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Alkaloids are lowmolecular weight nitrogen containing secondary plant metabolites, usually with heterocyclic structure. QBAs are group of alkaloids occurring in some species of Fumariaceae, Papaveraceae, Ranunculaceae and Rutaceae families. Extracts from some plants containing QBAs are used in folk medicine as antimicrobial, antifungal and antiinflammatory agents (for review see Šimánek, 1985). In addition to wide range of biological activities, QBAs also display interesting colour properties (from yellow to dark red) and fluorescence properties (Kovář et al., 1985; Slaninová et al., 2008). The chromophores responsible for fluorescence are highly condensed aromatic rings with electron-donor substituents containing oxygen. The other important property of these alkaloids is the ability to interact with DNA. The interaction of SA with DNA has been described by several authors during the last decade. Although the concept of intercalation is generally accepted, also other types of interactions have been considered, eg. out side binding of SA to the phosphate backbone of the double helical DNA or its binding to G C rich regions of DNA (for review see Slaninová et al., 2008). Recently we have described that macarpine (MA), chelirubine (CHR), sanguirubine (SR) and sanguinarine (SA) stain nucleic acids in living cells (Slaninová et al., 2007). We concluded that they can be used as supravital fluorescent DNA probes both in fluorescence microscopy and flow cytometry including multiparameter analysis of peripheral blood and bone marrow. The best DNA staining properties showed MA. MA binds DNA stoichiometrically and can report the DNA content. QBA are excitable using common argon lasers (488 nm) emitting at a range of 575 to 755 nm (i.e. fluorescence detectors FL2 to 5). In order to characterize QBA DNA interactions, we have carried out in vitro studies of binding of QBA to double stranded DNA (Urbanová et al., 2009) using fluorescence and UV-Vis spectroscopy. We have studied the spectral properties of SA, chelerythrine (CHE), CHR, SR, chelilutine (CHL), sanguilutine (SL) and MA in presence and in absence of calf thymus DNA. Generally, QBAs have at least four absorption maxims in UV Vis spectrum between 250 and 550 nm. First maximum is below 300 nm, second dominant peak is usually between 310 and 350 nm, the third absorption band is about 400 nm and merges to a wide band absorption between 450 and 520 nm (the last two bands are responsible for the colors of the alkaloids). Two different emission bands were found in the fluorescence spectra of the studied QBAs after excitation by light with a wavelength of about 330 nm (second excitation band). First, the high energetic band is situated between 400 and 500 nm, second, the low energetic band, is present between 500 and 700 nm. Intensities of these bands changes at the presence of DNA and these changes are characteristic for each alkaloid. Results from these experiments confirmed a great affinity of the studied alkaloids to DNA - association constants (logK11) are about 5. Although calculated association constants for all alkaloid-DNA systems were comparable, we observed dramatic differences in fluorescence emission of all studied alkaloids in presence of calf thymus DNA in comparison to fluorescence of free alkaloids. The most remarkable were changes in emission spectra of MA, CHR and SR (Urbanová et al., 2009). The results from all our studies, fluorescence spectroscopy, fluorescence microscopy and flow cytometry, confirmed great affinity of QBAs to double stranded DNA that is accompanied by changes in fluorescence properties. These characteristics of QBA provide possibilities to their use in analytical and bioanalytical chemistry and in fluorescence microscopy and flow cytometry.
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