Hanna Pruchnik

Interaction of selected anthocyanins with erythrocytes and liposome membranes

Anthocyanins are one of the main flavonoid groups. They are responsible for, e.g., the color of plants and have antioxidant features and a wide spectrum of medical activity. The subject of the study was the following compounds that belong to the anthocyanins and which can be found, e.g., in strawberries and chokeberries: callistephin chloride (pelargonidin-3-O-glucoside chloride) and ideain chloride (cyanidin-3-O-galactoside chloride). The aim of the study was to determine the compounds’ antioxidant activity towards the erythrocyte membrane and changes incurred by the tested anthocyanins in the lipid phase of the erythrocyte membrane, in liposomes composed of erythrocyte lipids and in DPPC, DPPC/cholesterol and egg lecithin liposomes. In particular, we studied the effect of the two selected anthocyanins on red blood cell morphology, on packing order in the lipid hydrophilic phase, on fluidity of the hydrophobic phase, as well as on the temperature of phase transition in DPPC and DPPC/cholesterol liposomes. Fluorimetry with the Laurdan and Prodan probes indicated increased packing density in the hydrophilic phase of the membrane in the presence of anthocyanins. Using the fluorescence probes DPH and TMA-DPH, no effect was noted inside the hydrophobic phase of the membrane, as the lipid bilayer fluidity was not modified. The compounds slightly lowered the phase transition temperature of phosphatidylcholine liposomes. The study has shown that both anthocyanins are incorporated into the outer region of the erythrocyte membrane, affecting its shape and lipid packing order, which is reflected in the increasing number of echinocytes. The investigation proved that the compounds penetrate only the outer part of the external lipid layer of liposomes composed of erythrocyte lipids, DPPC, DPPC/cholesterol and egg lecithin lipids, changing its packing order. Fluorimetry studies with DPH-PA proved that the tested anthocyanins are very effective antioxidants. The antioxidant activity of the compounds was comparable with the activity of Trolox®.

Effect of phenyltin compounds on lipid bilayer organization

Phenyltin compounds are known to be biologically active. Their chemical structure suggests that they are likely to interact with the lipid fraction of cell membranes. Using fluorescence and NMR techniques, the effect of phenyltin compounds on selected regions of model lipid bilayers formed from phosphatidylcholine was studied. The polarization of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) dipalmitoyl-L-phosphatidylethanolamine and desorption of praseodymium ions was used to probe the polar region, whereas the polarization of 1 - (4 - trimethylammoniumphenyl) - 6 - phenyl -1,3,5-hexatriene p-toluenesulfonate measured the hydrophobic core of the membrane. In addition, changes in the N-(5-fluoresceinthiocarbanoly)dipalmitoyl - L - α - phosphatidyl -ethanolamine fluorescence intensity indicated the amount of charge introduced by organotin compounds to the membrane surface. There were no relevant changes of measured parameters when tetraphenyltin was introduced to the vesicle suspension. Diphenyltin chloride causes changes of the hydrophobic region, whereas the triphenyltin chloride seems to adsorb in the headgroup region of the lipid bilayer. When the hemolytic activity of phenyltin compounds was measured, triphenyltin chloride was the most effective whereas diphenyltin chloride was much less effective. Tetraphenyltin causes little damage. Based on the presented data, a correlation between activity of those compounds to hemolysis (and toxicity) and the location of the compound within the lipid bilayer could be proposed. In order to inflict damage on the plasma membrane, the compound has to penetrate the lipid bilayer. Tetraphenyltin does not partition into the lipid fraction; therefore its destructive effect is negligible. The partition of the compound into the lipid phase is not sufficient enough, by itself, to change the structure of the lipid bilayer to a biologically relevant degree. The hemolytic potency seems to be dependent on the location of the compound within the lipid bilayer. Triphenyltin chloride which adsorbs on the surface of the membrane, causes a high level of hemolysis, whereas diphenyltin chloride, which penetrates much deeper, seems to have only limited potency.

