Biological Activity and Chemical Composition of Propolis from Various Regions of Poland.

Propolis is one of the bee products, with multiple biological properties used in numerous applications. The research objective was to determine the chemical composition and biological properties (antibacterial, antifungal, antiviral, antioxidant, and cytoprotective activity) of propolis extracts collected from various regions of Poland. The results indicated that the total content of phenols (116.16-219.41 mg GAE/g EEP) and flavonoids (29.63-106.07 mg QE/g EEP) in propolis extracts depended on their geographic origin. The high content of epicatechin, catechin, pinobanksin, myricetin, and acids: vanillic and syringic in propolis samples was confirmed by chromatographic analysis. Moreover, the presence of caffeic acid phenethyl ester was confirmed in all samples. The origin of propolis also influenced the biological properties of its extracts. The propolis extracts were characterized by moderate DPPH free radical scavenging activity (29.22-35.14%), and relatively low ferrous iron chelating activity (9.33-32.32%). The results indicated also that the propolis extracts showed high activity in the protection of human red blood cells against free radicals generated from 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AAPH). The extracts exhibited diversified activity against the tested pathogenic bacteria and limited activity against fungal strains. The research of selected propolis extracts showed that only 2 of 5 examined samples showed moderate activity against HPV (human papillomaviruses) and the activity depended on its geographical distribution.

Multifunctionality of Nanosized Calcium Apatite Dual-Doped with Li+/Eu3+ Ions Related to Cell Culture Studies and Cytotoxicity Evaluation In Vitro.

Li+/Eu3+ dual-doped calcium apatite analogues were fabricated using a microwave stimulated hydrothermal technique. XRPD, FT-IR, micro-Raman spectroscopy, TEM and SAED measurements indicated that obtained apatites are single-phased, crystallize with a hexagonal structure, have similar morphology and nanometric size as well as show red luminescence. Lithium effectively modifies the local symmetry of optical active sites and, thus, affects the emission efficiency. Moreover, the hydrodynamic size and surface charge of the nanoparticles have been extensively studied. The protein adsorption (lysozyme, LSZ; bovine serum albumin, BSA) on the nanoparticle surface depended on the type of cationic dopant (Li+, Eu3+) and anionic group (OH-, Cl-, F-) of the apatite matrix. Interaction with LSZ resulted in a positive zeta potential, and the nanoparticles had the lowest hydrodynamic size in this protein medium. The cytotoxicity assessment was carried out on the human osteosarcoma cell line (U2OS), murine macrophages (J774.E), as well as human red blood cells (RBCs). The studied apatites were not cytotoxic to RBCs and J774.E cells; however, at higher concentrations of nanoparticles, cytotoxicity was observed against the U2OS cell line. No antimicrobial activity was detected against Gram-negative bacteria with one exception for P. aeruginosa treated with Li+-doped fluorapatite.

Ligand-Sensitised LaF3 :Eu3+ and SrF2 :Eu3+ Nanoparticles and in Vitro Haemocompatiblity Studies.

Luminescent Ln3+ -doped nanoparticles (NPs) functionalised with the desired organic ligand molecules for haemocompatibility studies were obtained in a one-pot synthesis. Chelated aromatic organic ligands such as isophthalic acid, terephthalic acid, ibuprofen, aspirin, 1,2,4,5-benzenetetracarboxylic acid, 2,6-pyridine dicarboxylic acid and adenosine were applied for surface functionalisation. The modification of the nanoparticles is based on the donor-acceptor character of the ligand-nanoparticle system, which is an alternative to covalent functionalisation by peptide bonding as presented in our recent report. The aromatic groups of selected ligands absorb UV light and transfer their excited-state energy to the dopant Eu3+ ions in LaF3 and SrF2 NPs. Herein, we discuss the structural and spectroscopic characterisation of the NPs and the results of haemocompatibility studies. Flow cytometry analysis of the nanoparticles' membrane-binding is also presented.

Preparation and preliminary evaluation of bio-nanocomposites based on hydroxyapatites with antibacterial properties against anaerobic bacteria.

