Longitudinal Monitoring of Hair Cortisol Using Liquid Chromatography-Mass Spectrometry to Prevent Hypercortisolism in Patients Undergoing Glucocorticoid Replacement Therapy.

OBJECTIVE: Currently available methods for endogenous cortisol monitoring in patients with hormonal insufficiency rely on measurements of plasma levels only at a single time point; thus, any kind of chronic exposure to cortisol is challenging to evaluate because it requires collecting samples at different time points. Hair cortisol levels acquired longitudinally better reflected chronic exposure (both cortisol synthesis and deposition) and may significantly contribute to better outcomes in glucocorticoid replacement therapies. DESIGN: Twenty-two patients on cortisol substitution therapy were monitored for plasma, urinary, and hair cortisol levels for 18 months to determine whether hair cortisol may serve as a monitoring option for therapy setting and adjustment. METHODS: Plasma and urinary cortisol levels were measured using standardized immunoassay methods, and segmented (∼1 cm) hair cortisol levels were monitored by liquid chromatography coupled to mass spectrometry. A log-normal model of the changes over time was proposed, and Bayesian statistics were used to compare plasma, urinary, and hair cortisol levels over 18 months. RESULTS AND CONCLUSIONS: Hair cortisol levels decreased over time in patients undergoing substitutional therapy. The residual variance of hair cortisol in comparison to plasma or urinary cortisol levels was much lower. Thus, longitudinal monitoring of hair cortisol levels could prove beneficial as a noninvasive tool to reduce the risk of overdosing and improve the overall patient health.

Alkaloid Escholidine and Its Interaction with DNA Structures.

Berberine, the most known quaternary protoberberine alkaloid (QPA), has been reported to inhibit the SIK3 protein connected with breast cancer. Berberine also appears to reduce the bcl-2 and XIAP expression-proteins responsible for the inhibition of apoptosis. As some problems in the therapy with berberine arose, we studied the DNA binding properties of escholidine, another QPA alkaloid. CD, fluorescence, and NMR examined models of i-motif and G-quadruplex sequences present in the n-myc gene and the c-kit gene. We provide evidence that escholidine does not induce stabilization of the i-motif sequences, while the interaction with G-quadruplex structures appears to be more significant.

Naturally occurring quaternary benzo[c]phenanthridine alkaloids selectively stabilize G-quadruplexes.

In this work, the interaction of six natural benzo[c]phenanthridine alkaloids (macarpine, sanguilutine, sanguirubine, chelerythrine, sanguinarine and chelirubine) with parallel and antiparallel G-quadruplex DNA structures was studied. HT22 corresponding to the end of human telomeres and the modified promoter oncogene c-kit21 and Pu22 sequences have been used. Spectroscopically-monitored melting experiments and fluorescence titrations, competitive dialysis and nuclear magnetic resonance spectroscopy were used for this purpose. The results showed that these alkaloids stabilized G-quadruplex structures in terms of increments of Tm values (from 15 to 25 °C) with high selectivity over duplexes and unfolded DNA. The mode of binding was mainly by stacking on the terminal G-tetrads with stoichiometries of 1 : 2 (DNA : ligand). The presence of non-specific electrostatic interactions was also observed. Overall, the results pointed to a strong stabilization of G-quadruplex structures by these alkaloids.

Sanguinarine is reduced by NADH through a covalent adduct.

Sanguinarine is a benzo[c]phenanthridine alkaloid with interesting cytotoxic properties, such as induction of oxidative DNA damage and very rapid apoptosis, which is not mediated by p53-dependent signaling. It has been previously documented that sanguinarine is reduced with NADH even in absence of any enzymes while being converted to its dihydro form. We found that the dark blue fluorescent species, observed during sanguinarine reduction with NADH and misinterpreted by Matkar et al. (Arch. Biochem. Biophys. 2008, 477, 43-52) as an anionic form of the alkaloid, is a covalent adduct formed by the interaction of NADH and sanguinarine. The covalent adduct is then converted slowly to the products, dihydrosanguinarine and NAD+, in the second step of reduction. The product of the reduction, dihydrosanguinarine, was continually re-oxidized by the atmospheric oxygen back to sanguinarine, resulting in further reacting with NADH and eventually depleting all NADH molecules. The ability of sanguinarine to diminish the pool of NADH and NADPH is further considered when explaining the sanguinarine-induced apoptosis in living cells.

