Binary Co-Crystals of Quercetin: Synthesis, Structure, and Spectroscopic Characterization.

A mechanochemical method was used to obtain four new quercetin (QUE) co-crystals. Three co-formers have the systems of the heterocyclic rings with the oxygen and nitrogen atoms and they form co-crystals at the stoichiometric ratio of 1 : 2. In contrast, the QUE : o-dianisidine co-crystal represents the 1 : 1 stoichiometry and the former molecule is the aniline derivative. The X-ray crystallography and FT-IR and FT-Raman spectra revealed formation of the intermolecular O-H…N or N-H…O hydrogen bonds. The dynamics of the hydrogen bonds was investigated using the XPS method. The N 1s XPS spectra showed no proton transfer in the QUE : FEN and QUE : O-DIA co-crystal systems. The QUE : BZFP and QUE : EBZFP show the two-site static disorder across the proton transfer pathway to the pyridine ring, with the occupancies (C=N : C=NH+ ) of 72 : 28 and 77 : 23, respectively.

Potassium Complexes of Quercetin-5'-Sulfonic Acid and Neutral O-Donor Ligands: Synthesis, Crystal Structure, Thermal Analysis, Spectroscopic Characterization and Physicochemical Properties.

The coordination ability of QSA- ligand towards potassium cations was investigated. Potassium complex of quercetin-5'-sulfonate of the general formula [KQSA(H2O)2]n was obtained. The [KQSA(H2O)2] (1) was a starting compound for solvothermal syntheses of acetone (2) and dimethylsulfoxide (3) complexes. For the crystalline complexes 1-3, crystals morphology was analyzed, IR and Raman spectra were registered, as well as thermal analysis for 1 was performed. Moreover, for 1 and 3, molecular structures were established. The potassium cations are coordinated by eight oxygen atoms (KO8) of a different chemical nature; coordinating groups are sulfonic, hydroxyl, and carbonyl of the QSA- anion, and neutral molecules-water (1) or DMSO (3). The detailed thermal studies of 1 confirmed that water molecules were strongly bonded in the complex structure. Moreover, it was stated that decomposition processes depended on the atmosphere used above 260 °C. The TG-FTIR-MS technique allowed the identification of gaseous products evolving during oxidative decomposition and pyrolysis of the analyzed compound: water vapor, carbon dioxide, sulfur dioxide, carbonyl sulfide, and carbon monoxide. The solubility studies showed that 1 is less soluble in ethanol than quercetin dihydrate in ethanol, acetone, and DMSO. The exception was aqueous solution, in which the complex exhibited significantly enhanced solubility compared to quercetin. Moreover, the great solubility of 1 in DMSO explained the ease of ligand exchange (water for DMSO) in [KQSA(H2O)2].

Anticancer effects of sodium and potassium quercetin-5'-sulfonates through inhibition of proliferation, induction of apoptosis, and cell cycle arrest in the HT-29 human adenocarcinoma cell line.

In the present study, we compared the anticancer potential of quercetin (3,3',4',5,7-pentahydroxyflavone, I) and its sulfonic derivatives sodium/potassium quercetin-5'-sulfonates (described as II and III) against several human carcinoma cell lines. Quercetin (I) was used as a starting compound for synthesis of II and III. In this work, a modified and more efficient method of synthesizing derivatives II and III has been described. The molecular structures of the compounds were characterized in a solution and in the solid state using 1H NMR, 13C NMR, 2D NMR, and XPS spectroscopy, respectively. The stoichiometry of these complexes was determined by elemental analysis as well as thermogravimetric and X-ray fluorescence methods. The spectral data allowed complete characterization of the investigated compounds in the solution and in the solid state and unambiguous determination of the place of substitution of the sulfonic group in the phenyl ring in the C-5' position. Our in vitro studies revealed that II and III prominently reduced the viability of the HT-29 colon cancer cell line. Additionally, we observed that sulfonic derivatives decreased proliferation of colon (HT-29, LS180), lung (A549), and breast (T47D) cancer cell lines. Moreover, we detected a lower cytotoxic effect of II and III on several normal cell lines (colon epithelial CCD 841 CoTr, mouse subcutaneous connective tissue L-929, and human skin fibroblasts HSF cell lines) than that exerted by pure quercetin. The anticancer properties were especially evident in the HT-29 colon cancer cell line, where cell cycle inhibition in the G2-M phase and prominent apoptosis induced by II and III were observed. In conclusion, the sodium/potassium quercetin-5'-sulfonates prepared from quercetin showed promising anti-proliferative and pro-apoptotic activity against colon cancer cells. Therefore, we support the opinion that sodium/potassium quercetin-5'-sulfonates should be considered as promising organometallic compounds for possible clinical applications.