Hexagonal nanocrystalline powders of the non-doped Ca10(PO4)6(OH)2 as well as activated with Ag+ and Eu3+ ions were synthesized by using different wet chemistry methods. Moreover, the obtained hydroxyapatite was loaded with Ag0, as well as nitroimidazole antimicrobials: metronidazole and tinidazole. The structural properties of the products were analyzed by X-ray diffraction (XRD), scanning (SEM) and transmission (TEM) electron microscopy as well as infrared (IR) and Raman spectroscopy. The photoluminescence properties of the Eu3+ and Ag+ co-doped Ca10(PO4)6(OH)2 were characterized via the PL emission, excitation spectra and the luminescence decay curve. The antimicrobial activity of the obtained materials against Prevotella bivia and Parabacteroides distasonis was studied. The cytotoxicity assessment was carried out on the human osteosarcoma cell line (U2OS) as well as human red blood cells (RBC). The choice of the in vitro model was based on the fact that U2OS is a cancer cell line derived from bone tissue which is rich in apatites that play a pivotal role in the extracellular matrix formation. RBCs are the most abundant blood cells and they are used as a cell model in the study of biocompatibility of new prepared biocompounds with potential medical applications. The obtained multifunctional materials do not exhibit the haemolytic activity, therefore, they could be used as a promising antimicrobial agent and for anaerobic bacteria.

The Influence of Bee Venom Melittin on the Functioning of the Immune System and the Contractile Activity of the Insect Heart-A Preliminary Study.

Melittin (MEL) is a basic polypeptide originally purified from honeybee venom. MEL exhibits a broad spectrum of biological activity. However, almost all studies on MEL activity have been carried out on vertebrate models or cell lines. Recently, due to cheap breeding and the possibility of extrapolating the results of the research to vertebrates, insects have been used for various bioassays and comparative physiological studies. For these reasons, it is valuable to examine the influence of melittin on insect physiology. Here, for the first time, we report the immunotropic and cardiotropic effects of melittin on the beetle Tenebrio molitor as a model insect. After melittin injection at 10-7 M and 10-3 M, the number of apoptotic cells in the haemolymph increased in a dose-dependent manner. The pro-apoptotic action of MEL was likely compensated by increasing the total number of haemocytes. However, the injection of MEL did not cause any changes in the percent of phagocytic haemocytes or in the phenoloxidase activity. In an in vitro bioassay with a semi-isolated Tenebrio heart, MEL induced a slight chronotropic-positive effect only at a higher concentration (10-4 M). Preliminary results indicated that melittin exerts pleiotropic effects on the functioning of the immune system and the endogenous contractile activity of the heart. Some of the induced responses in T. molitor resemble the reactions observed in vertebrate models. Therefore, the T. molitor beetle may be a convenient invertebrate model organism for comparative physiological studies and for the identification of new properties and mechanisms of action of melittin and related compounds.

Antioxidant and cytotoxic activity of new di- and polyamine caffeine analogues.

A series of new di- and polyamine-caffeine analogues were synthesised and characterised by NMR, FT-IR, and MS spectroscopic methods. To access the stability of the investigated caffeine analogues, molecular dynamic simulations were performed in NAMD 2.9 assuming CHARMM36 force field. To evaluate the antioxidant capacity of new compounds, three different antioxidant assays were used, namely 1,1-diphenyl-2-picryl-hydrazyl free radical (DPPH•) scavenging activity, ferrous ions (Fe2+) chelating activity, and Fe3+→Fe2+reducing ability. In vitro, the ability of new derivatives to protect human erythrocytes against oxidative haemolysis induced by free radical from 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AAPH) was estimated. The cytotoxic activity was tested using MCF-7 breast cancer cells and human erythrocytes. All compounds showed the antioxidant capacity depending mostly on their ferrous ions chelating activity. In the presence of AAPH, some derivatives were able to effectively inhibit the oxidative haemolysis. Two derivatives, namely 8-(methyl(2-(methylamino)ethyl)-amino)caffeine and 8-(methyl(3-(methylamino)propyl)amino)caffeine, showed cytotoxic activity against MCF-7 breast cancer cells but not against human erythrocytes. Therefore, it is concluded that the selected di- and polyamine caffeine analogues, depending on their chemical structure, were able to minimise the oxidative stress and to inhibit the tumour cell growth. The confirmed antioxidant and cytotoxic properties of some caffeine derivatives make them attractive for potential applications in food or pharmaceutical industries.

Nicotine alkaloids as antioxidant and potential protective agents against in vitro oxidative haemolysis.

