6.1.4. Pharmacological Studies

The alcoholic extract from *B. antiacantha* fruits, and methanolic, hexanic, ethyl acetate and raw alcoholic extract from leaves were tested for antimicrobial, molluscicidal and antioxidant properties. All extracts were tested against clinically isolated *C. albicans* and *C. glabrata* strains and *C. albicans* (ATCC 90028), *E. coli* (ATCC 8739), *P. aeruginosa* (ATCC 9027) and *S. aureus* (ATCC 6538) reference strains. All strains tested were not affected by extracts, that is, none of the extracts showed antimicrobial or antifungal activity. The evaluated extracts were considered inactive in relation to the molluscicidal activity, since no significant effects were found at concentration of 400 μg/mL, a value above the maximum for this activity. To test the antioxidant activity, the performance of extracts against the DPPH radical was observed, showing unsatisfactory results, since only the extract from leaves with ethyl acetate obtained moderate performance, with 35% inhibition of radicals [182].

The methanolic (BAM), hexanic (BAH), dichloromethane (BAD), ethyl acetate (BAA) and hydromethanolic (BAHa) extracts from *B. antiacantha* leaves and fruits were submitted to antibacterial activity tests. Only BAM and BAD extracts from leaves and BAA extract from fruits inhibited the growth of *P. aeruginosa*, and the BAM extract from leaves showed activity on *E. coli* [193].

Daucosterol (**63**) is a saponin found in the methanolic extract from *B. antiacantha* leaves. This substance was the focus of a study that investigates its possible anti-inflammatory role on colitis induced by dextran sulfate sodium (DSS) in mice. Colitis is an inflammatory reaction in the large intestine, which source may be infectious or autoimmune. Pre or post-treatment with daucosterol (**63**) provided relief from the clinical symptoms of colitis, with reduction in the number of regulatory T cells, in the activity of Natural Killer (NK) cells and in the production of Immunoglobulin A (IgA), whose increase is characteristic of the disease. In addition, ROS inhibition and reduction in the expression of inflammatory cytokines such as TNF-<sup>α</sup>, IL-6, IL-1β and IFN-γ were observed, as well as increase in the anti-inflammatory cytokine IL-10 [198].

Induction of autophagic apoptosis in prostate cancer by daucosterol (**63**) was also analyzed, indicating anti-tumor activity. The action of this phytoconstituent on cancer cells promoted the

interruption of the cell cycle by activating the mitochondrial-dependent apoptotic signaling pathway that leads to increased expression of the pro-apoptotic proteins caspase 3 and 9 and Bax, in addition to reducing the expression of Bcl-2. The administration of the 3-methyladenine (3-MA) autophagy inhibitor attenuated the apoptotic e ffect triggered by daucosterol (**63**), indicating that its mechanism of action is the induction of autophagic apoptosis. This mechanism may also be related to the action of JNK protein kinases, known for the regulatory role of cell proliferation, survival and death. Daucosterol (**63**) increased the level of JNK proteins active in cancer cells, while the specific JNK inhibitor (SP600125) inhibited its action, which indicates that this phytoconstituent has tumor suppressive e ffect through the induction of autophagic apoptosis dependent on the activation of the JNK signaling [199].

The apoptotic action promoted by daucosterol (**63**) has been investigated by several studies that correlate it with the anti-tumor action on breast [200] and prostate [201] cancers. Esmaeili et al. [202] investigated the anti-tumor mechanism of daucosterol (**63**) on human breast adenocarcinoma cells and concluded that the apoptotic mechanism is associated with the mitochondrial pathway, with loss of the mitochondria membrane potential and release of cytochrome C being observed after reduction of the Bcl-2/Bax ratio through the increase in the levels of intracellular ROS and decrease in the levels of antioxidant protein GSH and MMP. In addition, the PI3K/AKT pathway is inhibited by daucosterol (**63**) by reducing the AKT expression, whose levels are increased in some types of tumor. This reduction occurs by increasing the expression of the phosphatase and tensin homolog (PTEN), a negative regulator of the PI3K/AKT pathway. Thus, the inhibition of the PI3K/AKT pathway, the increase in Bax expression and the reduction of Bcl-2 levels, promote apoptosis mediated by the mitochondrial pathway and activation of caspases 3 and 9 (Figure 7) [202].

**Figure 7.** Daucosterol mechanism on human breast adenocarcinoma cells: After treatment of tumor cells (MCF-7) with daucusterol, a phytosterol abundantly present in *Bromelia antiacantha* extracts, the positive regulation of Phosphatase and Tensin Homologue (PTEN) blocks Protein Kinase B (Akt) activation through PI3K. Daucusterol induces reactive oxygen species (ROS) synthesis that leads to mitochondrial oxidative stress and, subsequently, release of cytochrome C. Subsequently, the activation of caspases causes cell apoptosis [202]. Δψm, mitochondrial membrane potential; Akt, Protein Kinase B; Bax, BCL2 Associated X; Bcl2, B-cell lymphoma 2; Cyt C, cytochrome C; PI3K, phosphatidylinositol-3-kinase; PTEN, phosphatase and tensin homologue; ROS, reactive oxygen species.

Another study also investigated the anti-tumor property of daucosterol (**63**), which inhibits the migration and invasion of hepatocellular carcinoma cells using another mechanism, the Wnt/β-catenin signaling pathway, which regulates various physiological processes such as cell proliferation, apoptosis, di fferentiation, transcription and translation. Daucosterol (**63**) acts by significantly inhibiting the

expression of β-catenin, reducing the possibilities of cell proliferation, migration and invasion, which would occur through the Wnt/β-catenin pathway [203].

The neuroprotective effect of daucosterol (**63**) was also investigated in a study that reported the action of this compound as a modulator of the growth factor expression similar to insulin type 1 (IGF-1), which plays a neuroprotective role. Daucosterol (**63**) increased the level of AKT phosphorylation, resulting in more AKT in the active form and indicating that the AKT pathway was activated, resulting in protective effect on treated neurons, since the activation of the PI3K/AKT pathway favors cell survival, as it promotes the inactivation of GSK-3b, a pro-apoptotic protein, whose inhibition causes an increase in Mcl-1, which has the opposite effect, reducing the activity of caspase 3 [204].
