Evaluation of the antioxidant properties of propofol and its nitrosoderivative. comparison with homologue substituted phenols

Propofol (2,6-diisopropylphenol), some substituted phenols (2,6-dimethylphenol and 2,6-ditertbutylphenol) and their 4-nitrosoderivatives have been compared for their scavenging ability towards 1,1-diphenyl-2-picrylhydrazyl and for their inhibitory action on lipid peroxidation. These products were also compared to the classical antioxidants butylated hydroxytoluene and butylated hydroxyanisole. When measuring the reactivity of the various phenolic derivatives with 1,1-diphenyl-2-picrylhydrazyl the following order of effectiveness was observed: butylated hydroxyanisole > propofol > 2,6-dimethylphenol > 2,6-di-tertbutylphenol > butylated hydroxytoluene. In cumene hydroperoxide-dependent microsomal lipid peroxidation, propofol acts as the most effective antioxidant, while butylated hydroxyanisole, 2,6-di-tertbutylphenol and butylated hydroxytoluene exhibit a rather similar effect, although lower than propofol. In the iron/ascorbate-dependent lipid peroxidation propofol, at concentrations higher than 10 microM, exhibits antioxidant properties comparable to those of butylated hydroxytoluene and butylated hydroxyanisole, 2,6-Dimethylphenol is scarcely effective in both lipoperoxidative systems. The antioxidant properties of the various molecules depend on their hydrophobic characteristics and on the steric and electronic effects of their substituents. However, the introduction of the nitroso group in the 4-position almost completely removes the antioxidant properties of the examined compounds. The nitrosation of the aromatic ring of antioxidant molecules and the consequent loss of antioxidant capacity can be considered a condition potentially occurring in vivo since nitric oxide and its derivatives are continuously formed in biological systems.     

Evaluation of the Antioxidant Properties of Propofol and its Nitrosoderivative. Comparison with Homologue Substituted Phenols

Propofol (2,6-diisopropylphenol), some substituted phenols (2,6-dimethylphenol and 2,6-ditertbutylphenol) and their 4-nitrosoderivatives have been compared for their scavenging ability towards 1,1-diphenyl-2-picrylhydrazyl and for their inhibitory action on lipid peroxidation. These products were also compared to the classical antioxidants butylated hydroxytoluene and butylated hydroxyanisole. When measuring the reactivity of the various phenolic derivatives with 1,1-diphenyl-2-picrylhydrazyl the following order of effectiveness was observed: butylated hydroxyanisole>propofol>2,6-dimethylphenol>2,6-di-tertbutylphenol?>?butylated hydroxytoluene. In cumene hydroperoxide-dependent microsomal lipid peroxidation, propofol acts as the most effective antioxidant, while butylated hydroxyanisole, 2,6-di-tertbutylphenol and butylated hydroxytoluene exhibit a rather similar effect, although lower than propofol. In the iron/ascorbate-dependent lipid peroxidation propofol, at concentrations higher than 10?μM, exhibits antioxidant properties comparable to those of butylated hydroxytoluene and butylated hydroxyanisole. 2,6-Dimethylphenol is scarcely effective in both lipoperoxidative systems. The antioxidant properties of the various molecules depend on their hydrophobic characteristics and on the steric and electronic effects of their substituents. However, the introduction of the nitroso group in the 4-position almost completely removes the antioxidant properties of the examined compounds. The nitrosation of the aromatic ring of antioxidant molecules and the consequent loss of antioxidant capacity can be considered a condition potentially occurring in vivo since nitric oxide and its derivatives are continuously formed in biological systems.     

