BACKGROUND INFORMATION: Macarpine (MA) is a quaternary benzophenanthridine plant alkaloid isolated from Macleaya microcarpa or Stylophorum lasiocarpum. Benzophenanthridine alkaloids are interesting natural products that display antiproliferative, antimicrobial, antifungal and anti-inflammatory activities, and also fluorescence properties. In a previous study, we demonstrated that thanks to its ability to interact with DNA and its spectral properties MA could be used as a supravital DNA probe for fluorescence microscopy and flow cytometry including analyses of the cell cycle. In this study, we evaluated the suitability of MA as a DNA dye for time-lapse microscopy and flow-cytometric cell sorting. RESULTS: Living A-375 and MEF cells stained with MA were monitored by time-lapse microscopy for 24 h. Mitoses were observed at MA concentrations up to 0.5 μg/ml during the first 2-3 h. After this period of time, cells treated with MA at concentrations of 0.75 and 0.5 μg/ml underwent apoptosis. Cells cultivated with MA at concentration of 0.25 μg/ml or lower survived throughout the 24 h period. Toxicity of MA was dependent on light wavelength and frequency of image capturing. The intensity of MA fluorescence decreased during the incubation. MA concentration of 0.1 μg/ml was identified as the most suitable for live cell imaging with respect to fluorescence intensity and toxicity. MA at the concentration 10 μg/ml was used for sorting of enhanced green fluorescent protein (EGFP)-labelled neurons and fibroblasts yielding profiles similar to those obtained with DRAQ5. Contrary to DRAQ5, MA-stained cells survived in culture, and the sorted cells lost the MA signal suggesting reversible binding of the dye to the DNA. CONCLUSION: The results proved that MA may readily be used for chromosomes depicting and mitosis monitoring by time-lapse microscopy. In addition, MA has shown to be a suitable probe for sorting of EGFP-labelled cells, including neurons, that survived the labelling process. SIGNIFICANCE: In consideration of the results, we highly anticipate an onward use of MA in a broad range of applications based on live cell sorting and imaging, for example, cell synchronisation and monitoring of proliferation as an important experimental and/or diagnostic utility.
- MeSH
- benzofenantridiny analýza MeSH
- buněčné kultury MeSH
- buněčný cyklus fyziologie MeSH
- DNA analýza MeSH
- fluorescenční barviva analýza MeSH
- fluorescenční mikroskopie metody MeSH
- lidé MeSH
- průtoková cytometrie * metody MeSH
- separace buněk metody MeSH
- viabilita buněk MeSH
- zelené fluorescenční proteiny metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The metabolism of the benzo[c]phenanthridine alkaloids was studied using human hepatocytes which are an excellent model system for biotransformation studies. For analysis of the alkaloids and their metabolites, an electrospray quadrupole ion-trap mass spectrometry (ESI ion-trap MS) connected to a reversed phase chromatographic system based on cyanopropyl modified silica was used. The optimized experimental protocol allowed simultaneous analysis of the alkaloids and their metabolites and enabled study of their uptake into and interconversion in human hepatocytes. The results show that formation of the dihydro metabolite which may be followed by specific O-demethylenation/O-demethylation processes, is probably the main route of biotransformation (detoxification) of the benzo[c]phenanthridines in human hepatocytes. The structure of the main O-demethyl metabolite (2-methoxy-12-methyl-12,13-dihydro-[1,3]dioxolo[4',5':4,5]benzo[1,2-c]phenanthridin-1-ol; 336.1 m/z,) was proposed by the multi-stage MS and quadrupole time-of-flight MS methods using chemically synthesized standard.
- MeSH
- benzofenantridiny analýza metabolismus farmakokinetika MeSH
- biotransformace MeSH
- dospělí MeSH
- hepatocyty chemie metabolismus MeSH
- hmotnostní spektrometrie s elektrosprejovou ionizací metody MeSH
- isochinoliny analýza metabolismus farmakokinetika MeSH
- kultivované buňky MeSH
- lidé středního věku MeSH
- lidé MeSH
- oxidace-redukce MeSH
- reprodukovatelnost výsledků MeSH
- senioři MeSH
- tandemová hmotnostní spektrometrie metody MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Macleaya cordata (Willd.) (Papaveraceae) is used as an active component in the natural feed additive Sangrovit. Sangrovit contains mixture of the intact aerial parts and the fraction of quaternary benzo[c]phenanthridine alkaloids from M. cordata (FQBA). In a 90-day pilot toxicity trial, Sangrovit and the FQBA were tested for safety. Male Wistar rats were fed for 90 days with 100, 7000 or 14000mg of Sangrovit or 600mg of FQBA in 1kg of feed. Body and organ weights, clinical chemistry and hematology markers, oxidative stress parameters, morphological structure of tongue, liver, ileum, kidney and heart samples, and total cytochrome P450 in liver were monitored. The results showed no statistically significant alterations in any parameter between control and treated animals, except for the group treated with 14000ppm Sangrovit that resulted in elevation of reduced glutathione level and superoxide dismutase activity in liver.
- MeSH
- benzofenantridiny analýza MeSH
- dieta MeSH
- feces chemie MeSH
- isochinoliny analýza MeSH
- játra enzymologie účinky léků MeSH
- krevní obraz MeSH
- krmivo pro zvířata analýza toxicita MeSH
- krysa rodu rattus MeSH
- léky rostlinné čínské analýza toxicita MeSH
- oxidační stres účinky léků MeSH
- Papaveraceae chemie MeSH
- pilotní projekty MeSH
- potkani Wistar MeSH
- potravinářské přísady analýza toxicita MeSH
- přijímání potravy MeSH
- systém (enzymů) cytochromů P-450 analýza metabolismus MeSH
- tělesná hmotnost účinky léků MeSH
- tkáňová distribuce MeSH
- velikost orgánu účinky léků MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
