Concentrated green tea supplement: biological activity and molecular mechanisms.

AIM: This study was undertaken to determine the biological activity of a green tea supplement with respect to cells and erythrocyte membranes and the molecular mechanism of that activity. MAIN METHODS: The extract's activity was evaluated on the basis of its hemolytic, antioxidant and antiinflammatory actions. In addition, the extract's effect on the physical properties of the erythrocyte membrane was examined. We also conducted a detailed analysis of supplement ingredients using high-yield liquid chromatography, supplemented with standard tests of total content of polyphenols and flavonoids in the supplement. KEY FINDINGS: The study showed that green tea extract has a high antioxidant and anti-inflammatory capacity with no deleterious effect on red blood cells. The extract modifies the physical properties of the erythrocyte membrane, apparently by binding to its hydrophilic region, with consequent rigidity of the hydrophobic region, increased hydration and a moderate increase in its resistance to changes in tonicity of the medium. Because the extract's components anchor in the polar region of membrane lipids, they are able to effectively scavenge free radicals in the immediate vicinity of the membrane and hinder their diffusion into its interior. SIGNIFICANCE: Green tea supplement at concentrations markedly exceeding the blood plasma physiological polyphenol concentrations has no destructive effect on the erythrocyte membrane. Due to the high content of flavan-3-ols, the supplement exhibits high biological activity, which makes it an alternative source of those substances to the commonly used infusion of green tea leaves.

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.

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.

Physiological and hemolytic toxicity of some aminophosphonates.

The effect of novel synthesized aminophosphonates on membrane potential and electrical conductance of internodal cells of Nitellopsis obtusa and hemolysis of erythrocytes (RBC) was studied. It was found that those the organophosphorous compounds, when present at 10-100 microM concentrations, caused depolarization and increased electrical conductance of alga membranes. They also influenced fluidity of erythrocyte membranes. When used at higher concentrations aminophosphonates caused hemolysis of RBC. The changes observed depended on structural features of the aminophosphonates, i.e., substituents at the carbon, phosphorus and nitrogen atoms, and, most probably, may be the result of direct interaction of the aminophosphonates with the lipid phase of the plasma membrane and the induced structural changes. Two modes of interaction are proposed.

Influence of organic and inorganic ions on organolead-induced hemolysis of erythrocytes.

The influence of some inorganic (K+, Mg2+, and Al3+) and organic CnH(2n+1)SO3-, n = 12 and 14) ions was studied on the hemolysis of erythrocytes (RBC) caused by organolead compounds (tripropyllead--TPL, tributyllead--TBL and triphenyllead--TPhL chlorides). It was found that sulfonate anions increased the hemolytic effect induced by triorganoleads, while inorganic cations protected RBC against the triorganoleads action, especially when the latter were used at small concentrations. This protection was weaker when the concentration of organoleads increased and depended on the kind of ion. The protective efficacy sequence was like that: Mg2+ > Al3+ > K+. The less hemolytic of the triorganoleads studied was TPL. TBL was slightly more effective than TPhL. The efficacy of the sulfonate ions to increase the triorganolead chloride-induced hemolysis was practically the same for TPL and TBL. A weaker efficacy of C12H25SO3+ was observed when TPhL was used as RBC membrane modifier.

The hemolytic toxicity of some new aminophosphonates.

A series of ten aminophosphonate derivatives were assayed for their hemolytic activity as a preliminary screening for the detection of herbicides. The data obtained indicate: 1. A clear correlation between the hemolytic capacity of the test compounds and their plant growth inhibition and an increase in membrane fluidity was demonstrated. 2. It was found that the most active compounds revealed at least one of the following structural features: an iso-propyl substituent at the phosphorus atom, a tert-butyl group attached to their hexane ring or a long hydrocarbon chain. 3. Ring substituents at the phosphorus (phenyl ring), carbon or nitrogen atoms (hexane) removed the hemolytic activity of compounds. 4. It may be concluded that the hemolytic toxicity of the aminophosphonates studied is related to their ability to incorporate and fuse into the lipid phase of the erythrocyte membrane. The general conclusion is that both stereochemistry and hydrophobicity are deciding factors for the efficiency of the interaction of the studied compounds studied with erythrocytes, and that the most possible location of the aminophosphonates is in the lipid phase of the RBC membrane.

The membrane-disrupting activity of alpha-aminoalkanephosphonic acids and their derivatives.

The influence of a series of acyclic and cyclic aminophosphonates on the physicochemical properties of model (planar lipid membranes-BLM) and biological (erythrocytes-RBC) membranes was studied. The results obtained were compared with the results of physiological tests performed on the aquatic plant Spirodela oligorrhiza. It was found that the inhibition of plant growth by the compounds studied correlated, although not very highly, with the observed changes in the properties of membranes used. It was also found that both the biological activity of aminophosphonates and their efficiency at modifying the physicochemical parameters of membranes depended on their structural features.

Influence of triphenyllead chloride on biological and model membranes.