Inhibition of the redox cycling of vitamin K3 (menadione) in mouse liver microsomes

1. Concentration-dependent effects of vitamin K1, coenzyme Q10, butylated hydroxytoluene, nor-dihydroguaiaretic acid and Fe-initiated lipid peroxidation on redox cycling of vitamin K3 were studied in mouse liver microsomes in vitro. 2. The antioxidants (butylated hydroxytoluene, nor-dihydroguaiaretic acid) caused apparent non-competitive inhibition of vitamin K3 redox cycling. 3. Vitamin K1 and coenzyme Q10 caused competitive inhibition of the redox cycling (Ki = 33 and 46 microM, respectively). 4. Fe-initiated microsomal lipid peroxidation caused irreversible decrease of one-electron reduction of vitamin K3. 5. The role of NADPH:cytochrome P-450 reductase along with mechanisms of these inhibitions are discussed.     

The crocin assay for the determination of relative rate constants of alkoxyl radical reactions with the pyridoindole stobadine and with other antioxidants

Summary

The water-soluble carotenoid crocin is bleached in aqueous solutions by tert-butyloxy radicals (t-BuO^.) photolytically generated from tert-butyl hydroperoxide. Crocin bleaching as measured at 440 nm can be competitively inhibited by antioxidants. This method therefore allows the determination of the relative reaction rates of these compounds with the t-BuO^. radicals. In this test system the scavenging effect of the pyridoindole stobadine, a new antioxidant with potential for the treatment of tissue injury caused by oxidative stress, was compared with other known antioxidants. Generally good agreement was observed in two different approaches for the evaluation of the experimental data. The relative rate constants of the antioxidants tested increased in the order: stobadine, chlorpromazine, isoascorbic acid, trolox, ascorbic acid. Since it has been shown previously that stobadine can scavenge alkoxyl radicals also in a non-polar environment, this antioxidant property may be important in the inhibition of lipid peroxidation.     

Effects of Vitamin A and Its Analogs on Nonenzymatic Lipid Peroxidation in Rat Brain Mitochondria

Vitamin A (retinol) and some of its analogs exhibited varying degrees of inhibition on induced iron and ascorbic acid lipid peroxidation of rat brain mitochondria. Malonyldialdehyde production was used as an index of the extent of in vitro lipid peroxidation. The fat-soluble vitamins retinol, retinol acetate, retinoic acid, retinol palmitate, and retinal at concentrations between 0.1 and 10.0 mmol/L inhibited brain lipid peroxidation. Retinol and retinol acetate were the most effective inhibitors. It is concluded from this study that retinol and its analogs can be considered as potential antioxidant factors, more potent than some of the well-known antioxidants such as alpha-tocopherol and butylated hydroxytoluene.     

Lipid peroxidation associated protein damage in rat brain crude synaptosomal fraction mediated by iron and ascorbate

In crude synaptosomal fractions from rat brain exposed to iron and ascorbate, enhanced lipid peroxidation (more than 3-fold compared to control), loss of protein thiols up to the extent of 40% compared to control, increased incorporation of carbonyl groups into proteins (more than 4.5-fold compared to control) and non-disulphide covalent cross-linking of membrane proteins have been observed. The phenomena are not inhibited by catalase or hydroxyl radical scavengers like mannitol or dimethyl sulphoxide. However, chain breaking antioxidants like alpha-tocopherol and butylated hydroxytoluene prevent both lipid peroxidation and accompanying protein oxidation. It is suggested that in this system lipid peroxidation propagated by the decomposition of preformed lipid hydroperoxides by iron and ascorbate is the primary event and products of the peroxidation process cause secondary protein damage. In view of high ascorbate content of brain and availability of several transition metals, such ascorbate mediated oxidative damage may be relevant in the aetiopathogenesis of several neurodegenerative disorders as well as ageing of brain.     