Biophysical Mechanism of the Protective Effect of Blue Honeysuckle (Lonicera caerulea L. var. kamtschatica Sevast.) Polyphenols Extracts Against Lipid Peroxidation of Erythrocyte and Lipid Membranes

Abstract The aim of the present research was to determine the effect of blue honeysuckle fruit and leaf extracts components on the physical properties of erythrocyte and lipid membranes and assess their antioxidant properties. The HPLC analysis showed that the extracts are rich in polyphenol anthocyanins in fruits and flavonoids in leaves. The results indicate that both extracts have antioxidant activity and protect the red blood cell membrane against oxidation induced by UVC irradiation and AAPH. The extracts do not induce hemolysis and slightly increase osmotic resistance of erythrocytes. The research showed that extracts components are incorporated mainly in the external part of the erythrocyte membrane, inducing the formation of echinocytes. The values of generalized polarization and fluorescence anisotropy indicate that the extracts polyphenols alter the packing arrangement of the hydrophilic part of the erythrocyte and lipid membranes, without changing the fluidity of the hydrophobic part. The DSC results also show that the extract components do not change the main phase transition temperature of DPPC membrane. Studies of electric parameters of membranes modified by the extracts showed that they slightly stabilize lipid membranes and do not reduce their specific resistance or capacity. Examination of IR spectra indicates small changes in the degree of hydration in the hydrophilic region of liposomes under the action of the extracts. The location of polyphenolic compounds in the hydrophilic part of the membrane seems to constitute a protective shield of the cell against other substances, the reactive forms of oxygen in particular.

Changes Caused by Fruit Extracts in the Lipid Phase of Biological and Model Membranes

The aim of the study was to determine changes incurred by polyphenolic compounds from selected fruits in the lipid phase of the erythrocyte membrane, in liposomes formed of erythrocyte lipids and phosphatidylcholine liposomes. In particular, the effect of extracts from apple, chokeberry, and strawberry on the red blood cell morphology, on packing order in the lipid hydrophilic phase, on fluidity of the hydrophobic phase, as well as on the temperature of phase transition in DPPC liposomes was studied. In the erythrocyte population, the proportions of echinocytes increased due to incorporation of polyphenolic compounds. Fluorimetry with a laurdan probe indicated increased packing density in the hydrophilic phase of the membrane in presence of polyphenolic extracts, the highest effect being observed for the apple extract. Using the fluorescence probes DPH and TMA-DPH, no effect was noted inside the hydrophobic phase of the membrane, as the lipid bilayer fluidity was not modified. The polyphenolic extracts slightly lowered the phase transition temperature of phosphatidylcholine liposomes. The studies have shown that the phenolic compounds contained in the extracts incorporate into the outer region of the erythrocyte membrane, affecting its shape and lipid packing order, which is reflected in the increasing number of echinocytes. The compounds also penetrate the outer part of the external lipid layer of liposomes formed of natural and DPPC lipids, changing its packing order.

Butyltin(IV) 2-sulfobenzoates: Synthesis, structural characterization and their cytostatic and antibacterial activities

Three butyltin complexes with 2-sulfobenzoic acid [Sn(C(4)H(9))(2){O(3)SC(6)H(4)COO-2}(H(2)O)]·(C(2)H(5)OH) (1), [Sn(C(4)H(9))(3){O(3)SC(6)H(4)COOH-2}] (2) and [Sn(2)(C(4)H(9))(6){μ-O(3)SC(6)H(4)COO-2}] (3) have been synthesized and characterized by IR and (1)H, (13)C and (119)Sn NMR spectra. They show interesting properties in solid state and solutions because there are many modes of coordination of the Sbz ligand. The structure of complex 1 has been determined by X-ray crystallography. It is a chain compound with 2-sulfonatobenzoate coordinated to Sn atoms as a bridging and chelate ligand via O atoms of COO and SO(3) groups. In solutions the chains dissociate giving mainly mononuclear complexes. The NMR spectra and calculation at the DFT B3LYP/3-21G** level indicate that in solutions of compounds 1, 2 and 3 in polar solvents, many complexes showing dynamic properties are formed. Density functional theory (DFT) calculations showed that many five- and six-coordinate isomers and conformers can exist in equilibrium. All compounds effectively interact with AMP and ATP. The NMR spectra showed that nucleotides are coordinated to Sn atoms via PO(4) groups. The complexes are very active cytostatic agents against tumor strains. They are more effective than cisplatin. It is interesting that activity of 3 against non-tumor cell NHDF is lower than against tumor cells. Antibacterial activity of 1 and 2 has been investigated. Compound 2 is a very effective agent against Gram-positive bacteria. Antibacterial activity of 1 is lower than that of 2. Activity of 1 both against Gram-positive and Gram-negative bacteria is similar.