The physiological and hemolytic toxicities of triphenyllead chloride (TPhL) as well as its modifying influence on model lipid membranes were studied. The experiments allowed the determination of TPhL concentrations causing 50% inhibition of growth of Spirodela oligorrhiza, Lemna minor and Salvinia natans (EC50), 100% hemolysis of pig erythrocytes (C100) and destabilization of planar lipid membranes (CC). Also, fluidity of erythrocyte ghosts was measured by fluorescence technique and osmotic sensitivity of erythrocytes to the presence of TPhL. All parameters studied were found to be dependent on pH, of experimental solutions and the concentration of TPhL. Acidic conditions increased EC50 C100 and CC concentrations of TPhL. Fluorescence and osmotic measurements showed that osmotic stability and fluidity decreased with increasing trimethyllead concentration. A possible mechanism of TPhL toxicity is discussed. It is assumed that TPhL is interacting with the lipid phase of the models used. It is also assumed that there may exist various, ionic and nonionic, forms of TPhL as a result of its speciation under different experimental conditions. These species, due to their differentiated lipophilicity, may exert different effects on the model membranes studied.

Influence of amphiphilic compounds on membranes.

On the basis of Gortel & Grendel (J. Exp. Med., 1925, 41, 439-494) discovery, the importance of the lipid bilayer as an integral and indispensible component of the cell membrane is discussed. In particular, attention focuses on the interaction between membranes and amphiphilic substances. The effect on membranes of quaternary ammonium salts, both in the form of pesticides and oxidants as well as organic compounds of tin and lead are discussed in greater detail.

Antioxidative activity of some quaternary ammonium salts incorporated into erythrocyte membranes.

The antioxidative activity of two series of amphiphilic compounds from a group of quaternary ammonium salts has been investigated. They were so-called bifunctional surfactants synthesized to be used as common pesticides or as antioxidants. The latter application was to be ensured by providing the compounds studied with an antioxidant group. Studies on antioxidative possibilities of those compounds were performed on pig erythrocytes. Due to their hydrophobic parts, they anchor in the erythrocyte membrane and influence the degree of lipid oxidation in the erythrocyte membrane subjected to UV radiation. It was found that compounds of both series decreased the oxidation of the membrane lipids. The inhibition of this oxidation increased with the length of their hydrophobic chains up to fourteen carbon atoms. The compounds of the longest hydrophobic chains showed a somewhat weaker antioxidative activity. Of the two series studied compounds were more effective having bromide ions as counterions. The corresponding compounds of a second series (chlorides) protected erythrocyte significantly weaker against oxidation. The effect of the compounds on fluidity of the erythrocyte membrane has been studied in order to explain the oxidation results. Change in fluidity of the erythrocyte ghost membranes was found also dependent on length of the hydrophobic part of the compounds and was more pronounced in the case of bromide surfactants. The final conclusion is that the compounds studied can be succesfully used as antioxidant agents of good efficacy.

Physiological activity of some organophosphorus compounds and their effects on the physico-chemical properties of model membranes.

The effect of the newly synthesized phosphonic compound dibutyl 2-octylamino-2-propanephosphonate (DBOP) on the growth of the aquatic plant Spirodela oligorrhiza and stability of red blood cells (RBC) and planar lipid membranes (BLM) was studied to determine its physiological activity and, if possible, correlate this activity to compound-induced changes in the mechanical properties of the model membranes. The measure of the phytotoxicity was the DBOP concentration causing 50% plant growth retardation, while measures of stability of model membranes were 100% hemolysis of RBC and a critical concentration of DBOP causing BLM destruction in no more that 3 min. These data were compared with those for dibutyl 1-butylamino-1-cyclohexanephosphonate (DBBC) and diethyl 9-butylamino-9-fluorenephosphonate (DEBF) known for their physiological activities. Both DBBC and DEBF influenced Spirodela growth significantly less than DBOP Destabilization of the model membrane caused by DBBC and DBOP was similar whereas DEBF exerted a weak influence on RBC and BLM stability. The results indicate that the physiological activities of DBOP and DEBF are not limited to the lipid phase of biological membranes and may involve also disturbance of metabolic processes.

Protective effect of quaternary piperidinium salts on lipid oxidation in the erythrocyte membrane.

A new series of amphiphilic compounds with incorporated antioxidant functional group has been investigated. Piperidinium bromides, differing in the alkyl chain length (8, 10, 12, 14 and 16 carbon atoms in the chain) were synthesised to protect biological and/or model membranes against peroxidation and following negative consequences. Their antioxidant activity was studied with erythrocytes subjected to UV radiation. The salts used inhibited lipid oxidation in the erythrocyte membrane. The degree of this inhibition depended on the alkyl chain length of the bromide used and increased with increasing alkyl chain length. A comparison of the results obtained for piperidinium bromides with those obtained for the widely used antioxidant 3,5-di-t-butyl-4-hydroxytoluene-(BHT) revealed that only two shortest alkyl chain salts were less efficient than BHT in protecting erythrocyte membranes. A similar comparison with antioxidant efficiency of flavonoids extracted from Rosa rugosa showed that they protected the membranes studied more weakly than the least effective eight-carbon alkyl chain piperidinium bromide. The three compounds of longest alkyl chains were the most active antioxidants. Their activities did not differ significantly.