Influence of Antioxidants on the Peroxidative Swelling of Mitochondria in vitro

To clarify the role of prooxidative processes during in vitro swelling of freshly isolated rat liver mitochondria, the influence of different antioxidants and free-radical scavengers was tested. Ascorbate below 10 mmol/L without externally added Fe²⁺ acted as a prooxidant and enhanced swelling. Higher concentrations in the presence of Fe²⁺ showed antioxidant properties and a decrease in swelling and lipid peroxidation. Swelling was abolished by -tocopherol and reduced to 50% by butylated hydroxytoluene. Glutathione supplementation decreased both swelling and lipid peroxidation. Oxidized glutathione caused swelling without any effect on peroxidation. Hydrogen peroxide, cumene hydroperoxide and t-butyl hydroperoxide caused progressive decreases in glutathione and reduced niacinamide coenzyme levels, suggesting prooxidative changes. Dithiothreitol was found to abolish this effect. Thus, antioxidants reverse superoxide-induced mito chondrial swelling and lipid peroxidation in vitro.     

Antioxidant protection by haemopexin of haem-stimulated lipid peroxidation.

Haem (ferrous protoporphyrin IX) is a reactive low-molecular-mass form of iron able to participate in oxygen-radical reactions that can lead to the degradation of proteins, lipids, carbohydrates and DNA. Oxygen-radical reactions are likely to occur upon tissue damage. Extracellular fluids rely on antioxidant mechanisms different from those found inside the cell, and circulating proteins limit radical reactions by converting pro-oxidant forms of iron into less-reactive forms. Of the compounds tested, only apohaemopexin and the chain-breaking antioxidant butylated hydroxytoluene inhibited (by more than 90%) haemin-stimulated peroxidation as measured by formation of conjugated dienes, thiobarbituric acid-reactive material from linolenic acid or peroxidation-induced phospholipid fluorescence. Haptoglobin, the haemoglobin-binding serum protein, was ineffective. Conversely, only haptoglobin significantly inhibited haemoglobin-stimulated lipid peroxidation. Iron-salt-induced lipid peroxidation was inhibited only by apotransferrin and the iron-chelator desferrioxamine. All lipid peroxidations were inhibited by the radical scavengers butylated hydroxytoluene and propyl gallate. These findings support the concept that transport and conservation of body iron stores are closely linked to antioxidant protection.     

Antioxidant Activities of Aqueous Extract from Cultivated Fruit-bodies of Cordyceps militaris （L.） Link In Vitro

Biological antloxldants extracted from plants and fungi have potential abilities to scavenge free radicals and Inhibit lipid peroxldatlon, playing Important roles in preventing diseases, for example, cancer, and aging Induced by reactive oxygen species, which may cause oxidative damage to DNA, proteins and other macromolecules. The antloxldant potency of cultivated fruit-bodies of Cordyceps militarls （L.） Link was investigated In this study. Five established In vitro systems were employed, including the 1,1-dlphenyl-2- plcryldrazyl （DPPH） free radical scavenging, hydroxyl radical eliminating, iron chelating, Inhibition of Ilnolelc acid lipid peroxldatlon and reducing power. The aqueous extract from cultivated fruit-bodies was subjected to the test of amino acid, polysaccharlde and mannitol. Ascorblc acid （Vc）, butylated hydroxytoluene （BHT） and ethylenedlamlnetetraacetlc acid （EDTA） were used as positive controls for comparisons. Among the assays, the aqueous extract of C. mllltarls frult-bodles shows a significant scavenging effect on DPPH, eliminating the capability on hydroxyl radicals and the chelating effect on ferrous Iron. The extract also shows positive results of Inhibiting Ilnoleic acid lipid peroxldatlon and reducing power.     

Combining ursodeoxycholic acid or its NO-releasing derivative NCX-1000 with lipophilic antioxidants better protects mouse hepatocytes against amiodarone toxicity

