# Phytochemical Analysis and Amoebicidal Evaluation of Different Agave Species
## 3 Results and Discussion
### Ethanolic Extracts of Agave Leaves Inhibit the Growth of E. histolytica Trophozoites  
The in vitro amoebicidal activity of ethanolic extracts of the species _Agave tequilana_, _Agave angustifolia_, _Agave rhodacantha_, and _Agave maximiliana_ was evaluated. Our results showed that all the Agave species studied in the present investigation showed promising amoebicidal activity (Figure 2). The negative control (DMSO 0.4%) did not exhibit any significant differences compared with untreated cells. Remarkably, the highest amoebicidal activity was observed with _A. tequilana_; at 72 h, with concentrations of 300 and 600 ug/mL, a more than 90% decrease in the trophozoite count was observed compared to the negative controls (Figure 2D), with IC50 = 193 ug/mL (Table 3). With _A. angustifolia_, a growth inhibitory effect of more than 60% was seen only at 600 ug/mL during the entire exposure period (Figure 2F-H), with IC50 = 364 ug/mL (Table 3). The extracts with moderate activity were _A. rodlacantha_ and _A. maximiliana_ (Figure 2I-P); large and undefined IC50 values were set at 1220 and 1824 ug/mL, respectively, for purposes of illustration (Table 3). Our results correspond with those reported by [PERSON] et al. (2014), who showed the amoebicidal activity of _A. lechuguilla_[38]; however, our results showed that for _E. histolytica_, higher doses are necessary to achieve a reduction of more than 90%. In addition, we confirmed that other species of the Agave genus present varying degrees of amoebicidal activity. The phytochemical analysis (Table 2), showing differences in the presence and relative abundance of some metabolites, could explain the different efficacies of the Agave extracts when killing _E. histolytica_. In addition, in this research study, there was a positive correlation between the higher absorbance values of _A. tequilana_ and _A. angustifolia_ (Figure 1 and Table 1) and the higher amoebicidal properties observed for these species. Chromatographic separation, chromatography-mass spectrometry, and a UV-Vis analysis confirmed the abundant presence of quercetin, kaempfer, gallic acid, \(\beta\)-sitosterols, and steroidal saponins in _A. tequilana_ and _A. angustifolia_[36; 58; 59; 60; 61]. This variety of compounds has been associated with numerous medicinal uses and biological activities. In _E. histolytica_, studies by [PERSON] et al. (2018) demonstrated the amoebicidal activity of epicatechin, kaempfer, and quercetin [62]. Similar amoebicidal activity has been reported by [PERSON] et al. (2001) for \(\beta\)-sitosterol and \(\beta\)-sitosterol glucoside phenolic [63]. In the latter work, the authors described that pure compounds were less active than complete plant extracts, suggesting an important synergism activity with the presence of several metabolites. On the other hand, MTZ is considered the drug of choice for treating amoebiasis; in this study, at 24 h, 600 ug/mL of _A. angustifolia_ showed greater efficacy than MTZ (Figure 3A). After 48 h, _A. tequilana_ and _A. angustifolia_ were demonstrated to be more effective than MTZ (Figure 3B,C). Even though Agave extracts showed higher amoebicidal activity in comparison to MTZ, the plants use natural toxins as a defense mechanism. Additional studies are necessary to establish the toxicity or adverse health effects of Agave extracts for their use as amoebicidal alternatives. \begin{table}
\begin{tabular}{c c} \hline
**Ethanolic Extract** & **IC50** \(\mu\)g/mL * \\ \hline _A. tequilana_ & 193.47 \(\pm\) 22.16 \\ _A. angustifolia_ & 363.54 \(\pm\) 10.41 \\ \hline _A. maximiliana_ & 1219.93 \(\pm\) 18.05 \\ \hline _A. rhodanachta_ & 1824.10 \(\pm\) 7.7772 \\ \hline \end{tabular}  
* Data are expressed as mean concentration \(\pm\) SD (n = 3) values.  
\end{table} 
Table 3: IC50 value of Agave extracts on _E. histolytica_ growth after 72 h of incubation. Figure 2: Extracts of Agave inhibit _E. histolytica_ growth. Growth inhibition curves of _A. requilana_ (**A**), _A. angustifolia_ (**E**), _A. rhodacantha_ (**I**), and _A. maximiliana_ (**M**).