Antimicrobial Annona muricata L. (soursop) extract targets the cell membranes of Gram-positive and Gram-negative bacteria

Annona muricata has become an interesting subject in the search for new therapeutic agents. We investigated the bacterial mode of action of the methanolic extract of A. muricata leaves (AML). AML extract was tested against several bacteria strains by broth microdilution susceptibility method. The bacterial killing assay, bacterial abundance and membrane viability analysis were made using fluorescent probes. The nucleotide leakage and outer membrane (OM) permeability assays were used to verify membrane destabilization. The biochemical reaction profile was carried out on a VITEK(.)2 system. For UPLC-ESI-MS/MS Analysis an Acquity UPLC system was used. AML was active against both Gram-negative and Gram-positive bacteria, showing greater activity against S. aureus, S. typhimurium and E. faecalis. AML exhibited rapid time dependent kinetics of bacterial killing. DAPI staining revealed that AML inhibited the bacterial growth, while the LIVE/DEAD BacLight analysis showed that AML induced an increase in dead cells. AML increased nucleotide leakage and was also capable of increasing the OM permeability in the tested bacteria. Differences between the stressed clones and controls observed in the biochemical characterization were not enough to modify the strain identity. UPLC-ESI MS/MS analysis revealed the presence of the alkaloids anonaine, asimilobine, corypalmine, lirioderine, nornuciferine, xylopine and reticuline. Our findings demonstrate, for the first time, a broad spectrum of antibacterial activity for AML and identify that bacterial membranes (both plasma and outer membranes) are primary targets of this extract. Based on these observations, AML has a good potential for the design of novel antimicrobial agents.