The effect of some phenyltin compounds on the thermotropic phase behaviour and the structure of model membranes

The effects of diphenyltin dichloride (DPhT), triphenyltin chloride (TPhT) and tetraphenyltin (TTPhT) on the thermotropic phase behaviour of phosphatidylcholine bilayers were studied. All the phenyltin compounds investigated affected phase transitions differently. TTPhT broadened the main phase transition but it left the transition temperatures and enthalpy unchanged. TPhT reduced the transition temperatures and the enthalpies while DPhT showed a dual effect on the pretransition and the main transition. At low concentrations DPhT reduced the temperatures of the transitions slightly and at higher concentrations it increased them. Based on differential scanning calorimetry (DSC) and also 1 H NMR and 31 P NMR measurements, it is suggested that DPhT induces interdigitated gel phase formation and TPhT induces hexagonal phase formation. TTPhT seems to affect the structure only a little. The toxic activity of DPhT and TPhT seems to be connected with their ability to induce changes in the membrane structure.

Thermodynamics and biological properties of the aqueous solutions of new glucocationic surfactants.

Thermodynamic properties of aqueous solutions of newly synthesized compounds, namely, N-[2-(beta-D-glucopyranosyl)ethyl]-N,N-dimethyl-N-alkylammonium bromides with hydrophobic tails of 12 (C12DGCB) and 16 (C16DGCB) carbon atoms, determined as a function of concentration by means of direct methods, are reported here. Dilution enthalpies, densities, and sound velocities were measured at 298 K, allowing for the determination of apparent and partial molar enthalpies, volumes, and compressibilities. Changes in thermodynamic quantities upon micellization were derived using a pseudophase-transition approach. From a comparison with the corresponding acetylated compounds N-[2-(2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl)ethyl]-N,N-dimethyl-N-dodecylammonium bromide (C12AGCB) and N-[2-(2,3,4,6-tetra-O-acetyl-beta-D-glucopyanosyl)ethyl]-N,N-dimethyl-N-hexadecylammonium bromide (C16AGCB), the role played in the micellization process by the acetylated glycosyl moiety was inferred: it enhances the hydrophobic character of the molecule and lowers the change in enthalpy of micelle formation by about 1.5 kJ mol(-1). By comparing the volume of C12DGCB with those of DEDAB and DTAB, the volumes taken up by the (beta- d-glucopyranosyl)ethyl and beta- d-glucopyranosyl groups were found to be 133 and 99 cm3 mol(-1), respectively. Regarding the interaction with DPPC membranes, it seems that the sugar moiety of the hexadecyl deacetylated compound gives rise to hydrogen bonds with the oxygen atoms of the lipid phosphates, shifting the phase transition of DPPC from a bilayer gel to a bilayer liquid crystal to lower temperatures. C16AGCB induces significantly greater changes than C16DGCB in the structure of liposomes, suggesting the formation of domains. The interaction is strongly enhanced by the presence of water. Neither compound interacts strongly with DNA or compacts it, as shown by EMSA assays and AFM images. Only C16AGCB is able to deliver little DNA inside cells when coformulated with DOPE, as shown by the transient transfection assay. This might be related to the ability of C16AGCB to form surfactant-rich domains in the lipid structure.

The effect of the lipid bilayer state on fluorescence intensity of fluorescein-PE in a saturated lipid bilayer.