Studies on the interactions of neutral Galleria mellonella cecropin D with living bacterial cells.

Cecropins constitute an important family of insect antimicrobial peptides involved in humoral innate immune response. In comparison with the highly basic cecropins A and B, cecropins D are less cationic and more hydrophobic. Interestingly, cecropins D were described only in lepidopteran insects, e.g., the greater wax moth Galleria mellonella. In the present study, interactions of neutral cecropin D (pI 6.47) purified from hemolymph of G. mellonella with living Escherichia coli cells were investigated. Fluorescence lifetime imaging microscopy using fluorescein isothiocyanate-labeled cecropin D revealed very fast binding of the peptide to E. coli cells. Fourier transform infrared spectroscopy analyses showed that G. mellonella cecropin D interacted especially with E. coli LPS and probably other lipid components of the bacterial cell envelope and exhibited an ordering effect with regard to lipid chains. This effect is consistent with the peptide binding mechanism based upon its incorporation into the lipid phase of the cell membrane. The interaction resulted in permeabilization of the bacterial cell membrane. Upon cecropin D binding, the cells lost characteristic surface topography, which was accompanied by altered nanomechanical properties, as revealed by atomic force microscopy. The interaction of the peptide with the bacterial cells also led to intracellular damage, i.e., loss of the cell envelope multilayer structure, formation of membrane vesicles, and enlargement of periplasmic space, which eventually caused death of the bacteria. In summary, it can be concluded that amphipathic character of α-helices, exposure of small positively charged patches on their polar surfaces and hydrophobic interactions are important physicochemical characteristics related to effective binding to E. coli cells and antibacterial activity of neutral G. mellonella cecropin D.

A New Method for the Isolation of Ergosterol and Peroxyergosterol as Active Compounds of Hygrophoropsis aurantiaca and in Vitro Antiproliferative Activity of Isolated Ergosterol Peroxide.

In the present study, ergosterol peroxide and ergosterol were isolated for the first time from fresh fruit bodies of Hygrophoropsis aurantiaca (False Chanterelle). The substances were characterized mainly by spectroscopic methods (¹H-NMR, (13)C-NMR, DEPT-45, DEPT-90, DEPT-135, 2D-NMR). In our study, a new specific thin layer chromatographic method was developed for determination of ergosterol and ergosterol peroxide in H. aurantiaca extract. The method is based on the separation of n-hexane extract on silica gel (Silica Gel G) TLC plates using the optimized solvent system toluene/ethyl acetate (3:1; v/v). The main advantages of the developed method are the simplicity of operation and the low cost. The in vitro study results revealed the antiproliferative properties of ergosterol peroxide against LS180 human colon cancer cells. The described effect was attributed both to altered mitochondrial activity and decreased DNA synthesis. Additionally, in the same concentration range the investigated compound was not toxic to CCD 841 CoTr human colon epithelial cells. The present study suggests that fruit bodies of H. aurantiaca have great potential for producing substances and extracts with potential applications in medicine.

Identification of MMP-1 and MMP-9 inhibitors from the roots of Eleutherococcus divaricatus, and the PAMPA test.

The purpose of this study was the isolation of metalloproteinases MMP-1 and MMP-9 inhibitors from the chloroform extract of the Eleutherococcus divaricatus roots. Using GC-MS, (1)H and (13)C NMR, HMQC, HMBC, COSY and DEPT, (+)-sesamin has been identified as a new anti-MMP inhibitor. We report for the first time that (+)-sesamin inhibited MMP-1 and MMP-9 activity in 40% and 17%, respectively. The high inhibitory potential has been shown by ursolic acid (90.9% and 89.8% for MMP-1 and MMP-9). In the PAMPA test, the Pe value for sesamin was established as 17.4 × 10(-6) cm/s, that for ursolic acid as 30.0 × 10(- 6) cm/s. Verapamil and theophylline were used as a positive and negative control (Pe 42.1 and 2.9 × 10(-6) cm/s). To our best knowledge, no information was available on this activity of sesamin and other compounds. These studies provide a biochemical basis for the regulation of MMP-1 and MMP-9 by E. divaricatus compounds.