Nonalcoholic steatohepatitis (NASH) is a common and potentially severe form of liver disease. This study aimed to determine the effect of ursodeoxycholic acid and its NO-releasing derivative NCX-1000 alone or in combination with antioxidants on cultured mouse hepatocytes treated with amiodarone to mimic certain aspects of hepatocyte injury found in NASH. Isolated mouse hepatocytes were incubated with ursodeoxycholic acid or NCX-1000 (0-100 micromol/L) combined or not combined with the hydrophilic antioxidants butylated hydroxytoluene and ascorbic acid (0-100 micromol/L) or with the lipophilic antioxidant alpha-tocopherol (0-100 micromol/L) 15 min before adding amiodarone (50 micromol/L) to the culture medium. Twenty hours later, necrosis, apoptosis, superoxide anion production, and malondialdehyde levels were assessed in cultured cells. Amiodarone led to a dose-dependent decrease in cell viability with an LD50 of 50 micromol/L and increased production of superoxide anion and lipid peroxidation. NCX-1000 showed a better protective potential than ursodeoxycholic acid against the toxic effects of amiodarone. The hydrophilic antioxidants had no effect on the toxicity of amiodarone, whereas alpha-tocopherol at a concentration >100 micromol/L almost completely suppressed it. Ursodeoxycholic acid and NCX-1000 protection was additive only when they were combined with alpha-tocopherol, not with butylated hydroxytoluene or ascorbic acid. In addition, all the antioxidants tested reduced the superoxide anion detected, but only alpha-tocopherol prevented lipid peroxidation induced by amiodarone. The combination of lipophilic antioxidants with ursodeoxycholic acid or NCX-1000 enhances their protective potential and could represent an interesting therapeutic approach to explore for the treatment of NASH.     

Mechanism accounting for the induction of nonspecific permeability of the inner mitochondrial membrane by hydroperoxides

The effect of antioxidants on the nonspecific permeability of the inner mitochondrial membrane induced by cumene hydroperoxide or Ca(2+) has been studied. Butylated hydroxytoluene, butylated hydroxyanisole and 2,2,5,7,8-pentamethyl-6-chromanol, taken at a concentration up to 50 microM, suppress the cumene hydroperoxide-induced accumulation of lipid peroxidation products. In the same range of concentrations, these antioxidants inhibit the activation of nonspecific permeability by cumene hydroperoxide or Ca(2+). Propyl gallate, being less effective under such conditions, fails to affect the induction of nonspecific permeability. Additionally, 2,2,5,7,8-pentamethyl-6-chromanol at a concentration decreasing the accumulation of lipid peroxidation products by 70% has been shown not to increase the lag period of nonspecific permeability induction. Higher antioxidant concentrations, while leading to an increase in the lag period of nonspecific permeability induction, cause but minor suppression of lipid peroxidation. From the results obtained we can assume that free radicals formed in the course of hydroperoxide decomposition or on mitochondrial redox complex interact directly with a system responsible for nonspecific permeability or with regulating components of this system.     

Inhibition of the mono-oxygenase system by butylated hydroxyanisole and butylated hydroxytoluene

The commonly used food-additive antioxidants, butylated hydroxyanisole and butylated hydroxytoluene, are inhibitors of the hepatic microsomal mono-oxygenase system, as assayed by benzpyrene hydroxylase activity and demethylase activities. Generally, butylated hydroxyanisole is a more potent inhibitor than butylated hydroxytoluene. Both inhibitors bind to cytochrome P-450 and induce “type I” binding spectra. Cytochrome P-450 is tentatively assigned as the site of inhibition.     

Preventive effect of several antioxidants after oxidative stress on rat brain homogenates

