## 4 Discussion
Activated charcoal treatment and the partitioning of the methanolic extract into fractions of different polarities allowed for the broader detection of compounds. The bactericidal activity level of the _B. karatas_ fruits' ME was too low (the MBC values were 20 mg/mL for _E. coli_ and _E. faecalis_ and 40 mg/mL for _S. enteritidis_ and _S. flexneri_) for them to be considered sources of pure antibiotic activity [24]. However, being an edible fruit, the consumption of 10 fruits, with approximately 85 g of fruit juice (equivalent to 5 g of ME), may be enough to prevent mild bacterial infections, similar to the effect of consumption of cranberry fruit extracts [5; 6; 25]. However, this still needs to be evaluated. In the GC-MS analysis of the extract and of the fractions with bactericidal activity (ME and MEC, EAF, and MF), we found that the common compounds were: maleic anhydride (MA), 2,5 furandione, dihydro-3-methylene (titaconic anhydride, ITAN), and 5-hydroxymethylfurfural (5-HMF). An increase in the concentration of 5-HMF in the MEC could be observed (Figure 2), possibly caused by the removal of other compounds; furthermore, 5-HMF could be produced during the drying process (50 \({}^{\circ}\)C/ 24 h) [26]. Maleic anhydride (MA) has been reported to be present in the copolymer manufacturing of biopolymers with activity against pathogens such as _S. enteritidis_, _S. faecalis_, and _E. coli_[27]. However, the results of the present study indicated that it exhibits bactericidal activity as a monomer, and 5-HMF is known to be an intermediary in the Maillard reaction, formed by the degradation of sugars at high temperatures. It has been found in various food products, including nuts, fruit juices, caramel products, coffee, bakery products, malt, and vinegar [28; 29]. 5-HMF has anti-quorum sensing and anti-biofilm activity against _Pseudomonas aeruginosa_[30]. It is also reported to inhibit growth in _E. coli_ LY01 [31], which is consistent with our results (Table 1). MA, ITAN, and 5-HMF showed activity against the bacteria evaluated (Table 1). However, their activity levels were relatively low [24] and their concentrations were not sufficient (Figure 2) to explain the ME activity against the bacteria analyzed. Therefore, further studies are required to identify the other bactericidal compounds of the ME. We did not find evidence of the bactericidal activity of ITAN in the literature reports; however, it is reported to be an inhibitor of _M. tuberculosis_ isocitrate lyase (MitCL) [32]. These authors showed that 0.03 mg/mL of ITAN can inhibit 90% of MiCL. This enzyme can promote the persistence and virulence of bacteria in macrophages, develop resistance to antibiotics, and promote the growth and survival of _M. tuberculosis_ during its latent infection [33; 34; 35]. Considering their crucial roles, MtCLs are current inhibition targets for the development of new antibiotics to treat tuberculosis [35]. Considering that the structures of ITAN, MA, and 5-HMF have a certain degree of similarity, using molecular docking analysis, we evaluated whether MA and 5-HMF interact molecularly at the MtCL catalytic site as does ITAN. In Figure 3, it can be observed that MA, ITAN, and 5-HMF have similar binding patterns to the catalytic site. These results could suggest the possibility that MA and 5-HMF have roles in the inhibition of MtCL function. To fully understand this aspect, it is necessary to carry out enzyme activity assays; however, this was not the objective of this study. Metabolites from _B. karatas_ fruits have other biological activities in addition to their bactericidal activity (Table 3). This strengthens the proposal that the fruit of _B. karatas_ can be a functional food.