Antimycobacterial action of a new glycolipid-peptide complex obtained from extracellular metabolites of Raoultella ornithinolytica.

In this paper, an antimycobacterial component of extracellular metabolites of a gut bacterium Raoultella ornithinolytica from D. veneta earthworms was isolated and its antimycobacterial action was tested using Mycobacterium smegmatis. After incubation with the complex obtained, formation of pores and furrows in cell walls was observed using microscopic techniques. The cells lost their shape, stuck together and formed clusters. Surface-enhanced Raman spectroscopy analysis showed that, after incubation, the complex was attached to the cell walls of the Mycobacterium. Analyses of the component performed with Fourier transform infrared spectroscopy demonstrated high similarity to a bacteriocin nisin, but energy dispersive X-ray spectroscopy analysis revealed differences in the elemental composition of this antimicrobial peptide. The component with antimycobacterial activity was identified using mass spectrometry techniques as a glycolipid-peptide complex. As it exhibits no cytotoxicity on normal human fibroblasts, the glycolipid-peptide complex appears to be a promising compound for investigations of its activity against pathogenic mycobacteria.

Analysis of nanomechanical properties of Borrelia burgdorferi spirochetes under the influence of lytic factors in an in vitro model using atomic force microscopy.

BACKGROUND: Atomic force microscopy (AFM) is an experimental technique which recently has been used in biology, microbiology, and medicine to investigate the topography of surfaces and in the evaluation of mechanical properties of cells. The aim of this study was to evaluate the influence of the complement system and specific anti-Borrelia antibodies in in vitro conditions on the modification of nanomechanical features of B. burgdorferi B31 cells. MATERIAL AND METHODS: In order to assess the influence of the complement system and anti-Borrelia antibodies on B. burgdorferi s.s. B31 spirochetes, the bacteria were incubated together with plasma of identified status. The samples were applied on the surface of mica disks. Young's modulus and adhesive forces were analyzed with a NanoScope V, MultiMode 8 AFM microscope (Bruker) by the PeakForce QNM technique in air using NanoScope Analysis 1.40 software (Bruker). RESULTS/CONCLUSION: The average value of flexibility of spirochetes' surface expressed by Young's modulus was 10185.32 MPa, whereas the adhesion force was 3.68 nN. AFM is a modern tool with a broad spectrum of observational and measurement abilities. Young's modulus and the adhesion force can be treated as parameters in the evaluation of intensity and changes which take place in pathogenic microorganisms under the influence of various lytic factors. The visualization of the changes in association with nanomechanical features provides a realistic portrayal of the lytic abilities of the elements of the innate and adaptive human immune system.

Synergistic action of Galleria mellonella apolipophorin III and lysozyme against Gram-negative bacteria.

Insect immune response relies on the humoral and cellular mechanisms of innate immunity. The key factors are the antimicrobial polypeptides that act in concert against invading pathogens. Several such components, e.g. apolipophorin III (apoLp-III), lysozyme, and anionic peptide 2, are present constitutively in the hemolymph of non-challenged Galleria mellonella larvae. In the present study, we demonstrate an evidence for a synergistic action of G. mellonella lysozyme and apoLp-III against Gram-negative bacteria, providing novel insights into the mode of action of these proteins in insect antimicrobial defense. It was found that the muramidase activity of G. mellonella lysozyme considerably increased in the presence of apoLp-III. Moreover, apoLp-III enhanced the permeabilizing activity of lysozyme toward Escherichia coli cells. As shown using non-denaturing PAGE, the proteins did not form intermolecular complexes in vivo and in vitro, indicating that the effect observed was not connected with the intermolecular interactions between the proteins. Analysis of AFM images of E. coli cells exposed to G. mellonella lysozyme and/or apoLp-III revealed evident alterations in the bacterial surface structure accompanied by the changes in their biophysical properties. The bacterial cells demonstrated significant differences in elasticity, reflected by Young's modulus, as well as in adhesive forces and roughness values in comparison to the control ones. The constitutive presence of these two defense molecules in G. mellonella hemolymph and the fact that apoLp-III enhances lysozyme muramidase and perforating activities indicate that they can be regarded as important antibacterial factors acting at the early stage of infection against Gram-negative as well as Gram-positive bacteria.