Identification of metabolites of selected benzophenanthridine alkaloids and their toxicity evaluation.

Selected benzo[c]phenathridine alkaloids were biotransformed using rat liver microsomes and identified by liquid chromatography and mass spectrometry. While the metabolites of commercially available sanguinarine and chelerythrine have been studied in detail, data about the metabolism of the minor alkaloids remained unknown. Reactions involved in transformation include single and/or double O-demethylation, demethylenation, reduction, and hydroxylation. Two metabolites, when isolated, purified and tested for toxicity, were found to be less toxic than the original compounds.

Introduction of macarpine as a novel cell-permeant DNA dye for live cell imaging and flow cytometry sorting.

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.

Modulatory Effects of Eschscholzia californica Alkaloids on Recombinant GABAA Receptors.

The California poppy (Eschscholzia californica Cham.) contains a variety of natural compounds including several alkaloids found exclusively in this plant. Because of the sedative, anxiolytic, and analgesic effects, this herb is currently sold in pharmacies in many countries. However, our understanding of these biological effects at the molecular level is still lacking. Alkaloids detected in E. californica could be hypothesized to act at GABAA receptors, which are widely expressed in the brain mainly at the inhibitory interneurons. Electrophysiological studies on a recombinant α 1 ß 2 γ 2 GABAA receptor showed no effect of N-methyllaurotetanine at concentrations lower than 30 µM. However, (S)-reticuline behaved as positive allosteric modulator at the α 3, α 5, and α 6 isoforms of GABAA receptors. The depressant properties of aerial parts of E. californica are assigned to chloride-current modulation by (S)-reticuline at the α 3 ß 2 γ 2 and α 5 ß 2 γ 2 GABAA receptors. Interestingly, α 1, α 3, and α 5 were not significantly affected by (R)-reticuline, 1,2-tetrahydroreticuline, codeine, and morphine-suspected (S)-reticuline metabolites in the rodent brain.

Hydroxyl radicals' production and ECG parameters during ischemia and reperfusion in rat, guinea pig and rabbit isolated heart.

Ischemic and reperfusion injury is a serious condition related to numerous biochemical and electrical abnormalities of the myocardium. It has been repeatedly studied in various animal models. In this study, the production of hydroxyl radicals and electrophysiological parameters were compared in three species. Rat, guinea pig and rabbit isolated hearts were perfused according to Langendorff under strictly identical conditions. The heart rate and arrhythmia were monitored during ischemia and reperfusion periods at defined time intervals; the production of hydroxyl radical was determined by HPLC as 2.5-dihydroxybenzoic acid (2.5-DHBA) formed by salicylic acid hydroxylation. Relationship between arrhythmias and production of 2.5-DHBA was studied. The inter-species differences were observed in timing of arrhythmias onset and their severity, and in the production of 2.5-DHBA in both ischemia and reperfusion. The most considerable changes were observed in rats, where arrhythmias appeared early and with highest severity during ischemia on one side and the regular rhythm was restored early and completely during reperfusion. The corresponding changes in the production of 2.5-DHBA were observed. It can be concluded that rat isolated heart is the most suitable model for evaluation of ischemia/reperfusion injury under given experimental conditions.

Alkaloid chelirubine and DNA: blue and red luminescence.

Extracts from Himalayan herb Dicranostigma lactucoides containing alkaloid chelirubine have been used for centuries in Chinese herbal medicine. We have found a new utilization for the alkaloid: it can be used as a DNA fluorescent probe showing blue (free form) and red (intercalated to DNA) luminescence emission after irradiation by near-UV light. Besides quantification of DNA (LOD = 6 ng ml(-1)) it can also be used as a supravital cell probe because chelirubine molecules can effectively enter into the living cell through the cell membrane.

Necroptosis modulated by autophagy is a predominant form of melanoma cell death induced by sanguilutine.

