# Antibacterial Activity of Crude Extract and Purified Acetogenins from _Amona muricata_ Seeds
## 3 Results and Discussion
### Antibacterial Assays of Crude Extract and Purified Acetogenins
This is attributed to the fact that the crude extracts may contain different bioactive compounds with antimicrobial activity and that some bacteria are susceptible not only to a compound but to a number of them; in addition, a crude extract contains a complex mixture of secondary metabolites that could be exerting a synergistic effect against a certain type of bacteria [25]. In addition, at high doses (4000 mg/mL), the CE exhibited a greater antibacterial effect (92.15%) against _L. monocytogenes_ than the P-ACGs (84.31%). This is consistent with previous reports, which demonstrate that CE may exceed the antibacterial activity of purified compounds. This is attributed to it containing different bioactive compounds with antimicrobial activity, and to the fact that some bacteria are susceptible not only to one compound but to several of them. Therefore, a CE contains a complex mixture of secondary metabolites that could exert a synergistic effect against certain types of bacteria [25]. Thus, \begin{table}
\begin{tabular}{c c c} \hline \hline
**Bacterial Strain** & **Crude Extract** & **Purified ACGs** \\ \cline{2-3}  & **MIC (ug/mL)** & **MIC (ug/mL)** \\ \hline _Streptococcus mitis_ & 12.50 & 0.36 \\ _Listeria monocytogenes_ & 0.51 & 0.49 \\ _Staphylococcus aureus_ & 1000 & 800 \\ _Enterococcus faecalis_ & 0.78 & 0.67 \\ _Streptococcus mutans_ & NI & 0.87 \\ _Streptococcus salivularis_ & 12.5 & 12.5 \\ _Burkholderia encocpacia_ & 0.02 & 0.01 \\ _Salmonella pantyaphi_ & 0.67 & 0.12 \\ _Klebsiella pneumoniae_ & 1.62 & 1.30 \\ _Aeronomas hydrophillia_ & 12.5 & 12.5 \\ _Escherichia coli_ & 0.24 & 0.009 \\ _Salmonella chloressius_ & 0.51 & 0.09 \\ \hline \hline \end{tabular}  
* MIC = Minimum inhibitory concentration; NI = No inhibition.  
\end{table} 
Table 3: Antibacterial activity of crude extract and purified acetogenins on several bacterial strains. different compounds working against _L. monocytogenes_ could exist in greater quantities in the CE at that specific dose; however, in a minor concentration, the effectiveness of the CE decreases. Regarding _S. paratyphi_ and _E. coli_, the antibacterial activity was higher when P-ACGs was used (67.84% and 35.55%, respectively) at 800 ug/mL and 400 ug/mL compared to those observed with CE in the exact quantities. Congruent with the antibacterial effect reported, P-ACGs present an inhibition of 63% for _S. paratyphi_ and 56.25% for _E. coli_[38]. Figure 2 shows that both the CE and P-ACGs present the lowest inhibitory effect against _E. faecalis_, having the highest inhibition percentage (30.95%) at 2000 ug/mL; this is similar to previously published results [39], informing a mortality rate of _E. faecalis_ near 25% using a reconstituted solution of dried fractions from a leaf extract of _A. muricata_ (1000 ug/mL). According to [PERSON] et al. [19], some bacteria may be resistant to some natural extracts, which is associated with their ability to produce biofilm. Measurement of the Membrane Potential of E. faecalis, Listeria monocytogenes, Salmonella paratyphi, and Escherichia coli Treated with the Crude Extract and Purified Acetogenins  
Flow cytometry is a fast and cost-effective technology for assessing the viability of microbial cells under the effect of natural extracts/bioactive compounds [40; 41]. In this study, bacterial death was investigated using the fluorescent viability technique (Live/Dead(r)BacLight). This clearly shows the loss of the integrity of the plasma membrane. Damaged membranes render cells incapable of maintaining an electrical potential and these cells are considered dead. With the Live/Dead(r)BacLight technique, fluorochrome enters and stains dead cells with damaged membranes (Green fluorescence). In contrast, living cells do not pick up fluorochrome because their membrane is intact and can be distinguished as stained or unstained (Red fluorescence) [16; 41].