# Antibacterial Activity of Crude Extract and Purified Acetogenins from _Amona muricata_ Seeds
## 3 Results and Discussion
### Antibacterial Assays of Crude Extract and Purified Acetogenins
The cell viability ratio is expressed as follows: a high red/green ratio means that bacterial cells retain their membrane potential, whereas a low red/green ratio means that bacterial cells do not maintain their membrane potential because they are damaged. The cell viability  
Figure 2: Percentage inhibition of crude extract and purified acetogenins on the most common pathogenic bacteria in food: (**a**) _Enterococcus faecalis_; (**b**) _Listeria monocytogenes_; (**c**) _Salmonella paratyphi_ and (**d**) _Escherichia coli_. Different letters (a, b) indicate significant statistical differences between treatments (\(\alpha\) = 0.05) by each concentration. measured by flow cytometry and expressed as a red/green fluorescence ratio is shown in Figure 3. Compared to traditional approaches, the BacLight membrane potential kit (Molecular Probes B34950, Carlsbad, CA, USA) allowed for a significantly more quantitative determination of the cell viability of CE and P-ACG-treated cells. This allowed for the calculation of the percentages of polarized and depolarized cells within a population of bacteria. As expected, 10 M CCCP did not increase the potential of the treated cell membrane. However, the antibacterial effect was more significant in the cells exposed to P-ACGs (red/green ratio of 0.09 to 0.18) than in those exposed to CE (red/green ratio of 0.17 to 1.22), and this depended on the type of bacteria. Figure 3 describes that bacteria treated with the P-ACGs induced a significant decrease in cell viability and increased bacterial cell death. Cell death is related to the effect of programmed cell death and lysis [16]. As previously mentioned, the CE and P-ACGs presented a more significant effect on Gram-positive bacteria than Gram-negative bacteria. Since Gram-negative bacteria have relatively thin cell walls composed of a single layer of peptidoglycan, lipoproteins, lipopolysaccharide, and the outer membrane, which acts as an efficient permeability barrier against hydrophobic antibacterial agents like ACGs, these results may be related to the structure of Gram-negative bacteria [16; 42]. Therefore, the outer membrane reduces the access of ACGs, thus preventing these compounds from effectively infiltrating this intact membrane of Gram-negative bacteria. However, they may permeate the membrane of specific defective mutants of the outer membrane, which are damaged by chelators or polycations [16]. In this regard, [PERSON] et al. [16] used the LIVE/DEAD BacLight approach to assess the impact of _A. muricata_ leaf extract on the cell viability of _S. aureus_, _S. typhimuriumium_, and _E. faecalis_. These authors showed the potential antibacterial impact of a leaf extract from _A. muricata_, increasing the number of dead cells in a concentration-dependent response. They reported that the leaf extract promoted changes at the cellular level in the tested bacteria that are indicative of cell death, mostly linked to the degradation of plasma membrane integrity. Furthermore, these authors attributed the  
Figure 3: The membrane potential of pathogenic bacterial strains treated and untreated with antibacterial extracts. antimicrobial effect to several alkaloids included in the extract. They suggested a synergy with compounds present in the extract, such as acetogenins. Lethality and Sublethal Injury on Enterococcus faecalis, Listeria monocytogenes, and Escherichia coli Caused by the Crude Extract and Purified Acetogenins  
According to the results, all treatments decreased the plate count (_p_ < 0.05) for all tested bacteria (initial bacterial load \(\approx\) 7.74-8.16 log CFU/mL) (Table 4). The lowest log reduction was observed using CE (0.35-1.27 log CFU/mL), while the P-ACGs induced the highest log reduction (0.38-1.81 log CFU/mL). The P-ACGs had a high effect on _L. monocytogenes_ (1.81 log CFU/mL) and _E. faecalis_ (1.27 log CFU/mL) due to the presence of ACGs with unsaturations in the aliphatic chain that cause bacterial inhibition, [37] and the increase in purity [13].