## 4 Discussion  
The extract of _A.__nuvasiorum_ obtained by UAE was active against _Staphylococcus aureus_, yeasts, and molds tested. It suggests that _A.__nuvasiorum_ leaf extract has a broad-spectrum antimicrobial effect. The green extraction methods employed here, such as ultrasound extraction, minimize the use of solvents and energy [1; 2; 3]. We chose ethanol as a solvent because it is one of the most environmentally friendly and biodegradable compared to other organic solvents [32]. [PERSON] et al. (2017) compared the energy consumption and CO\({}_{2}\) generation of three extraction methods: UAE requires the lowest energy consumption (0.25 kW\(\cdot\)h vs. 6.0 kW\(\cdot\)h for maceration, and 8.0 kW\(\cdot\)h for Soxhlet) and generates the lowest amount of CO\({}_{2}\) per 100 g of solid extract (200 g vs. 3600 g for maceration, and 6400 g for Soxhlet). So, the UAE method represents an eco-friendly alternative for obtaining bioactive plant extracts. Also revealed is the fact that the UAE process may involve single or combined mechanisms. The possible mechanisms are fragmentation, erosion, capillarity, detetxturation, and sonoporation leading to cell disruption and increased mass transfer [4]. Additionally, ultrasonic waves improve concentration and synthesis processes using so-called sonochemical methods [33; 34; 35]. The extract presented MIC values \(\leq\)1.25 mg/mL for all the microorganisms tested. These data suggest that the extract contains promising antimicrobial molecules according to the criteria established by [PERSON] and [PERSON] (2005) [36], and is important for further identification or isolation of bioactive molecules. Remarkably, the antibacterial activity  
Figure 1: Antimicrobial effect of _Agave__nuvasiorum_ leaf extract. (**A**) Turbidity curves of bacterium and yeasts (**a**) and mold microorganisms (**b**). Values represent means \(\pm\) SE for three replicates. (**B**) Growth percentages were obtained from a comparison of the areas under the curves (AUC) of microorganisms compared to the control (AUC established as 100%). AUC of bacterium and yeasts (**a**) and mold microorganism (**b**). Significant differences *** \(p<0.001\), in comparison with the control group, were detected by one-way analysis of variance (ANOVA)-[PERSON] ’s multiple comparison test. of _A.__nusaviorum_ against _S. aureus_ (MIC 0.5 mg/mL) was higher than those previously reported for _Agave_ species, e.g., _A.__pita_ and _A.__sisalana_ showed MIC values for _S. aureus_ of 7.0 mg/mL and 10 mg/mL, respectively [37; 38]. Likewise, the antifungal activity against _Candida_ species (MIC 0.25-1 mg/mL) was higher than those reported in the literature, e.g., _A.__sisalana_ exhibited MIC values \(>\) 10 mg/mL [38; 39]. In addition, the antifungal activity against _Trichophyton_ species (0.4-1 mg/mL) was higher than the data reported in the literature in other _Agave_ species that showed MIC values \(>\) 3.5 mg/mL [37]. Interestingly, _C.__parapsilosis_ was more sensitive to the extract evaluated here than _C. albicans_, which suggests that the extracts tested could be a more effective therapeutic alternative for species other than _C. albicans_. A similar effect was observed on _T.__nentagrophytes_ (CFQ-H-92) and _T.__nurbum_ (CFQ-H-93). Furthermore, the extract inhibited the growth of all clinical isolates of _Trichophyton_ (Figure 1B(a,b)). However, a positive correlation between in vitro and in vivo studies is not always observed [40], so it is important to continue in vivo evaluations of _A.__nusaviorum_ extract to validate in vitro findings. A possible explanation for the higher antimicrobial activity observed by the extract of _A.__nusaviorum_ obtained by UAE may be that these bioactive metabolites are found in higher amounts in the evaluated extract due to the green extraction method used. UAE generates a higher heat and mass transfer, increases the extraction yield, and improves the quality of the extract [2; 3; 4].