Synergistic action of Galleria mellonella anionic peptide 2 and lysozyme against Gram-negative bacteria.

Lysozyme and antimicrobial peptides are key factors of the humoral immune response in insects. In the present work lysozyme and anionic defense peptide (GMAP2) were isolated from the hemolymph of the greater wax moth Galleria mellonella and their antibacterial activity was investigated. Adsorption of G. mellonella lysozyme on the cell surface of Gram-positive and Gram-negative bacteria was demonstrated using immunoblotting with anti-G. mellonella lysozyme antibodies. Lysozyme effectively inhibited the growth of selected Gram-positive bacteria, which was accompanied by serious alterations of the cell surface, as revealed by atomic force microscopy (AFM) imaging. G. mellonella lysozyme used in concentrations found in the hemolymph of naive and immunized larvae, perforated also the Escherichia coli cell membrane and the level of such perforation was considerably increased by GMAP2. GMAP2 used alone did not perforate E. coli cells nor influence lysozyme muramidase activity. However, the peptide induced a decrease in the turgor pressure of the bacterial cell. Moreover, in the samples of bacteria treated with a mixture of lysozyme and GMAP2 the sodium chloride crystals were found, suggesting disturbance of ion transport across the membrane leading to cell disruption. These results clearly indicated the synergistic action of G. mellonella lysozyme and anionic peptide 2 against Gram-negative bacteria. The reported results suggested that, thanks to immune factors constitutively present in hemolymph, G. mellonella larvae are to some extent protected against infection caused by Gram-negative bacteria.

Synthesis, structure elucidation and antitumour activity of N-substituted amides of 3-(3-ethylthio-1,2,4-triazol-5-yl)propenoic acid.

New N-substituted amides of 3-(3-ethylthio-1,2,4-triazol-5-yl)propenoic acid (2-12) were designed and prepared by the condensation reaction of exo-S-ethyl-7-oxabicyclo-[2.2.1]-hept-5-ene-2,3-dicarbonyl isothiosemicarbazide (1) with primary amines. The chemical structure of all compounds was confirmed by IR, (1)H NMR, (13)C NMR spectra, the X-ray crystallography (for compounds 8, 11, 12) and elemental analysis. Moreover, compounds 9-11 were screened for their anticancer activity. Compounds 9 (in concentrations of 0.32 mM and 0.16 mM), 10 (in concentrations of 0.28 mM and 0.14 mM), and 11 (in concentrations of 0.35 mM and 0.17 mM) were found to be evidently effective in vitro against lung cell line (IC50). The distinctly marked antiproliferative effect of compounds 9 and 10 in breast carcinoma cells in vitro was ascertained. Moreover, the lowest cytotoxicity of compound 9 in concentrations of 0.16 mM and 0.03 mM against the normal skin fibroblast cell line and breast carcinoma cell in vitro after 24- and 48-h periods of incubation was noticed in this study.

Pinostrobin--an anti-leukemic flavonoid from Polygonum lapathifolium L. ssp. nodosum (Pers.) Dans.

AIM OF STUDY: Search for plant compounds possessing anti-leukemic properties. RESULTS: We have shown that 5-hydroxy-7-methoxy flavanone (pinostrobin) isolated from Polygonum lapathifolium ssp. nodosum quickly penetrates through cytoplasm to the cellular nucleus of the cultured cells, and gives intensive apoptotic response in stimulating leukemic cells in vitro. The number of apoptotic cells increased with the concentration of pinostrobin: 10 nM - 25% and 60%; 100 nM - 45% and 76%; 1 microm - 70% and 88% for Jurkat and HL60 cell lines, respectively CONCLUSION: Pinostrobin may be considered as a good candidate for a leukemia chemopreventic agent.