Analyzing the Carotenoid Composition of Melilot (Melilotus officinalis (L.) Pall.) Extracts and the Effects of Isolated (All-E)-lutein-5,6-epoxide on Primary Sensory Neurons and Macrophages

Melilotus officinalis is known to contain several types of secondary metabolites. In contrast, the carotenoid composition of this medicinal plant has not been investigated, although it may also contribute to the biological activities of the drug, such as anti-inflammatory effects. Therefore, this study focuses on the isolation and identification of carotenoids from Meliloti herba and on the effect of isolated (all-E)-lutein 5,6-epoxide on primary sensory neurons and macrophages involved in nociception, as well as neurogenic and non-neurogenic inflammatory processes. The composition of the plant extracts was analyzed by high performance liquid chromatography (HPLC). The main carotenoid was isolated by column liquid chromatography (CLC) and identified by MS and NMR. The effect of water-soluble lutein 5,6-epoxide-RAMEB (randomly methylated-β-cyclodextrin) was investigated on Ca²⁺-influx in rat primary sensory neurons induced by the activation of the transient receptor potential ankyrin 1 receptor agonist to mustard-oil and on endotoxin-induced IL-1β release from isolated mouse peritoneal macrophages. (all-E)-Lutein 5,6-epoxide significantly decreased the percent of responsive primary sensory neurons compared to the vehicle-treated stimulated control. Furthermore, endotoxin-evoked IL-1β release from macrophages was significantly decreased by 100 µM lutein 5,6-epoxide compared to the vehicle-treated control. The water-soluble form of lutein 5,6-epoxide-RAMEB decreases the activation of primary sensory neurons and macrophages, which opens perspectives for its analgesic and anti-inflammatory applications.     

Novel quantitative electrophoretic analysis of endotoxins on microchips

A novel microchip electrophoresis method was developed and applied for sensitive detection and quantitative analysis of endotoxins extracted from Gram-negative bacteria. The method provides a fast and quantitative differentiation of smooth and rough endotoxins based on the solubilization and complexation of the lipopolysaccharides with dodecylsulfate, and then with a fluorescent dye. The migration of the complexes was followed by LIF detection. The novel method is able to replace the SDS-PAGE with the advantage of high speed and better sensitivity, and by avoiding the laborious gel-preparation and silver staining.     

Creep Behavior of Polystyrene/Fluorohectorite Micro‐ and Nanocomposites

Summary: Pristine FH is incorporated into a PS matrix by melt blending with and without latex precompounding of PS and FH. Direct melt blending results in microcomposites, whereas the latex‐mediated (masterbatch) technique results in PS/FH nanocomposites. The tensile creep response of the micro‐ and nanocomposites are determined in short‐term creep tests. The resistance to creep is improved with increasing dispersion of FH in the PS matrix. Master curves (creep compliance vs. time), constructed based on isothermal creep tests performed in the temperature range between 5 and 45 °C, show that the FH reinforcement affects mostly the initial creep compliance (interphase effect). On the other hand, the stable creep is matrix (bulk) dominated. It is established that the Williams‐Landel‐Ferry equation is fairly applicable to the creep results.

Scheme of the change of creep compliance as a function of time for micro‐ and nanocomposites with an amorphous matrix.     

Surface Corrosion Studies on High-Purity Quartz Vessels for Digestive Sample Preparation

 High purity quartz is the most common material for pressure vessels of high pressure digestion systems. Such systems are in worldwide use especially for matrix decomposition and mineralization procedures with either conventional heating or heating in a microwave field. Usually, the quartz vessels are used for hundreds of decompositions and corrosive attack is often already visible with the bare eye. On the other hand it is well known that adsorption as well as leaching phenomena increase with rising surface roughness of a material. This is especially critical in trace and ultratrace work. For the latter such interactions with the vessel wall might even become the limiting factor in ultratrace determinations at the ng/g-level and below. Therefore, systematic morphological and compositional investigations by high resolution scanning electron microscopy were performed on the inner surfaces of decomposition vessels which were subjected to a rising number of digestions under pressure in a Multiwave digestion apparatus. Milk powder was digested with nitric acid for these investigations. Rising corrosive attack was observable but did not lead to severely damaged surfaces. The latter were found on a quartz pressure vessel for an HPA-digestion apparatus which was in intensive use over a long time. Recrystallization was also observable in this case. In addition to investigations by scanning electron microscopy, FT-NIR-Raman spectroscopy was also applied for the surface characterization of the various quartz vessels. The onset of recrystallization is detected much earlier with this method than by morphological changes in the scanning electron microscope (SEM). It appears that the combination of surface morphological and topochemical investigations in the SEM with vibrational spectroscopy is ideal for an optimal characterization of corroded quartz surfaces. Received January 15, 2001. Revision April 14, 2001.     

