4.1.5. Toxicity Studies

Furanocoumarins, the major constituents of *B. gaudichaudii* roots, are phototoxic substances that the plant uses as a protection mechanism against phytopathogenic microorganisms and herbivores. The mechanism of action is intercalation in the double helix of the DNA structure and in molecular complexation, and when activated by light, they react with pyrimidine bases, mainly with thymine, which can promote toxic, mutagenic and carcinogenic e ffects [113].

The genotoxic activity of *B. gaudichaudii* was evaluated using aqueous and methanolic extracts on *Salmonella typhimurium* strains, which showed an increase in chromosomal aberrations in cultures submitted to the methanolic extract in the G1/S and S phases of the cell cycle. For the aqueous extract, results were not significant, which is due to the lower amount of furanocoumarins extracted by the aqueous extract compared to the methanolic extract [114].

Lourenço, [91] performed toxicity tests for the aqueous and methanolic extract from *B. gaudichaudii* root cortex and obtained through the MTT technique (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide), cytotoxicity indexes (IC50) of 4.65 mg/mL and 1.31 mg/mL, for each extract, respectively. The mutagenicity assay was also performed with *Salmonella typhimurium*, which generated the mutagenicity ratio (MR) through the observation of revertants, that is, the number of individuals whose natural phenotype induced by mutation, was restored. The ratio is calculated using the average number of revertants in the test plate, spontaneous or induced, divided by the average number of revertants per negative control plate, that is, spontaneous, with the sample being considered positive with ratio greater than or equal to 2. MR was higher for the aqueous and methanolic extracts in the TA102 strain in the presence of microsomal fraction S9, which reveals whether the substance or sample is mutagenic in its original form or whether it needs to be metabolized or activated to become mutagenic, in this case, it has been metabolized.

A study determined the acute toxicity of the extract from *B. gaudichaudii* root bark orally and intraperitoneally administered in mice. Oral administration led some animals to death, and the median lethal oral dose (LD50) was 3517.54 mg/kg and intraperitoneally, LD50 was 2871.76 mg/kg. Up to 2000 mg/kg, 40% of animals had diarrhea, and increasing the dose, some of them presented, in addition to diarrhea, dry eyes, eyelid occlusion, ocular hemorrhage, epistaxis, weight loss, tachypnea and death at the lethal dose. Dead animals were analyzed and showed dilation and hemorrhage, in addition to increase in the amount of hemosiderin in the spleen, indicating previous hemorrhage and destruction of erythrocytes [80].

Using dry *B. gaudichaudii* extract, the acute and subacute toxicities of this species were analyzed, with an estimated lethal dose (DLA) of 3800 mg/kg. In the acute study, 14 days after administration, leukopenia and hemosiderin accumulation were observed in the spleen, and in the subacute, after 30 days, changes in the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine and total proteins were observed, indicating hepatotoxicity and nephrotoxicity for the dose of 100 mg/kg, in addition to leukopenia and renal hemorrhage [115].

These clinical signs of hemorrhage may be related to the anticoagulant property of some coumarins found in large amounts in this species. To exert such activity, coumarins act as competitive inhibitors of epoxide reductase, an enzyme that reduces vitamin K, oxidized by participating as co-factor in the synthesis of coagulation factors II, VII, IX and X. With the inhibition of epoxide reductase, this regeneration does not occur, depleting the levels of active vitamin K and consequently inhibiting the synthesis of coagulation factors (Figure 4) [116].

**Figure 4.** Proposed mechanism for inducing hemorrhage by coumarins: Coumarins act as competitive epoxide reductase inhibitors. This enzyme reduces oxidized vitamin K during its participation as co-factor in the synthesis of coagulation factors II, VII, IX, and X. With epoxide reductase inhibition, the reduction that occurs to regenerate vitamin K is blocked, depleting its levels and, consequently, inhibiting the synthesis of coagulation factors, causing hemorrhage.
