# Antibacterial Activity of Crude Extract and Purified Acetogenins from _Amona muricata_ Seeds
## 3 Results and Discussion
### Antibacterial Assays of Crude Extract and Purified Acetogenins
Due to the chelation capacity of calcium, A.G.C.s induce the formation of pores, modifying the solubility of the membrane and changing the structure and functionality of the membrane phospholipid bilayer [14; 32]. Therefore, the chelating activity of ACGs plays an important role by increasing the membrane permeability of these microorganisms. Gram-positive and Gram-negative bacteria treated (CE and P-ACGs) with an inhibition halo are shown in Figure 1; the P-ACGs and CE presented a substantial IH, depending on the concentration and the type of bacteria evaluated. Minimum Inhibitory Concentration (MIC) of the Crude Extract and Purified Acetogenins against Gram-Positive and Gram-Negative Bacteria  
The MIC values of the CE and P-ACGs tested on Gram-positive and Gram-negative bacteria demonstrated significantly different antibacterial activity (\(p<0.05\)) (Table 3). The MIC values were observed in a range of 0.009 to 12.50 \(\mathrm{\SIUnitSymbolMicro m}\)/mL, except for the case of the CE on _S. mutans_, where no inhibition was observed, associated with the low concentration of ACGs in the crude extract, which is higher in the P-ACGs [25]. In previous reports of purified compounds, MIC values of 10 \(\mathrm{\SIUnitSymbolMicro m}\)/mL to > 100 \(\mathrm{\SIUnitSymbolMicro m}\)/mL of crude extracts are reported, which present a good antimicrobial effect [33]. The CE and P-ACGs evaluated in this study showed characteristics of antibacterial agents, except for the P-ACGs on _S. aureus_ (2.40-4.01 \(\mathrm{\SIUnitSymbolMicro m}\)/mL). The MIC values of the CE and P-ACGs against the tested Gram-positive and Gram-negative bacteria are completely described in Table 3.  
The MIC values reported in the present work are higher than for the methanolic, ethanolic, and aqueous extracts of _A. muricata_ bark, leaf, and root (65.25, 125, and 250 mg/mL, respectively) of _E. coli_ and _S. pantyaphi_[34]. Reports describe that combining antibiotics with _A. muricata_ seed extract may increase drug uptake, reducing the efflux and biofilm activity of biofilm-forming Methicilline-resistant _S. aureus_[19]. Additionally, the MIC values for _E. coli_ applying _A. muricata_ extracts (leaves, seeds, roots, pulp) were between 256 and 1024 mg/mL were previously reported [35]. Comparatively, the MIC values reported for _E. coli_ in this work with the CE (0.24 mg/mL) and P-ACGs (0.009 mg/mL) are much lower, indicating its greater antibacterial effect. Inhibition Percentage of the Most Prevalent Harmful Bacteria Found in Foods: Enterococcus faecalis, Salmonella pantyphi, Escherichia coli, and Listeria monocytogenes by the Crude Extract and Purified Actegeins  
_E. faecalis_, _S. pantyaphi_, _E. coli_, and _L. monocytogenes_ were selected as the most prevalent harmful bacteria found in foods [36] to determine the percentage of bacterial inhibition of the CE and P-ACGs. Figure 2 shows statistically significant differences (_p_ < 0.05) between the concentration-dependent CE and P-ACGs. The P-ACGs showed greater inhibition in _L. monocytogenes_ (98.66%) when using 800 mg/mL. The number of unsaturations present in the aliphatic chain of ACGs is a structural feature that significantly influences antibacterial activity. ACGs insert into the cell membrane, affecting the cellular functionality and viability of the bacteria [37]. Furthermore, at higher doses (4000 mg/mL), the CE exhibited a higher antibacterial effect (92.15%) against _L. monocytogenes_ than P-ACGs (84.31%). This can be explained through previous reports. It has been reported that high concentrations of crude extracts can overcome antibacterial activity (depending on the individual bacteria) compared to some purified compounds.