4.1.4. Pharmacological Studies

The pharmacological use of *B. gaudichaudii* is widely explored due to its repigmentating property, clinically used for the treatment of vitiligo, a skin pigmentation anomaly. This property is attributed to the presence of furanocoumarins, molecules found mainly in the *B. gaudichaudii* root cortex, which have photosensitizing action, and associated with ultraviolet (UV) radiation, is used to treat not only vitiligo, but also other skin disorders such as psoriasis, systemic lupus erythematosus and mycoses [101] (Figure 3).

The mechanism of action of furanocoumarins for the UV phototherapy technique is not ye<sup>t</sup> defined. However, previous studies have proposed that there is induction of apoptosis of cells that participate in the generation of disorders, such as T cells, mast cells and keratinocytes, in addition to

increased proliferation of melanocytes and reduced histamine release by mast cells and basophils [102]. Lang et al. [103] reported an increase in CD8 + T lymphocytes specific for the skin in a patient with vitiligo and that this may be related to the etiology of the disease, since these cytotoxic T lymphocytes may participate in the reduction in the number of melanocytes observed in the disease (Figure 3).

**Figure 3.** Use of furanocoumarins in the technique of extracorporeal photopheresis for the treatment of systemic or multifocal diseases: Leukocytes obtained by apheresis are exposed to 8-metoxipsoraleno (8-MOP), which is activated by UVA radiation and covalently binds to the DNA of these cells, causing damage and inducing apoptosis within 48 h. Pre-apoptotic leukocytes are reintroduced into the peripheral circulation, being recognized and phagocyted by antigen-presenting cells in phagolysosomes. This recognition induces tolerogenic anti-inflammatory response, which reduces the production of pro-inflammatory cytokines like IL-6, IL-12, IL-23, and TNF-α and increases the production of anti-inflammatory cytokines like IL-10, TGF-β1, and PGE2 [104]. 8-MOP, 8-metoxipsoraleno; IL-6, interleukin 6; IL-10, interleukin 10; IL-12, interleukin12; IL-23, interleukin 23; TGF-β1, Transforming growth factor beta 1; TNF-<sup>α</sup>, Tumor Necrosis Factor-Alpha. Another technique that uses furanocoumarins is extracorporeal photopheresis (ECP), which treats systemic or multifocal diseases such as Crohn's disease, type 1 diabetes mellitus, multiple sclerosis, and rheumatoid arthritis. In this technique, the most used furanocoumarin is 8-methoxypsoralen (8-MOP), to which leukocytes, obtained by apheresis, are exposed. 8-MOP is activated by radiation and covalently binds to leukocyte DNA, leading to apoptosis within 48 h. These pre-apoptotic leukocytes are reintroduced into the peripheral circulation, where are recognized and phagocyted by antigen-presenting cells in phagolysosomes. This recognition induces a tolerogenic anti-inflammatory response that leads to a reduction in the production of pro-inflammatory cytokines IL-6, interleukin-12 (IL-12), interleukin-23 (IL-23) and TNF-α and induces the production of anti-inflammatory cytokines such as interleukin-10 (IL-10), transforming growth factor beta 1 (TGF-β1) and prostaglandin E2 (PGE2) (Figure 3) [102,104].

Lourenço, [105] tested the possible antibacterial activity of the ethanolic extract of *B. gaudichaudii* leaves and stem bark against *S. aureus* from clinical samples, beta-hemolytic streptococci, *P. aeruginosa*, *E. coli*, *Citrobacter* sp. and *Proteus* sp. Both extracts showed antimicrobial activity against all bacterial strains, including multi-resistant strains, with variable percentage of bacterial growth inhibition. However, the bioactive compounds responsible for this activity have not ye<sup>t</sup> been identified.

The ethanolic extract from *B. gaudichaudii* stem bark was analyzed for antifungal properties on *Candida albicans* and *Candida* sp. This activity was observed for *C. albicans* at concentration of 200 mg/mL and for *C. non albicans* at concentrations of 100 mg/mL and 200 mg/mL [96]. The hydroalcoholic extract from *B. gaudichaudii* leaves was also tested for trypanocidal activity. Mice previously infected with the blood form of *Trypanosoma cruzi* received the extract at different concentrations. The infection was

assessed by counting parasites present in 10 μL of blood between the 5th and the 11th day after infection, showing reduction in the number of trypomastigotes at concentration of 100 mg/kg. Although the chemical compound responsible for this activity has not been identified, it is possible to associate it with furanocoumarins, which are the main components of the extract that may have acted through the production of oxide and superoxide radicals [106].

