Bioprospecção de alcaloides em extratos de fungos endofíticos de Duroia macrophylla Huber (Rubiaceae)

Abstract

Endophytic microorganisms have enormous potential for the production of bioactive substances and are an alternative for the development of new drugs against various diseases, including cancer and infectious diseases. The plants of the Rubiaceae family have a great biotechnological potential, such as Duroia macrophylla, which has already revealed the presence of an alkaloid with antitumor activity and low toxicity to nonneoplastic cells and eight alkaloids with antimycobacterial activity, which resulted in two patents for this research group. For these reasons it was proposed the study of the biotechnological potential of the endophytic microorganisms of this plant. A total of 47 endophytic fungi were isolated and 21 morphologically different fungi were selected for the chemical and biological exploration of their extracts from the mycelium and fermented broth. Among the methanolic extracts, none showed antioxidant activity and 18 showed antibacterial activity. The extracts obtained from the ethyl acetate phase of the fungi Dm SB 43 and Dm BDA 12c were active against A. salina, presenting LC50 of 109.5 and 605.5 μg/mL, respectively. Evidences of the presence of phenolic substances, alkaloids, terpenes and sugars were found. Four fungi showed evidence of alkaloid production. One of them was selected for a new crop with more replicates to obtain larger amounts of extracts. This fungus was identified as Xylaria sp. and all extracts showed high or moderate toxicity against A. salina. Only two presented no antibacterial activity and no extract showed antioxidant activity. The extracts myceliumdichloromethane, mycelium-ethylacetate and phase-dichloromethane showed evidence of alkaloids in the CCDC and they were fractionated. From these fractions were isolated cytochalasin D and two other substances that are in elucidation phase. Due to the low mass of isolated substances, only cytochalasin D was submitted to biological and chemical tests, which was strongly cytotoxic against A. salina (31.4 μg/mL) and showed no antibacterial and no antioxidant activity at the concentrations tested. The higher antitumor activity of extracts of Xylaria sp. and cytochalasin D was against the HCT 116 cell line (53.5, 56.9, 58.4 and 77.28% of cell viability). On the MCF7 and DU145 cells, absence or low activity was observed. Against the MRC-5 healthy cell line, cytotoxic D did not show cytotoxicity. This result may be useful in relation to the toxicity of the substance in possible correlations with other biological activities. Therefore, this study demonstrated the diversity of chemical classes and the potential of biological activities that the endophytic fungi of D. macrophylla present, in addition to the first report of Xylaria sp. in this plant, which showed to be a major producer of cytochalasin D.