Estudo fitoquímico de Pilocarpus spicatus subsp. aracatensis (Rutaceae) e avaliação da atividade moduladora da resistência bacteriana de cumarinas isoladas dessa espécie

Abstract

Research in the area of natural products has grown in recent years due to the great demand of the population for less offensive and more viable therapeutic alternatives. Bacterial drug resistance is a public health problem that can be solved with the use of resistance modifying agents obtained from natural sources. The species Pilocarpus spicatus sbsp. aracatensis Kaastra is known in folk medicine as jaborandi-da-restinga, belongs to the Rutaceae family and is distributed in Northeast Brazil. Previous studies have reported the potential of P. spicatus as a source of biologically active metabolites. The purpose of this work is the phytochemical investigation of this species and evaluation of the modulating activity of bacterial resistance against Staphylococcus aureus. The aerial parts of P. spicatus were collected in Maturéia-PB. One specimen (7428) was deposited at the Herbarium Prof. Lauro Pires Xavier (UFPB). The plant material was dehydrated, crushed and extracted with 95% EtOH. The extractive solution was concentrated in a rotary evaporator to obtain the crude ethanol extract (CEE). A portion of the CEE (100 g) was subjected to liquid-liquid partition using the solvents hexane, chloroform and ethyl acetate. The hexane phase (4 g) was subjected to column chromatography (CC) using silica gel which resulted in 20 fractions. The PAH03 fraction was subjected to a new CC to obtain xanthoxylin. The PAH12 fraction was subjected to preparative high performance liquid chromatography (HPLC) to obtain bergaptol, heraclenol, pabularinone and isosaxalin. PAH13 was subjected to semi-preparative HPLC, resulting in the isolation of more isosaxaline and 8-(3-ethoxy-2-hydroxy-3-methylbutyloxy)-psoralen. The chloroform phase (5.5 g) was subjected to column chromatography (CC) using silica gel resulting in 25 fractions which were grouped in 12. The PACF fraction was identified as heraclenol. The PACE fraction was subjected to semi-preparative HPLC, resulting in the isolation of scopoletin and isopimpineline. The structural identification of the isolated chemical constituents was performed through analysis of the 1H and 13C NMR, HMBC, HSQC, COSY and NOESY spectra, as well as high resolution mass spectra. The drug resistance modulating effect of two isolated coumarins (heraclenol and isoxalin) on norfloxacin and ethidium bromide against an efflux strain of S. aureus was evaluated. Heraclenol reduced the MIC of norfloxacin two-fold and isosaxalin reduced it eight-fold. The reduction of the MIC of ethidium bromide demonstrated that these compounds are putative inhibitors of the efflux system in bacteria. This work contributed to the expansion of chemical knowledge of the P. spicatus species, demonstrating its potential as a source of coumarins.