β-Amyrin (**38**), a triterpene found in *B. gaudichaudii* root bark promotes sleep modulation through the activation of the GABAergic system. For the analysis, a pentabarbital-induced sleep model was used in mice, and it was observed that, after the administration of 1, 3 or 10 mg/kg of β-amyrin (**38**), the time for the beginning of sleep was reduced and the sleep duration increased significantly, which effects were inhibited after administration of a type A gamma-aminobutyric acid antagonist receptor (GABAA), demonstrating that this property is associated with the GABAergic system. In addition, the levels of gamma-aminobutyric acid (GABA) in the brain were analyzed, which increased after the administration of β-amyrin (**38**), which could be related to the blocking of GABA transaminase, inhibiting GABA degradation and, consequently, increasing its available concentration [107].

A study was carried out on the role of β-amyrin (**38**) in attenuating the neurodegeneration of Parkinson's disease using *Caenorhabditis elegans* strain. The analysis describes a protective effect of β-amyrin (**38**) against neurotoxicity induced by 6-hydroxydopamine (6-OHDA), which was related to the possible antioxidant role of β-amyrin (**38**). This study also investigated its anti-apoptotic activity on the expression of pro-apoptotic genes in *C. elegans*, which were not significantly altered after treatment with β-amyrin (**38**). The aggregation of α-synuclein protein is one of the mechanisms associated with Parkinson's disease. The effects of β-amyrin (**38**) on this mechanism were compared to the effect of the medicine clinically used to treat the disease, L-dopa and both substances significantly reduced the α-synuclein aggregation [108].

Sunil et al. [109] analyzed the antioxidant activity of β-amyrin (**38**) in Wistar rats with oxidative stress induced by carbon tetrachloride (CCl4). The effect was positive in the elimination of DPPH radicals, hydroxyl, nitric oxide (NO), superoxide radicals and had strong reducing and suppressive power of lipid peroxidation. The increase in free radical levels also leads to elevated levels of hepatic enzymes serum glutamic oxalacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT) and LDH, which after administration of β-amyrin (**38**), had their levels reduced. In addition, the levels of the antioxidant enzymes SOD, CAT, GSH and GPx, were high.

A study analyzed the β-amyrin (**38**) activity on inflammation induced by lipopolysaccharide (LPS) and IFN-γ in the microglial cells of rats. β-amyrin (**38**) reduced the expression of pro-inflammatory factors such as TNF-<sup>α</sup>, IL-1β, IL-6, PGE-2 and cyclooxygenase-2 (COX-2) and increased the expression of arginase-1. The reduction of factors was attributed to the activation of the cannabinoid receptor, since antagonists of these receptors inhibit the anti-inflammatory effects of β-amyrin (**38**). Through another not ye<sup>t</sup> identified mechanism, β-amyrin (**38**) reduces the activity of COX-2 and, consequently, the production of PGE-2, commonly inhibited by the action of non-steroidal anti-inflammatory drugs. Enzymes arginase-1 and NO synthase compete for the same substrate, L-arginine, with the overexpression of arginase-1, observed by the increase in its product, urea, NO production is compromised, since the availability of substrate for NO synthase is reduced [110].

Marmesin (**33**), a coumarin found in *B. gaudichaudii* root bark, is the target of several studies that investigate its medicinal properties, especially its anti-tumor properties. The in vitro and in vivo activity of marmesin (**33**) was evaluated on cells with human leukemia and healthy human monocytes. The results indicate that marmesin (**33**) exerts dose-dependent anti-tumor activity and that the cytotoxic effect on healthy monocytes was lower, which is essential for the safety of a probable treatment, since it predicts that the compound has action selectivity, reducing the possibility of adverse effects. Marmesin (**33**) increased the expression of pro-apoptotic protein Bax and reduced the expression of anti-apoptotic protein Bcl-2, increasing the Bax/Bcl-2 ratio, which promotes activation of caspase 3, leading to apoptosis, with mechanism of action similar to other chemotherapeutic drugs. Marmesin (**33**) also causes an increase in intracellular ROS and reduces the mitochondrial membrane potential

(MMP), which was related to the reduced migration of cells with leukemia, important in inhibiting metastases [111].

Another analysis of the same property associated with marmesin (**33**), but on lung cancer and tumor angiogenesis, reports anti-tumor activities of marmesin (**33**) mediated by the inactivation of mitogenic signaling pathways and negative regulation of proteins related to cell signaling, including vascular endothelial growth factor-2 receptor (VEGFR-2), integrin β1, integrin-linked kinase (ILK) and matrix metalloproteinases-2 (MMP-2), nullifying mitogen-stimulated proliferation and invasion in cells expressing p53 or not [112].