The capacity of eleven nicotine alkaloids to reduce oxidative stress was investigated. In order to provide a structure-activity relationships analysis, new nicotine derivatives with a substituent introduced into the pyrrolidine ring were synthesized and investigated together with nicotine and its known analogs. All newly synthesized compounds were characterized by (1)H, (13)C NMR and EI-MS technique. The antioxidant properties of nicotine, its known analogs and newly produced derivatives, were evaluated by various antioxidant assays such 1,1-diphenyl-2-picryl-hydrazyl free radical (DPPH(•)) scavenging, ferrous ions (Fe(2+)) chelating activity and total reducing ability determination by Fe(3+) â†’ Fe(2+) transformation assay. The protective effects of all compounds tested against 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AAPH) and tert-butyl hydroperoxide (t-BuOOH)-induced oxidative haemolysis and morphological injury of human erythrocytes, were estimated in vitro. The results showed that nicotine alkaloids exhibited various antiradical efficacy and antioxidant activity in a structure- and a dose-dependent manner. In addition, the capacity of nicotine alkaloids to protect erythrocytes from AAPH- and t-BuOOH-induced oxidative haemolysis, was dependent on its incubation time with cells. Our findings showed that chemical and biological investigations conducted simultaneously can provide comprehensive knowledge concerning the antioxidant potential of nicotine alkaloids. This knowledge can be helpful in better understanding the properties of nicotine alkaloids under oxidative stress conditions.

Potentiation of natural killer cell activity with myricetin.

BACKGROUND/AIM: Myricetin is a flavanoid that can be found in a variety of food sources, including red wine. Several reports have indicated that flavonoids may reduce the disease risk of cancer. The aim of the present study was to investigate the effect of some flavonols on natural killer (NK) cell activity. MATERIALS AND METHODS: A time-resolved fluorometric assay (TDA-labeled K562 target cells) was used for measuring the cytotoxic activity of NK-cells pre-treated with different flavonoids. RESULTS: A limited number of flavanoids was tested for their ability to enhance the NK activity. Pre-treating NK cells with myricetin, could potentiate their ability to kill K562 erythroleukemia cells. This enhancement of the NK activity was observed in a dose-dependent manner. Similar treatments with the structurally similar molecule quercetin, that lacks one hydroxyl group, did not have any impact at all on NK activity. CONCLUSION: The enhanced cytotoxic activity observed with myricetin-pretreated-NK cells might shed some light on human studies indicating a preventive role of flavonols against cancer.

Synthesis and haemolytic activity of novel salts made of nicotine alkaloids and bile acids.

A series of novel salts made of nicotine alkaloids and bile acids were synthesized and their haemolytic activity was examined in vitro using human erythrocytes. All compounds were characterized by spectroscopic methods. The novel salts show membrane-perturbing properties inducing the erythrocyte shape alterations and haemolysis in dose-dependent manner. Nicotine decreases the membrane interacting potential of bile acids in the novel compounds. The presence of sulfur or selenium atom in the nicotine molecule affects the haemolytic activity of its novel salts depending on the hydrophobicity of bile acids.

Spontaneous curvature of ganglioside GM1--effect of cross-linking.

The membrane-curvature dependent lateral distribution of outer leaflet ganglioside GM1 (GM1) and the influence of GM1 cross-linking induced by fluorophore-tagged cholera toxin subunit B (CTB) plus anti-CTB was analysed in cell membranes by fluorescence microscopy. Data are presented indicating that cross-linked GM1-ligand patches accumulated at the tips of human erythrocyte echinocytic spiculae induced by Ca(2+)/ionophore A23187. However, when lipid fixative osmium tetroxide was added prior to the ligand no accumulation in spiculae occurred. GM1-staining remained here distributed over the spheroid cell body and in spiculae. Similarly, osmium tetroxide completely prohibited CTB plus anti-CTB-induced GM1 patching in representatives for flat membrane, i.e. discoid erythrocytes and K562 cells. Our results demonstrate that GM1 per se shows low membrane curvature dependent distribution and therefore holds flexible spontaneous curvature. In contrast, the cross-linked GM1-ligand complex has a strong preference for highly outward curved membrane and possesses overall positive spontaneous curvature. Osmium tetroxide efficiently immobilises GM1.

Increased sensitivity of cholera toxin B treated K562 cells to natural killer cells.

Cholera toxin B-subunit (CTB) treatment of K562 erythroleukemia cells increased their sensitivity to be killed by NK-92 cells with more than 10%, compared to untreated cells. A similar treatment of non-T, non-B acute lymphoblastic REH leukemia cells, known to be unsensitive to NK cell mediated cytotoxicity, did not have any impact at all. Visualization of the cross-linked ganglioside(M1) (GM(1)) using fluorescent labeled CTB, indicated accumulation of the fluorescence to one cap and a few smaller patches in both type of cells. Additional cross-linking using anti-CTB antibodies further accentuated capping and increased lysis in the case of K562 cells. Blocking experiments performed with anti-MICA/B, ULBP-2 and/or CD59 antibodies could not inhibit the increased sensitivity mediated by CTB.

