Suppression of radical-induced lipid peroxidation in a model system by alkyl esters of cinnamate quaternary ammonium salts.

Three new groups of phenolic antioxidants, quaternary ammonium salts with a phenol ring and alkyl chains of different length (pyrrolidine ethyl esters of 3,5-di-t-butyl-4-hydroxydihydrocinnamic acid n-alkoxymethylchlorides (PYE-n) or n-alkylbromides (PYA-n) and 2-dimethylaminoethyl ester n-alkylbromides (PPA-n), were synthesized. Some of them were previously found to efficiently protect yeast cells against oxidants and to inhibit the production of thiobarbituric acid-reactive substances in whole yeast cells and in isolated membrane lipids. The new antioxidants (at 1-100 microM) abolished or diminished peroxidation of olive oil emulsions caused by the OH*-producing Fe2+ and RO* and ROO*-producing tert-butylhydroperoxide (TBHP) and the azo compounds 2,2'-azobis-(amidinopropane)dihydronitrile (AAPH) and 1,1'-azobis-(1-cyclohexanecarbonitrile) (ACHN): all present at 10 mM. The efficiency of individual both antioxidants was examined in relation to the type of lipid peroxidation inducer, the site of antioxidant incorporation into the emulsion lipid phase, the length of the alkyl chain, and the maximum concentration of effective antioxidant monomers given by its critical micelle concentration. PYA-n class compounds were highly efficient against all peroxidation inducers and their efficiency did not depend on the position of their molecules in the lipid phase and/or on the aliphatic chain length. In contrast, the efficiency of PYE-n and PPA-n class compounds depended both on the type of oxidant and on the length of their aliphatic chain. Their potency against Fe2+ and ACHN increased with increasing alkyl chain length whereas with AAPH it dropped with increasing alkyl chain length. A similar pattern was found with the action of PYE-n against TBHP whereas in the PPA-n group an extending alkyl chain reduced the anti-TBHP efficiency. These relationships may not be entirely straightforward and other factors (chemical nature of each compound, its possible interaction with fluorescent probes used for diagnostics, etc.) may play a considerable and not yet quite clear role. PPA-n class antioxidants have the lowest critical micelle concentration, which may limit their efficiency. Nevertheless, these phenolic antioxidants can be conveniently employed as highly efficient inhibitors of lipid peroxidation.

The antioxidant activity of BHT and new phenolic compounds PYA and PPA measured by chemiluminescence.

The antioxidative properties of two series of new phenolic, amphiphilic compounds were evaluated using the chemiluminescence (CL) method. 2,2'-Azobis (2-amidinopropane) dihydrochloride (AAPH) was used as a source of free radicals, to obtain high and prolonged CL. Three different kinds of buffers (organic and inorganic) were tested. The CL level varied only slightly depending on the buffer but increased significantly with the pH. Twelve newly synthesised compounds were compared with butylated hydroxytoluene (BHT), a commercially used antioxidant. The new antioxidants included two classes of quaternary ammonium salts with a phenol substituent functioning as an antioxidant. The salts were synthesised by quaternarization of pyrrolidine ethyl esters of dihydrocinnamic acid by n-alkoxymethyl bromides (PYA-n) or quaternarization of 2-dimethylaminoethyl esters by n-alkyl bromides (PPA-n). All the tested compounds quenched CL proportionally to their concentrations. In our experimental conditions 8.5 microM BHT quenched 50% of the CL. The PYA and PPA compounds had IC50 two to six times lower than BHT. CL inhibition was proportional to the pH for all antioxidants. The relationships between the structure and activity of the tested compounds are discussed.

Spontaneous and radical-induced plasma membrane lipid peroxidation in differently oxidant-sensitive yeast species and its suppression by antioxidants.

Formation of thiobarbituric acid-reactive substances (TBRS; nmol/mg lipids) indicative of lipid peroxidation was measured in whole cells and in isolated plasma membrane lipids from three yeast species differing in oxidant sensitivity (Schizosaccharomyces pombe, Saccharomyces cerevisiae and Rhodotorula glutinis) after exposure to the Fenton reagent, FeII, H2O2, tert-butyl hydroperoxide (TBHP) and azo compounds (AAPH, ACHN). In whole cells, spontaneous TBRS formation rose in the sequence S. pombe < S. cerevisiae < R. glutinis (1:approximately 5:approximately 7). Oxidants increased the TBRS production 13-18 fold in the sequence FeII approximately TBHP > AAPH approximately ACHN approximately Fe-Fenton > H2O2. This increase need not be solely due to increased lipid peroxidation. In isolated plasma membrane lipids from all three species, the spontaneous TBRS production referred to 1 mg lipids was 9-13-fold higher than in whole cells. In S. pombe lipids, only TBHP increased the TBRS production. In lipids from S. cerevisiae and R. glutinis, all added oxidants increased the spontaneous TBRS production 2-3 times in the sequence TBHP > ACHN > AAPH > FeII > Fe-Fenton > H2O2. Oxidant-induced TBRS production in both whole cells and isolated membrane lipids was partially suppressed by the lipid peroxidation inhibitors 2,6-di-tert-butyl-4-methylphenol ("butylated hydroxytoluene"; BHT) and the newly synthesized PYA12 compound. Both agents were more effective in isolated lipids than in whole cells and against OH.-producing than against ROO.- or RO.-producing oxidants. Yeast membrane lipids, which are generally poor in polyunsaturated fatty acids, are thus subject to perceptible lipid peroxidation.

