Figure 1.
Phylogenetic tree depicting the relationships between UFMGCB-555 and related species.
The tree was constructed by neighbor-joining analysis (Kimura-2 parameter) of their ITS domain sequences. Bootstrap percentage from 1000 replicates is shown. The GenBank accession numbers are indicated for each reference sequence. The ITS sequence of Alternaria (AY673074) was used as an outgroup.
Table 1.
Biological activities of crude extract, fraction, and compounds isolated from Cochliobolus sp. (UFMGCB-555) tested at a single dose of 20 µg mL−1.
Figure 2.
Flowchart illustrating the isolation of cochlioquinone A and isocochlioquinone A.
The fractionation was guided by the bioassay with the enzyme trypanothione reductase, in which the crude extracts, fractions, and isolated compounds were tested at 20 µg mL−1.
Figure 3.
Dose response curves for 1 (diamonds) and 2 (squares) in the assay with amastigote-like forms of L. amazonensis.
The EC50 (effective concentration to kill 50% of the parasites) values were found to be 1.7 µM (95% confidence interval = 1.5 to 1.9 µM) and 4.1 µM (95% confidence interval = 3.6 to 4.7 µM), respectively. The data in the plots are derived from at least two independent experiments.
Figure 4.
ESI-MS-MS in the positive ion mode showing the main fragments generated by cochlioquinone A (panel A) and isocochlioquinone A (panel B).
Both compounds show sodiated quasi-molecular ion peaks [M+Na]+ at 555 daltons and the loss of acetic acid (Δ m/z = 60 daltons) from the side chain. In addition, the hydroxyl group in the pyrane ring of cochlioquinone A is responsible for the observed loss of water (Δ m/z = 18 daltons).
Figure 5.
Chemical structures of compounds isolated from Cochliobolus sp. UFMGCB-555.
The numbering in these structures follows that used in [42].
Table 2.
1H-NMR and HSQC spectral data of 1 and comparison with literature data for cochlioquinone A.
Table 3.
1H-NMR and HSQC spectral data of 2 and comparison with literature data for isocochlioquinone A.
Table 4.
13C-NMR and DEPT spectral data of compounds 1 and 2 and comparison with literature data for Cochlioquinone A and Isocochlioquinone.
Figure 6.
(A) Schematic view of the chemical and enzymatic reactions involved in the TryR assay resulting in the reduction of the Elmman's reagent (DTNB) to the yellow-colored 2-nitro-5-thiobenzoic acid (TNB); (B) Same as above, showing the interference of cochlioquinone A resulting in a false positive readout due to its reaction with the reduced form of trypanothione, interrupting the chemical reduction of DTNB.