV, and MS spectra of the F5 and F6 subfractions purified from dichloromethane fraction (DCM) of Kielmeyera membranacea. (A) F5 chromatogram and UV spectrum of the peak with t_R 12.72 min (podocarpusflavone A—PCFA). (B) F5 mass spectrum of the peak with t_R 12.72 min. (C) F6 chromatogram and UV spectra of the peak with t_R 11.31 and 12.76 min. (D) F6 mass spectrum of the peak with t_R 11.31 min. (E) PCFA structure.

Growth inhibition of Mycobacterium marinum Wasabi by Kielmeyera membranacea. (A) ethanolic extract (EE); (B) dichloromethane fraction (DCM); (C) podocarpusflavone A (PCFA; F5); and (D) F6. Microplate assay with green-fluorescent bacteria after 5 days of incubation. Fluorescence reading was performed using excitation at 493 nm and emission at 509 nm. M. marinum treated with rifampicin (RIF) at a concentration of 2 µg/mL was used as a positive control, and untreated M. marinum was used as a negative control. *** p <0.001, ** p < 0.01, and * p < 0.05 when compared to the untreated group (0).

Growth inhibition of Mycobacterium strains by podocarpusflavone A (PCFA). Microplate assay after 5 days of incubation in the presence of PCFA (16–128 µM). Growth of Mycobacterium tuberculosis strains H37Rv and M299 was evaluated by the MTT test. The growth of M. marinum E11 mCherry red-fluorescent bacteria was assessed by measuring bacterial fluorescence (excitation at 588 nm and emission at 610 nm). The untreated Mycobacterium strains were used as a negative control, and rifampicin (RIF) was used as a positive control, with MIC values for each of the strains equal to 0.3 µg/mL for M299; 1 µg/mL for H37Rv and 4 µg/mL for E11 mCherry, respectively.

Effect of podocarpusflavone A (PCFA), isoniazid (INH), and INH-PCFA combinations on Mycobacterium tuberculosis H37Rv strain growth in THP-1-derived macrophages. (A) THP-1 macrophages were infected and treated with PCFA at 32, 64, and 128 µM and INH at 0.5× MIC (0.032 µg/mL) and 1× MIC (0.063 µg/mL). (B) THP-1 macrophages infected and treated with combinations of INH-PCFA. The intracellular viability of the bacteria was determined by CFU counting. Values were reported as mean ± error. The mean value of each group was significantly different from the mean value of the controls, as indicated in the graph after the two-way ANOVA analysis. *** p < 0.001, ** p < 0.01, and * p < 0.05 compared to no drug. ^### p < 0.001 and ^## p < 0.01 compared to INH 0.5× MIC. ⁺⁺⁺ p < 0.001 and ⁺ p < 0.05 compared to INH 1× MIC.

Viability of zebrafish larvae treated with Kielmeyera membranacea samples. The wild-type zebrafish larvae were treated with ethanolic extract (EE) (A), dichloromethane fraction (DCM) (B), and PCFA (C) for 2, 24, and 48 hpf, and viability was evaluated by microscopy-based morphological analysis. CTL (negative control, dilution water) and VPA (positive control, valproic acid—15 μM).

Effect of treatment with the ethanolic extract (EE), dichloromethane fraction (DCM) (32 µg/mL), and podocarpusflavone A (PCFA) (32 µM) on mycobacterial growth (mCherry E11) in 4- and 5-day-old Tg(mpeg1:eGFP)gl22 larvae after yolk sac injection of Mycobacterium marinum. (A) Red-fluorescent mycobacteria measured by COPAS analysis after 24 h, and (B) 48 h. (C) Green-fluorescent macrophages were measured by COPAS analysis after 24 h and (D) 48 h. UN: uninfected; UNT: untreated; RIF: rifampicin. *** p < 0.001 ** p < 0.01 and * p < 0.05 when compared to the infected and untreated group (UNT).

Caudal vein injection of Mycobacterium marinum and effect of treatment on infected larvae. Fluorescence microscopy images of fluorescent zebrafish and M. marinum. (A) Transgenic zebrafish larva with red fluorescent macrophages Tg(mpeg1:mCherry)gl23 infected with green fluorescent M. marinum (Wasabi), untreated or treated with PCFA (podocarpusflavone A) at 32 µM after 3 days. (B) Mean values of relative fluorescence units of green-fluorescent bacteria after 3 days with PCFA (32 µM) and rifampicin (RIF) (32 and 100 µM) treatment. Scale bar: 250 µm. *** p < 0.001 compared to the infected and untreated group (UNT).

Molecular docking of podocarpusflavone A (PCFA) and PtpB of Mycobacterium tuberculosis. The carbon atoms of the molecule are colored pink at the ATP-binding site of PtpB. The amino acid residues in green show the interactions between van der Waals forces, the residues depicted in yellow are those involved in hydrogen bonding, π-π stacking is indicated in light blue, oxygen atoms are shown in red, and nitrogen atoms are shown in dark blue. Yellow dashed lines indicate hydrogen bonding. Picture made with Pymol, version 0.99.

Amino acid sequence alignment of PtpB of Mycobacterium tuberculosis and M. marinum, * refers to the same amino acid residues in protein alignment, showing the consistency of the results. Image from the server Praline (https://www.ibi.vu.nl/programs/pralinewww/) accessed on 29 October 2024.

Expression levels of cytokines and macrophage markers in zebrafish larvae infected with Mycobacterium marinum determined by RT-qPCR. The Tg(mpeg1:mCherry)gl23 zebrafish larvae, infected (+) or non-infected (−) with M. marinum, were treated with podocarpusflavone A at 32 µM (PCFA32), rifampicin at 32 or 100 µM (RIF32 and RIF100) or were left untreated (CTRL) for 5 days, and relative expression of mRNA was measured for IL-12, IL-6, TNF-α, IL-10, and CXCR3.2. The expression of each target gene was related to the expression of the housekeeping ppial gene. The data were expressed as the mean ± SEM. * p < 0.05 and ** p < 0.01 compared to the CTRL infected group and # p < 0.05, ## p < 0.01 and ### p < 0.001 as compared to the CTRL non-infected group.