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.

New phenolic antioxidants of PYA and PYE class increase the resistance S. cerevisiae strain SP4, its SOD- and catalase-deficient mutants to lipophilic oxidants.

Was demonstrate the protection ability against reactive oxygen species afforded to S. cerevisiae (wild-type strain SP4 and its mutants deficient in major antioxidant enzymes--catalase T and A, CuZnSOD) by PYA and PYE, new groups of phenolic antioxidants (quaternary ammonium salts of dihydrocinnamic acid amino esters with different alkyl chains; synthesized in this laboratory). The survival of strains exposed to the lipophilic oxidation inducers tert-butyl hydroperoxide (TBHP) and 1,1'-azobis(4-cyclohexane carbonitrile) (ACCN) with or without antioxidant pretreatment was determined by plating. S. cerevisiae mutant deficient in SOD was found to be hypersensitive to TBHP and ACCN while the sensitivity of the strain deficient in catalase T and A was about the same as in the wild-type strain. A 1-h preincubation of cells of both the wild-type and the mutant strains with the phenolic antioxidants prior to exposure to TBHP or ACCN substantially increased the cell survival. The magnitude of protection depended on the strain and the length of the alkyl chain of the antioxidant; the best average protection against TBHP was provided by PYE and PYA compounds with 12- and 16-membered alkyl chains whereas PYE-8 and PYA-12 derivatives afforded the best average protection against ACCN.

Inhibition of lipid peroxidation in the erythrocyte membrane by quaternary morpholinium salts with antioxidant function.

The antioxidant activity of five new compounds from a group of quaternary ammonium salts with antioxidant function has been investigated. The effect of the compounds on the degree of lipid oxidation in the erythrocyte membrane subjected to UV radiation was studied. It was found that all the salts used decreased oxidation of the lipids in the erythrocyte membrane. The antioxidant activity of the ammonium salts studied increased with their alkyl chain length. Three compounds with the longest alkyl chains were the most active antioxidants, and their antioxidant properties were comparable to those of a flavonoid extracted from hawthorn. The antioxidant effectiveness of the ten-carbon alkyl chain compound was comparable with that of the known antioxidant 3,5-di-t-butyl-4-hydroxytoluene (BHT). The least effective antioxidant studied proved to be the eight-carbon alkyl chain compound. The effect of these compounds on fluidity of the erythrocyte membrane has been studied, and for all an increase of fluidity of the membrane was observed. The changes in erythrocyte ghosts fluidity depended both on concentration and type of compound. A fluorimetric study also indicated that rigidity of the erythrocyte membrane increased with degree of its oxidation, but with antioxidants present membrane rigidity increased less.

Effects of C8 alkoxymethylene trimethylammonium chloride on Saccharomyces cerevisiae.

The influence of the C8 alkoxymethylene trimethyloammonium chloride on the growth of Saccharomyces cerevisiae and activity of mitochondria was studied. It was shown that the compound at low concentration inhibited growth on glycerol medium, but considerably higher concentration is involved in the inhibition of growth on glucose medium. C8-ATC also exerted another inhibitory effect on genotypically different yeast strains: it appeared that rho- strain is more sensitive than rho+ strain. C8-ATC compound was not capable itself of inducing petite mutations, but is able of retarding the petite inducing activity of the mutagen ethidium bromide. The result pointed out the role of mitochondria in the expression of sensitivity to the investigated compound.

Biological effect of alkoxymethylene trimethylammonium chlorides on yeast Saccharomyces cerevisiae.

Six compounds of the group of quaternary ammonium salts have been tested for their biological activity using yeasts as a biological system. They have an inhibitory effect on respiration, cell growth and amino acid transport. A destroying action on protoplast regeneration and respiration has been also observed. The studied chemicals appear to have very pleiotropic action, focused on a damage of mitochondrial and cell plasma membranes.