Influence of counterions on the interaction of pyridinium salts with model membranes.

The interaction of pyridinium salts (PS) with red blood cells and planar lipid membranes was studied. The aim of the work was to find whether certain cationic surfactant counterion influence its possible biological activity. The counterions studied were Cl-, Br-, I-, ClO4-, BF4- and NO3-. The model membranes used were erythrocyte and planar lipid membranes (BLM). At high concentration the salts caused 100% erythrocyte hemolysis (C100) or broke BLMs (CC). Both parameters describe mechanical properties of model membranes. It was found that the efficiency of the surfactant to destabilize model membranes depended to some degree on its counterion. In both, erythrocyte and BLM experiments, the highest efficiency was observed for Br-, the lowest for NO3-. The influence of all other anions on surfactant efficiency changed between these two extremities; that of chloride and perchlorate ions was similar. Some differences were found in the case of BF4- ion. Its influence on hemolytic possibilities of PS was significant while BLM destruction required relatively high concentration of this anion. Apparently, the influence of various anions on the destructive action of PS on the model membrane used may be attributed to different mobilities and radii of hydrated ions and hence, to different possibilities of particular anions to modify the surface potential of model membranes. This can lead to a differentiated interaction of PS with modified bilayers. Moreover, the effect of anions on the water structure must be taken into account. It is important whether the anions can be classified as water ordering kosmotropes that hold the first hydration shell tightly or water disordering chaotropes that hold water molecules in that shell loosely.

The role of counterions in the protective action of some antioxidants in the process of red cell oxidation.

A number of new surfactants with an incorporated antioxidant functional group were synthesized in order to be used as agents protecting biological and/or model membranes against lipid peroxidation. Hydrophobic parts of these amphiphilic antioxidants ensured their incorporation into the membranes studied, viz. pig erythrocytes. Proper concentrations of the antioxidants were used to avoid erythrocyte membrane destruction during the experiments. The work contains the results of studies on the protective effect of two groups of antioxidants. They differed in the chain length incorporated into the membrane hydrophobic part and in the kind of counterion (chloride and bromide). The role of these factors in the protective action of the compounds studied is discussed as well as their practical application.

The role of counterions in the interaction of bifunctional surface active compounds with model membranes.

Interaction of two series of bifunctional surfactants (bromides and chlorides) with red blood cells and planar lipid membranes was studied. The aim of the work was to determine the role of counterions in the mechanism of interaction of bifunctional cationic surfactants with model membranes. In each case bromides influenced model membranes to a greater degree than the corresponding chlorides. The possible explanation of the obtained results is presented. It seems that the greater ability of bromides to destabilize model membranes in comparison with chlorides can be attributed to the greater mobility and the smaller radius of the hydrated bromide ion. This may underlie the greater ease that this anion can modify the surface potential of the lipid bilayer, thus enhancing the interaction of the cationic surfactant with such a modified bilayer.

Hemolytic activity of aminoethyl-dodecanoates.

The effect of new lysosomotropic compounds on red blood cell hemolysis and erythrocyte membrane fluidity has been investigated. In earlier studies it was shown that the compounds inhibit the growth of yeast and plasma membrane H(+)-ATPase activity. The study was performed with eight aminoethyl esters of lauric acid variously substituted at nitrogen atom. Esters of dodecanoic acid were chosen for study because at that chain length dimethylaminoethyl esters showed maximum activity. The hemolytic activity of the substances studied exhibits diversified activity in their interaction with the erythrocyte membrane: they differ in hemolytic activity and affect membrane fluidity differently. Erythrocyte membrane fluidity changes under the effect of those compounds which possess highest hemolytic activity. The hemolytic activity of the aminoesters investigated was found to follow a sequence that depended on basicity (i.e. ability of the protonated form formation) of the compound and its polar head group size. The most active are the compounds that possess not more than four carbon atoms substituted at nitrogen and highest pKa value.

Electrostatic inhibition of hemolysis induced by organotin compounds.

The effect of cations on the kinetics of hemolysis caused by organotin compounds was studied. The ions used in the investigation diminish or totally inhibit hemolysis of red cells induced by organotin compounds. The degree of inhibition depends both on the kind of ion and the compounds that induce hemolysis. The ions Zn2+, Co2+, and Cd2+ present in the medium at 50 microM concentration totally protect the erythrocytes against hemolysis induced by the compound (C3H7)3SnCl. The study has also shown the monovalent ions K+ and trimethyldodecylammonium bromide are less potent inhibitors of hemolysis than divalent ions, which is not the case for two non-ionic organotin compounds only. The studies performed indicate that hemolysis induced by organotin compounds is inhibited due to electrostatic interaction between the cations selected and erythrocyte membrane.