Fluorescein-PE is a fluorescence probe that is used as a membrane label or a sensor of surface associated processes. Fluorescein-PE fluorescence intensity depends not only on bulk pH, but also on the local electrostatic potential, which affects the local membrane interface proton concentration. The pH sensitivity and hydrophilic character of the fluorescein moiety was used to detect conformational changes at the lipid bilayer surface. When located in the dipalmitoylphosphatidylcholine (DPPC) bilayer, probe fluorescence depends on conformational changes that occur during phase transitions. Relative fluorescence intensity changes more at pretransition than at the main phase transition temperature, indicating that interface conformation affects the condition in the vicinity of the membrane. Local electrostatic potential depends on surface charge density, the local dielectric constant, salt concentration and water organisation. Initial increase in fluorescence intensity at temperatures preceding that of pretransition can be explained by the decreased value of the dielectric constant in the lipid polar headgroups region related in turn to decreased water organisation within the membrane interface. The abrupt decrease in fluorescence intensity at temperatures between 25 °C and 35 °C (DPPC pretransition) is likely to be caused by an increased value of the electrostatic potential, induced by an elevated value of the dielectric constant within the phosphate group region. Further increase in the fluorescence intensity at temperatures above that of the gel-liquid phase transition correlates with the calculated decreased surface electrostatic potential. Above the main phase transition temperature, fluorescence intensity increase at a salt concentration of 140 m M is larger than with 14 m M . This results from a sharp decline of the electrostatic potential induced by the phosphocholine dipole as a function of distance from the membrane surface.

Antifungal activity of organotin compounds with functionalized carboxylates evaluated by the microdilution bioassay in vitro

We investigated the susceptibility of 96 well-characterized strains of yeast-like and filamentous fungi towards new organotin compounds: (1) [Sn(C4H9)3(OOCC6H4SO3H-2)], (2) Sn(C4H9)3{OOC(CH2)3P(C6H5)3}]Br, and (3) [Sn(C6H5)3[OOC(CH2)3N(CH3)3}]Cl. In the case of yeast-like fungi, the in vitro susceptibility tests were carried out according to the Clinical Laboratory Standards Institute (CLSI, formerly NCCLS) reference method M27-A2, while for filamentous fungi the investigations were conducted according to the M38-A and M38-P methods. The organotin complexes 1, 2 and 3 are active antifungal agents. Minimal inhibitory concentrations (MIC) were in the range of 0.25-4.68 microg/ml for all tested fungal strains. Considerably larger differences were found for minimal fungicidal concentrations (MFC). In the case of yeast-like fungi, the fungicidal effect was generally observed at organotin compounds concentrations of 2.34-9.37 microg/ml. The MFC values for filamentous fungi were considerably higher and were in the range of 18.74-50 microg/ml. In conclusion, organotin compounds 1, 2 and 3 showed high fungistatic and fungicidal activities against different species of pathogenic and nonpathogenic fungi. However, they were also highly cytotoxic towards two mammalian cell lines.

Effect of chlorogenic acid on the phase transition in phospholipid and phospholipid/cholesterol membranes

Chlorogenic acid (CGA) is present in many plants, especially in green coffee, dry plums, and bilberries. It is an important bioactive polyphenol. Studies showed that CGA has an antioxidative, bacteriostatic, anticancer, antiviral, and anti-inflammatory activity. Despite great interest in this compound, its interaction with the lipid model membrane has not yet been investigated. To better understand the relationship between the biological activity of CGA and its interaction with biological membranes, the thermotropic behavior of model lipid membranes was investigated. The effect of CGA on the model lipid membrane, specifically on the lipid bilayer phase transitions, was examined by the combined methods: differential scanning calorimetry and fluorescence spectroscopy. In particular, the degree of packing order of the hydrophilic phase of the lipid bilayer was determined using the fluorimetric method with Laurdan and Prodan probes, while the fluorescence anisotropy of the hydrophobic phase with the DPH and TMA-DPH probes. The results of the study show that CGA incorporates mainly into the hydrophilic part of membrane, changing the packing order of the polar heads of lipids. No significant changes were recorded in membrane fluidity of the hydrophobic membrane region, for the fluorescence anisotropy practically did not change. One can thus infer that CGA does not penetrate deep into the hydrophobic area of the membrane.