Contents lists available at ScienceDirect Industrial Crops & Products journal homepage: www.elsevier.com/locate/indcrop Antimicrobial Annona muricata L. (soursop) extract targets the cell membranes of Gram-positive and Gram-negative bacteria Nícolas de C.C. Pinto a , Lara M. Campos a , Anna Carolina S. Evangelista a , Ari S.O. Lemos a , Thiago P. Silva b , Rossana C.N. Melo b , Caroline C. de Lourenço c , Marcos J. Salvador c , Ana Carolina M. Apolônio d , Elita Scio a , Rodrigo L. Fabri a, ⁎ a Bioactive Natural Products Laboratory, Department of Biochemistry, Biological Sciences Institute, Federal University of Juiz de Fora, Juiz de Fora, Brazil b Laboratory of Cell Biology, Department of Biology, Institute of Biological Sciences, Federal University of Juiz de Fora, Juiz de Fora, Brazil c Department of Plant Biology, Institute of Biology, State University of Campinas, Campinas, São Paulo, Brazil d Laboratory of Bacterial Physiology and Molecular Genetics, Department of Parasitology, Microbiology and Immunology, Institute of Biological Sciences, Federal University of Juiz de Fora, Juiz de Fora, Brazil ARTICLE INFO Keywords: Annona muricata Antibacterial activity Membrane permeability Alkaloids Plant extract ABSTRACT 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 re- action 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 re- ticuline. 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. 1. Introduction The emergence and spread of microbial resistance is growing each day, thereby necessitating the development of new antimicrobials (Malik et al., 2017). Hospital-acquired infections are becoming a growing concern (WHO, 2015). Based on this, studies have shown that natural products appear to be resources for valuable resistance breaking molecules (Newman and Cragg, 2012). Plant derived compounds are of particular interest, based on their already proven antimicrobial activity (Jamkhande et al., 2016; Mocan et al., 2015; Mocan et al., 2016; Vlase et al., 2014). In this context, Annona muricata (Fig. 1) has become an interest subject in the search for new therapeutic agents. This species belongs to the Annonaceae family, which consists of 29 genera and 390 species with tropical and subtropical distribution worldwide (Joly, 1979). It is popular known as soursop or prickly custard apple, and the fruits are edible and commonly used to make ice cream, teas and other drinks (Degnon et al., 2013). Several parts of this plant are traditionally used to treat cancer, diabetes, hypertension, fever, gastrointestinal disorders, and parasitic infections (Adjanohou, 1996). Various pharmacological studies have confirmed the traditional uses of Annona muricata (Adewole and Caxton-Martins, 2006; N’gouemo et al., 1997). In particular, the antimicrobial properties of Annona muricata leaves (AML) have attracted attention due to their potential to also treat viral infections (Florence et al., 2014), hyperli- pidemia (Adewole and Ojewole, 2008) and neglected human diseases, http://dx.doi.org/10.1016/j.indcrop.2017.05.054 Received 19 December 2016; Received in revised form 12 May 2017; Accepted 28 May 2017 ⁎ Corresponding author at: Bioactive Natural Products Laboratory, Department of Biochemistry, Biological Sciences Institute, Federal University of Juiz de Fora, Juiz de Fora, Minas Gerais CEP 36036 900, Brazil. E-mail addresses: rodrigo.fabri@ufjf.com.br, rodrigolfabri@yahoo.com.br (R.L. Fabri). Industrial Crops & Products 107 (2017) 332–340 0926-6690/ © 2017 Elsevier B.V. All rights reserved. MARK