We show that the plant quaternary benzo[c]phenanthridine alkaloid sanguilutine (SL) is a strong inducer of caspase-independent non-apoptotic death in human melanoma cells. Necrostatin-1, a specific inhibitor of necroptosis, completely reversed the cytotoxic effect of SL, suggesting that necroptosis was a predominant type of cell death induced by SL in these cells. In addition, we showed that SL can trigger an autophagic response, as confirmed by GFP-LC3 puncta formation and LC3-II accumulation. Interestingly, we observed a significant decrease in the viability of melanoma cells treated with combination of autophagy inhibitors (3-methyladenine, bafilomycin-A1 and LY294002) and SL. Our results further indicated that autophagy may serve as a pro-survival mechanism, delaying the induction of necroptosis in melanoma cells. The ability of SL to induce caspase-independent non-apoptotic cell death (necroptosis) suggests its possible therapeutic potential in the treatment of apoptosis-resistant melanoma tumours. Furthermore, SL might serve as a useful tool for studying the mechanisms of necroptosis and autophagy induction and the interplay between these two processes.

Isolation of quaternary benzo[c]phenanthridine alkaloids from Macleaya microcarpa (MAXIM.) FEDDE: comparison of maceration, Soxhlet extraction and pressurised liquid extraction.

INTRODUCTION: Macleaya microcarpa (Papaveraceae family) has been of considerable interest in recent years as a prospective source of quaternary benzo[c]phenanthridine alkaloids (QBAs) related to many pharmaceutical beneficial effects. For this purpose, a quantitative, efficient and fast method to isolate the QBAs from the plant material is required. OBJECTIVE: To optimise and compare pressurised liquid extraction (PLE) with Soxhlet extraction and maceration in order to estimate extraction conditions for fast and efficient isolation of QBAs contained in the roots of Macleaya microcarpa. METHODOLOGY: The QBAs were extracted by PLE, Soxhlet extraction and maceration at different conditions (solvent, time, etc.). Reversed phase HPLC with diode-array detector was utilised for their determination and quantification. To optimise the PLE procedure, the variable parameters, including temperature (40-150 °C), sample-to-inert material ratio, extraction time (5-30 min) and number of extraction cycles (1-4), were also tested. RESULTS: Quantitative determination of QBAs resulted in 0.2-2.8 mg/g, 0.3-2.5 mg/g and 0.3-3.1 mg/g for PLE, Soxhlet extraction and maceration. To produce the yields mentioned above, PLE required only up to 30 min compared with 21 h for Soxhlet extraction and 49 days for maceration. CONCLUSION: PLE provided an effective and fast extraction of QBAs from M. microcarpa roots and can be recommended as an alternative isolation method to conventional techniques for QBAs from the plant sources.

Seasonal variation of bioactive alkaloid contents in Macleaya microcarpa (Maxim.) Fedde.

Macleaya microcarpa (Maxim.) Fedde belongs to the genus Macleaya, family Papaveraceae. Together with the better known and more frequently studied species M. cordata (Willd.) R. Br. it is a main source of quaternary benzo[c]phenanthridine alkaloids. Using HPLC we determined the content of eight isoquinoline alkaloids in the aerial and underground parts of 1-, 2-, 12- and 13-year old plants and followed their changes during the vegetative period. The dominant alkaloid of all samples collected in the end of this period was allocryptopine (3.8-13.6 mg/g for aerial parts, 24.2-48.9 mg/g for underground parts). Chelerythrine, sanguinarine and protopine were also present in both parts of the plant. Additionally, measurable concentrations of chelilutine (CL), chelirubine (CR), macarpine (MA) and sanguirubine (SR) were detected in underground parts. The most important finding was that contents of CR, CL, SR and MA in the 12- and 13-year old plant roots were significantly higher (approximately 3-fold for CR, 6-fold for CL, 5-fold for SR, and at least 14-fold for MA) than in 1- or 2-year old plants. The proportion of individual alkaloids in aerial and underground parts thus changed significantly during the vegetative period.

Benzo[c]phenanthridine alkaloids exhibit strong anti-proliferative activity in malignant melanoma cells regardless of their p53 status.

