Prospection of bioactive substances produced by Bacillus, isolated from cave.

Cancer is one of the most deadly diseases in the world. This disease is qualified with the disordered growth of altered cells in the body that can lead the individual to death. One of the great challenges of science is finding new substances to combat the irregular growth of these cells. Compounds found in nature show a huge variety of structure and pharmacological activity. Microorganisms from extreme environments, such as the seabed, fountains and caves, the conditions of competition for resources in which they live, show as a promising source of these compounds. In 2014, microbial ecology work in the Pedra da Cachoeira cave, a preserved environment in the dense Ombrófila Forest in the Eastern Amazon, located in the municipality of Altamira-PA, resulted in the isolation of 49 bacterial strains. Thus, this study aimed to perform the biomonitored study of two strains, CV26A and CV26B, against the tumor cell B16F10 (melanone) and cell 3T3 (fibroplast) via the MTT method. Bacteria were identified by analysis of ribosomal RNA coding DNA sequences. Bacteria were cultivated for biomass production in Luria-Bertani (LB) liquid culture medium. For the choice of the ideal period of microbial development aiming at the production of biocompounds, an optimization was made from a growth curve. From this, organic extracts were produced after 30 h (stationary phase) and 40 h (decline phase) of cultivation. Four extracts were obtained by liquid - liquid partition of the residual culture medium and four extracts from the bacterial cells after cultivation. The extracts obtained (CV26AM30h, CV26AI30h, CV26BM30h, CV26BI30h CV26AM30h, CV26AI40h, CV26BM40h and CV26BI40h) were submitted to cytotoxic and fractional assays via classical exclusion chromatography using methanol as the mobile phase. Cell viability of the extracts was evaluated by measuring the IC50 at concentrations of 6.25 μg / mL, 12.5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL and 400 μg / mL. Samples resulting from chromatographic fractionation were subjected to 1 H and 13 C NMR spectroscopic analyzes. From the phylogenetic analyzes of the DNA sequences of both CV26A and CV26B strains, grouping with the Bacillus subtilis species was verified. In the cell viability study, CV26AI30H, CV26AM40H and CV26BM30H extracts were highlighted as a function of the minimum inhibitory concentration (IC50) and selectivity index (SI) measurements (Table 1). Chromatographic fractionation led to the isolation and identification of a diketopiperazine.

Table 1. Inhibitory concentration (IC50) and selectivity index (SI)

Sample lineage IC50 (g/mL) IS
CV26AI30H B16F10 83,99 > 4,7
CV26AI30H 3T3 > 400
CV26AM40H B16F10 121,89 2,12
CV26AM40H 3T3 229,1
CV26BM30H B16F10 171,8 1,49
CV26BM30H 3T3 256,4

Keywords: Bioactive Compound, Bacillus, Cancer.