Resveratrol oligomers are potent MRP1 transport inhibitors.

BACKGROUND: Knowledge of the structure-activity relationships of multidrug resistance protein 1 (MRP1, ABCC1) inhibitors may aid in developing potent inhibitors that can be used to circumvent MRP1-mediated multidrug resistance. MATERIALS AND METHODS: Six stilbenes were examined for their ability to inhibit MRP1-mediated transport of 2',7'-bis-(carboxypropyl)-5(6)-carboxyfluorescein (BCPCF) from human erythrocytes and into inside-out erythrocyte membrane vesicles (IOVs). The concentrations of stilbenes decreasing BCPCF transport by 50% during 60 min of incubation at 37 degrees C (IC50) were determined from dose-response curves. RESULTS: Stilbenes inhibited BCPCF transport in cells in the rank order (+)-alpha-viniferin (IC50 = 0.8 microM) > sophorastilbene A (IC50 = 3.1 microM) > (-)-epsilon-viniferin (IC50 = 8.9 microM) > piceatannol (IC50 = 57 microM). Resveratrol and rhaponticin were ineffective. (+)-alpha-Viniferin (IC50 = 0.8 microM), sophorastilbene A (IC50 = 3.7 microM) and (-)-epsilon-viniferin (IC50 = 3.5 microM) were also efficient BCPCF transport inhibitors in IOVs. CONCLUSION: Stilbenes may efficiently inhibit MRP1-mediated organic anion transport. This inhibitory potency of stilbenes increases with oligomerisation. The membrane is not a strong barrier for the inhibitory activity of the trimeric stilbenes.

Inhibition of MRP1-mediated efflux in human erythrocytes by mono-anionic bile salts.

BACKGROUND: Multidrug resistance-associated protein 1 (MRP1 or ABCC1) -mediated transport is an important mechanism in multidrug resistance during cancer treatment. One strategy for the reversal of MRP1-mediated multidrug resistance is inhibition of this efflux pump. Therefore, efficient inhibitors are searched for and the structure-activity relationships of inhibitors are studied. In the present work, the ability of a series of mono-anionic bile salts to inhibit MRP1-like substrate transport was examined. MATERIALS AND METHODS: The effect of bile salts on the efflux of the MRP1 substrate 2,7'-bis-(carboxypropyl)-5(6)-carboxyfluorescein (BCPCF) from human erythrocytes was fluorimetrically monitored. RESULTS: All bile salts inhibited the BCPCF efflux. The most efficient inhibitor, lithocholic acid, decreased the BCPCF efflux by 50% (IC50) at 4 microM during 60 min of incubation at 37 degrees C. The most efficient bile salt inhibitors showed high haemolysis start/IC50 concentration ratios and did not induce membrane bending or phosphatidylserine (PS) exposure at IC50. CONCLUSION: The overall hydrophobicity, as well as the orientation of the hydroxyl groups and conjugation with glycine or taurine per se, affect the inhibitory potency of bile salts. The most efficient inhibitors apparently interact with MRP1 in a specific way.

Flavonoids as inhibitors of MRP1-like efflux activity in human erythrocytes. A structure-activity relationship study.

The potency of flavonoids (isoflavones, flavones, and flavanones) to inhibit efflux of 2',7'-bis-(carboxypropyl)-5(6)-carboxyfluorescein (BCPCF) from human erythrocytes was investigated. Structure-activity relationship analysis showed that the strongest inhibitors were found among flavanones bearing a hydrophobic prenyl, geranyl, or lavandulyl group at position 8 (and hydroxyl groups at 5 and 7) in ring A. A prenyl group at position 5' or stilbene at positions 4'-5' in ring B further seemed to increase inhibitor potency. The most efficient flavanones, euchrestaflavanone A and sophoraflavanone H, were approximately 20 times more efficient than genistein, and induced 50% inhibition of BCPCF efflux (IC50) at 3 microM (60 min, 37 degrees C). This is comparable to IC50 of benzbromarone (4 microM) and lower than IC50 of indomethacin (10 microM), both known MRP1 (ABCC1) inhibitors. It is suggested that BCPCF efflux is mainly due to MRP1 activity. Our results indicate that flavonoid molecular structure provides a promising base for development of potent MRP1 inhibitors.