Mumie constituents and their biological activity: modulation of reactive oxygen species (ROS) production of macrophages

摘要

For investigation of mumie constituents, the isolation of substances was performed by the classical method of humic substances fractionation that is based on their different solubility in water at different pH and ion-exchange properties. The elution patterns were investigated by size-exclusion chromatography using Sephadex G25, and the fractions obtained were characterized by UV-Vis absorbance, fluorescence, and infrared (IR) spectroscopy. The characteristic absorption bands typical for humic substances are observed in IR spectra of fulvic (FA), humic (HA), and hymatomelanic (HymA) acids. The chromatographic investigation has revealed the presence of two groups of characteristic fluorescent organic matters in the FA and HA fractions. The first group responsible for the long wavelength band of the fluorescence emission was assigned to FA and HA (excitation 485 nm; emission 535 nm). The second one contributing to the emission spectra in the short wavelength region (excitation 355 nm; emission 460 nm) was caused presumably by the presence of non-humic substances. Fractions enriched with the short wavelength fluorescent substances were obtained from HA and FA fractions on extraction by absolute ethanol. The organic matters in ethanolic extracts from FA is referred to as the fluorescent fraction of FA (FFFA) in the present study. We suppose that FFFA consists of coumarin derivatives, which are probably responsible for the fluorescence. FA, FFFA, and polysaccharide fractions (PFs) from mumie have been tested for their ability to modulate reactive oxygen species (ROS) production of murine peritoneal macrophages. Intracellular phorbol-12-myristate-13-acetate (PMA) stimulating the ROS formation was determined by fluorescence with the use of 2',7'-dichlorofluorescein diacetate. The dose dependent activation in ROS production was observed with increasing concentrations of FA and PF I (fraction desorbed from DEAE cellulose by 0.5 M NaCl), and duration of cell pre-incubation with these mumie constituents. On the contrary, FFFA significantly suppressed the macrophage activity.