BACKGROUND: Search for new substances with antiproliferative activity towards melanoma cells is important since malignant melanoma is notoriously resistant to conventional chemotherapy. Benzo[c]phenanthridine alkaloids (BAs) are natural products with significant anti-proliferative activities, therefore they are considered as agents promising for cancer therapy. OBJECTIVES: The effects of five BAs (sanguinarine, chelerythrine, chelidonine, sanguilutine, and chelilutine) on human malignant melanoma cell lines were compared. The study focused on BAs effects on DNA, anti-apoptotic and p53 protein levels; and the involvement of p53 in cellular responses to alkaloids treatment. METHODS: Melanoma cell lines, two wild types and two with dysfunctional p53 derived from one of them were used. The mechanism of anti-proliferative and pro-apoptotic effects and the effect on DNA was investigated using MTT assay, flow cytometry, Western blot analysis, fluorescence and electron microscopy. RESULTS: All tested alkaloids exhibit strong anti-proliferative activity. CHL, CHE and SA induced apoptosis, which was probably mediated by decreasing levels of anti-apoptotic proteins (Bcl-xL, Mcl-1, XIAP) and was accompanied by mitochondrial membrane potential decrease as well as caspase-3 and PARP cleavage. Although all alkaloids caused DNA damage, which was demonstrated by induction of H2AX phosphorylation, none of the tested alkaloids stabilised p53 and their toxicity in cells with non-functional p53 was comparable to wild type cells. CONCLUSION: Despite the profound similarity of BAs molecular structures, it is clear that the mechanism of cell death induction is different for each alkaloid. Our results indicate that BAs could be effective in malignant melanoma treatment, including tumours which have lost wild type p53.

Extractions of isoquinoline alkaloids with butanol and octanol.

Six different isoquinoline alkaloids (sanguinarine, chelerythrine, berberine, coptisine, allocryptopine, and protopine) were extracted by butanol and octanol from aqueous solution, pH 4.5. The samples were analyzed by HPLC. Butanol extraction was non-selective, alkaloids passed into organic phase in 83-98%. Octanol extraction provided more selective yields: sanguinarine 99%, chelerythrine 94%, berberine 18%, coptisine 16%, allocryptopine 7.5%, protopine 7%. Further, we tested octanol treatment of extract from Dicranostigma lactucoides. The octanol extraction yields were also selective: sanguinarine 98%, chelerythrine 92%, chelirubine 92.5%, protopine 6% and allocryptopine 3.5%. 6-Butoxy-5,6-dihydrosanguinarine and 6-butoxy-5,6-dihydrochelerythrine were prepared and their NMR and MS data are reported and discussed.

Fluorescence properties of selected benzo[c]phenantridine alkaloids and studies of their interaction with CT DNA.

The spectral, especially fluorescence properties, of seven selected quaternary benzo[c]phenantridine alkaloids (sanguinarine, chelerythrine, chelirubine, sanguirubine, chelilutine, sanguilutine, and macarpine) were studied in presence and in absence of double-stranded DNA. This study has proved 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 are changes in emission spectra of macarpine, chelirubine, and sanguirubine. Association constants (logK) for interaction of all studied alkaloids with CT DNA were calculated.

Effects of aqueous extracts from five species of the family Papaveraceae on selected aquatic organisms.

The effects of aqueous root extracts from five species of the family Papaveraceae on the growth of cyanobacteria, algae, and other non-target aquatic organisms were investigated to evaluate their potential use as algicides or cyanocides in the aquatic environment. Dicranostigma lactucoides and Sanguinaria canadensis featured the highest toxicity while Macleaya microcarpa was found to be the least toxic to all aquatic organisms tested. The Chelidonium majus extract had the best properties as a potential algicide or cyanocide because of its significant toxicity to phytoplankton and lower toxicity to non-target aquatic organisms as compared with the other Papaveraceae family members.

Quaternary benzo[c]phenanthridine alkaloids--novel cell permeant and red fluorescing DNA probes.