Glycoform Heterogeneity of Human Serum α1-Acid Glycoprotein Determined by CZE in Malignant Diseases

A novel capillary zone electrophoresis method was developed to investigate the glycoform heterogeneity of human serum α1-acid glycoprotein (AGP). The simultaneous application of a dimethyl polysiloxane coated capillary and oligoamine additives, particularly spermidine resulted in a more detailed separation of AGP glycoforms than reported previously. The relative distribution of AGP glycoforms in CZE was determined by baseline integration of peak areas and verified by peak-fitting analysis. Providing high purity of AGP samples suitable for CZE a schedule of isolation and purification steps including sample preparation and an improved technique of ion exchange chromatography was applied. Based on data obtained by CZE and on the serum AGP levels measured the serum concentrations of AGP glycoforms were calculated in cancer patients with Hodgkin and non-Hodgkin lymphoma, ovary carcinoma and melanoma compared to healthy donors. Results presented here demonstrated a significant increase in the serum concentration of the more acidic AGP fractions also indicating the overproduction of these glycoforms in cancer. In conclusion, our observations may raise the clinical diagnostic relevance of changes in the molecular heterogeneity of AGP detected by CZE in the various forms of malignant diseases.     

Optimization of Conditions for Culture of Test Bacteria used for Direct Bioautographic TLC Detection. 2. Gram-Negative Test Bacterium: Escherichia coli

JPC – Journal of Planar Chromatography – Modern TLC - Direct bioautography is a potent means of obtaining information about the antimicrobial activity of a compound separated from a...     

Characterization of complex,heterogeneous lipid A samples using HPLC–MS/MS technique II. Structural elucidation of non‐phosphorylated lipid A by negative‐ion mode tandem mass spectrometry

Non‐phosphorylated lipid A species confer reduced inflammatory potential for the bacteria. Knowledge on their chemical structure and presence in bacterial pathogens may contribute to the understanding of bacterial resistance and activation of the host innate immune system. In this study, we report the fragmentation pathways of negatively charged, non‐phosphorylated lipid A species under low‐energy collision‐induced dissociation conditions of an electrospray ionization quadrupole time‐of‐flight instrument. Charge‐promoted consecutive and competitive eliminations of the acyl chains and cross‐ring cleavages of the sugar residues were observed. The A‐type fragment ion series and the complementary X‐type fragment(s) with corresponding deprotonated carboxamide(s) were diagnostic for the distribution of the primary and secondary acyl residues on the non‐reducing and the reducing ends, respectively, of the non‐phosphorylated lipid A backbone. Reversed‐phase liquid chromatography in combination with negative‐ion electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry could provide sufficient information on the primary and secondary acyl residues of a non‐phosphorylated lipid A. As a standard, the hexa‐acylated ion at m/z 1636 with the Escherichia coli‐type acyl distribution (from E. coli O111) was used. The method was tested and refined with the analysis of other non‐phosphorylated hexa‐ and several hepta‐, penta‐, and tetra‐acylated lipid A species detected in crude lipid A fractions from E. coli O111 and Proteus morganii O34 bacteria. Copyright © 2016 John Wiley & Sons, Ltd.     

DSC analysis of human synovial fluid samples in the diagnostics of non-septic and septic arthritis

The synovial fluid analysis is an important method in diagnosing and managing septic arthritis. To reach a quick diagnosis, preferably faster than the microbiological cultures, could be a great advantage in the therapy. The differential scanning calorimetry (DSC) has recently been found useful in the differential diagnosis of septic and non-septic periprosthetic conditions. The aim of this study was to evaluate whether there is a specific pattern in the different grades of arthritis, and the effect of three different bacterial strains inoculated in synovial fluid. The sensitivity of our method was also determined by using synovial fluid samples with the different bacterial concentrations. Authors developed a standardized, experimental model to assess the denaturation characteristics of non-septic and inoculated synovial fluid, infected by different bacterial strains. The thermal characteristics [maximal denaturation temperature (T _m) and calorimetric enthalpy change (ΔH _cal)] were monitored by SETARAM Micro DSC-II calorimeter. The denaturation scans clearly demonstrated specific, representative curves in the case of different grades of arthritis, as well as with each individual bacterial strain. Therefore, thermoanalyses of human synovial fluid samples by DSC could be a useful tool in the staging of osteoarthritis and the diagnostics of septic arthritis.