Uso de técnicas hifenadas (CLAE-DAD/CL-EM) na identificação de metabólitos secundários da atemoia (Annona cherimola x Annona squamosa) e avaliação da atividade citotóxica

Abstract

Atemoieira is a hybrid resulting from the crossing of two species of the Annona genus (A. cherimola and A. squamosa) that are known for the richness of biologically active compounds. Due to the increasing number of new cases of cancer each year and aggressive and often inefficient treatments, it is necessary to search for new effective cytotoxic compounds. The use of hyphenated techniques of separation and identification of plant matrix molecules has been highly valued, since they can accelerate searches for these potentially active compounds. Therefore, the objective of this work was to analyze the cytotoxic potential of extracts and phases of different parts of the atemoia plant and to correlate the results with their chemical composition defined by means of different isolation and identification techniques. For this, plant leaves were collected in Petrolina-PE and extracted with hexane followed by methanol to obtain crude extracts hexane (EHB-F) and methanolic (EMB-F), respectively. The EMB-F was subjected to an acid-base extraction to obtain the alkaloidal fraction. The common chromatographic techniques used led to the isolation of a novel alkaloid (here called dehidro-N-oxide anomuricine) and the anonaine, asimilobine, lanuginosine, liriodenine, lisicamine, pronuciferine, stefarine and anomuricine alkaloids whose identifications were made by analysis of spectra masses, uni and bidimensional Nuclear Magnetic Resonance and by comparison with literature data. Anomuricine and dehydro-N-oxide-anomuricine had their fragmentations proposed. In addition, the aerial parts of the plant (mixture of stems and leaves), went through the same procedure that leaves to obtain the hexane crude extracts (EHB-PA), methanol (EMB-PA) and alkaloidal fraction (FAT-PA) which was further subjected to extraction solid-liquid to obtain subfractions - hexane (FAT-Hex), chloroform (FAT-CHCl3), ethyl acetate (FAT-AcOEt) and methanol (FAT-MeOH). All the extracts, fractions and subfractions obtained were tested for their cytotoxicity. The results were compared to liquid chromatography and gas chromatography, both coupled to mass spectrometer (LC-MS and GC-MS, respectively), for chemical correlation with biological potential. All procedures for access to genetic heritage and associated traditional knowledge were carried out and the project was registered with SisGen (Record # ABD9AA7). As result of analysis by LC-MS was possible to identify the presence of anomuricine, dehydro-N-oxide-anomuricine, scoulerine, reticuline, isocorydine, norisocorydine, asimilobine, nornuciferine, liriodenine, anonaine in the EMB-PA. The analysis of EHB-PA by GC-MS led to the identification of 29 compounds, with spatulenol being the major. This extract (EHB-PA) inhibited more than 90% of the growth of glioblastoma cells tested (SF-295). It was also possible to observe that the EMB-PA had its activity potentiated when fractioned, with a difference of up to 41%, approximately. Anomuricin was tested against nine cell lines and presented a cytotoxic potential against leukemic cells (HL-60) with selectivity index of 1.66. The different parts of the hybrid showed high cytotoxic potential and therefore this plant can be considered a promising source of cytotoxically active compounds.