BACKGROUND: Quaternary benzo[c]phenanthridine alkaloids (QBAs) are naturally occurring compounds isolated from plants in the Fumariaceae, Papaveraceae, Ranunculaceae, and Rutaceae families. In addition to having a wide range of biological activities, they are also attractive for their fluorescent properties. We observed interesting fluorescent characteristics in the QBAs-macarpine (MA), sanguirubine (SR), chelirubine (CHR), sanguilutine (SL), chelilutine (CHL), sanguinarine (SA) and chelerythrine (CHE) after interaction with living cells. METHODS: Water stock solutions of the alkaloids (10-100 microg/ml) were added to intact cells, and after a brief incubation the cells were observed. Human cell lines HL60 (human promyelocytic leukemia), HeLa (human cervix adenocarcinoma), and LEP (human lung fibroblasts), and piglet blood were used in the experiments. Blood cells were stained with MA in combination with FITC-conjugated anti-CD45 surface marker antibody. Cells were analyzed by fluorescence microscopy and by flow cytometry. RESULTS: All tested alkaloids immediately entered living cells with MA, CHR, and SA binding to DNA. MA showed the best DNA staining properties. Fluorescence microscopy of MA, CHR, and SA stained cells described the nuclear architecture and clearly described chromosomes and apoptotic fragments in living cells. Moreover MA can rapidly represent the cellular DNA content of living cells at a resolution adequate for cell cycle analysis. QBAs were excitable using common argon lasers (488 nm) emitting at a range of 575-755 nm (i.e. fluorescence detectors FL2-5). Spectral characteristics of MA allow simultaneous surface immunophenotyping. CONCLUSIONS: It was shown that MA, CHR, and SA stain nucleic acids in living cells. 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. MA binds DNA stochiometrically and can provide information on DNA content.

HPLC quantification of seven quaternary benzo[c]phenanthridine alkaloids in six species of the family Papaveraceae.

The content of the seven quaternary benzo[c]phenanthridine alkaloids (QBA) sanguinarine (SA), chelerythrine (CHE), chelirubine (CHR), chelilutine (CHL), sanguilutine (SL), sanguirubine (SR) and macarpine (MA) was determined in the underground part of six plant species of the family Papaveraceae (Sanguinaria canadensis L., Dicranostigma lactucoides HOOK.f.et THOMS, Chelidonium majus L., Macleaya cordata (Willd.), Macleaya microcarpa (Maxim) and Stylophorum lasiocarpum (Oliv.)). HPLC method with reversed phase column Synergi Max-RP C-12 Phenomenex was used, mobile phase consisted of heptanesulfonic acid (0.01 mol/l) with triethanolamine (0.1 mol/l) in redistilled water, pH 2.5, acetonitrile gradient 25-60% during 25 min. Detection was performed at 280 nm. The highest content of SA and CHE was found in the roots of D. lactucoides (1.99%, resp. 3.43% of the dried roots). In rhizomes of S. canadensis was their content more then two times lower.

Mass spectrometry of 1,3- and 1,5-dicaffeoylquinic acids.

Samples of 1,3- (1) and 1,5-dicaffeoylquinic acid (2) and their hexaacetate derivatives were examined using positive and negative electrospray ionization mass spectrometry and tandem mass spectrometry. Differences in the various spectra allow the discrimination of each of the isomers. Specific losses in the spectra of 2 also permit the identification of the site of substitution of one of the caffeic acid moieties as being at the 5-position. The spectra of 3,5- (3) and 4,5-dicaffeoylquinic (4) acids and their hexaacetate derivatives were compared with those of 1 and 2 and their derivatives, and differences in ion abundances or the presence/absence of specific ions can be used to identify uniquely each of the compounds.

Main flavonoids in the root of Scutellaria baicalensis cultivated in Europe and their comparative antiradical properties.

The content of the main flavonoids in the root of Scutellaria baicalensis Georgi cultivated in Central Europe was evaluated using the new simple RP-HPLC method with gradient of acetonitrile in mobile phase. The main components of the roots were baicalin (8.12% of dry root mass) and wogonin glucuronide (2.52%). The content of flavonoids was comparable with the content in plants cultivated in natural localities. Five main flavonoids were evaluated for their scavenging ability with DPPH radical-generating system and due to limited solubility only two flavonoids were investigated for their ability to scavenge hydroxyl radical by the aromatic hydroxylation method. The total extract was also tested in both the experimental arrangements. In experiments with DPPH, only baicalin and baicalein displayed a significant scavenging effect, while the production of OH radicals generated by UV photolysis of H(2)O(2) was considerable decreased in the presence of baicalin and wogonin glucuronide. After comparison with results obtained for the total extract, it was concluded, that the scavenging activity of the extract against DPPH is mainly derived from baicalin. On the other hand, baicalin, wogonin glucuronide and probably other flavonoids participate in scavenging OH radical.