Isolamento de compostos e atividades biológicas de Simaba maiana Casar. e análise funcional de citocromos P450 envolvidos na biossíntese de monoterpenóides em Arabidopsis thaliana

Abstract

Secondary metabolites are compounds that are not necessary for the survival of the organism, but which are to be related to the organism's interaction with its environment. This work studied compounds of secondary metabolism through the study of chemical and biological extracts and fractions Simaba Maiana Casar. and the involvement of candidate genes (TPS10, TPS14, and CYP71B31 CYP76C3) in the metabolism of monoterpenes in Arabidopsis thaliana. The extracts from roots and stems of Simaba Maiana were tested in antioxidant activity, molluscicide, inhibition of lymphocyte proliferation, inhibition of NO production, anti-Leishmania amazonensis and anti-Trypanosoma cruzi. The four candidate genes (CYP76C3, CYP71B31, TPS10 and TPS14) were selected with the CYPedia, which calculates the coexpression between genes based on the Arabidopsis Affymetrix ATH1 microarray. The extracts and fractions Simaba Maiana showed a lower antioxidant activity by DPPH method, low concentrations of total phenols measured by Folin-Ciocalteu, however, a good antioxidant activity by TBARS method, using three agents of oxidative damage (AAPH, FeSO4 and H2O2). The extract showed cytotoxic activity and molluscicidal concentration of 100 mg/mL. The crude extract of the stem was not active for leishmanicidal and trypanocidal. This extract did not inhibit NO production, but showed a high percentage of inhibition of lymphoproliferation. The skimmianine furoquinoline alkaloid and pellopterin furanocoumarin were isolated from chloroform fraction. The candidate genes showed a similar pattern of expression of the stamen, more specifically the top of the filaments. Heterologous expression in transiently expressed in leaves of Nicotiana benthamiana, in the volatiles of TPS10 TPS14 (alone) were found enantiomers R-(-)-linalool and S-(+)-linalool. In the extraction buffer leaf discs were found that CYP76C3 converts linalool to E-8-hydroxy-linalool and E-8-oxo-linalool, and CYP71B31 in 1,2-epoxy-linalool. The analysis of the methanol extract of the discs incubated in S-(+)-linalool showed the use of this substrate by P450s converting to lilac alcohol for both P450s. Analysis of flowers of Arabidopsis mutants showed minor differences, analyzes with extracts of fresh flowers in UPLC-MS/MS MRM mode, has been found a compound having the same signature as linalool, however with different retention time and may be is an indication of linalool bound.