Brain homogenate was used as a model system to study antioxidant properties of several natural and synthetic antioxidants under oxidative stress. Oxidative stress was induced by Fe/ascorbate system and lipid peroxidation as well as protein modification were studied. Thiobarbituric acid reactive substances (TBARS) were used as a marker of lipid peroxidation. The preventive effect concerning lipid peroxidation decreased in the order: buthylated hydroxytoluene (BHT) (3.5), stobadine (ST) (35), serotonin (54), trolox (98), U 74389G (160), melatonin (3100), (the numbers in the brackets represent IC50 in micromol/l). Methylprednisolone had no effect, and spin traps interfered with TBARS determination. Concerning creatine kinase (CK) activity as a selected marker of oxidative modification of proteins, the preventive effect of antioxidants (30 micromol/l) decreased in the order: BHT (30), trolox (75), stobadine (ST) (77), alpha-phenyl-N-tert-buthylnitrone (PBN) (87), sodium salt of N-tert-buthyl-C-(phenyl-2-sulfone) nitrone (SPBN) (90), (the numbers in the brackets represent the loss of CK activity in percentages, when 100% was the loss of CK activity in the absence of any antioxidant). The nonglucocorticoid steroid U 74389G, methylprednisolone and serotonin had no preventive effects, while melatonin had antioxidant effect only in a higher concentration (1 mmol/l).     

Enzymatic lipid peroxidation and oxidative metabolism of aminazine in brain microsomal fractions]

NAD (P) H-dependent enzymic systems, both of lipid peroxidation and chlorpromazine oxidative metabolism are shown to be localized in the microsomal fractions from human and rat brain. Hydroxy-derivatives of chlorpromazine (e.g. 7-OH-chlorpromazine) formed in the course of enzymic NADPH-dependent metabolism possess antioxidant activity and inhibit lipid peroxidation in the brain microsomes. The properties of enzymic NAD (P) H-dependent oxigenase systems in the membranes of the microsomal reticulum of the liver and brain are compared.     

The role of iron in ferritin- and haemosiderin-mediated lipid peroxidation in liposomes.

Ferritin and haemosiderin were shown, by the measurement of malondialdehyde production and loss of polyunsaturated fatty acids, to stimulate lipid peroxidation in liposomes. At pH 7.4 ascorbate was additionally required to achieve peroxidation; however, peroxidation occurred at pH 4.5 in the presence of iron-proteins alone. The damage was completely inhibited by the incorporation of chain-breaking antioxidants (alpha-tocopherol and butylated hydroxytoluene) into the liposomes. Metal chelators (desferrioxamine and EDTA) also completely inhibited lipid peroxidation. These and further results indicate that, at pH 4.5, even in the absence of a reducing agent, iron is released from haemosiderin and can mediate oxidative damage to a lipid membrane.     

Peroxidation of the dried thin film of lipid by high-energy alpha particles from a cyclotron

High-energy alpha particles produced a dose-dependent linear increase in different lipid peroxidation products (e.g., malondialdehyde (MDA), conjugated dienes, and hydroperoxides) in the dried thin film state. An inverse dose-rate effect was observed when the dose rate was varied by changing either the alpha-particle fluence rate or the alpha-particle energy. The antioxidants alpha-tocopherol and butylated hydroxytoluene (BHT) suppressed the alpha-particle-induced lipid peroxidation in the dried thin film state, and in this respect alpha-tocopherol was found superior to BHT. It was found that alpha-tocopherol was equally efficient in inhibiting lipid peroxidations by alpha particles and ultraviolet light.     

Antioxidant activity of L-adrenaline: a structure-activity insight

L-adrenaline belongs to a group of the compounds known as catecholamines, which play an important role in the regulation of physiological process in living organisms. The antioxidant activity and antioxidant mechanism of L-adrenaline was clarified using various in vitro antioxidant assays including 1,1-diphenyl-2-picryl-hydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), N,N-dimethyl-p-phenylenediamine (DMPD(+)), and superoxide anion radicals (O(2)(-)) scavenging activity, hydrogen peroxide (H(2)O(2)), total antioxidant activity, ferric ions (Fe(3+)) and cupric ions (Cu(2+)) reducing ability, ferrous ions (Fe(2+)) chelating activity. L-adrenaline inhibited 74.2% lipid peroxidation of a linoleic acid emulsion at 30 microg/mL concentration. On the other hand, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), alpha-tocopherol and trolox displayed 83.3, 82.1, 68.1 and 81.3% inhibition on the peroxidation of linoleic acid emulsion at the same concentration, respectively. BHA, BHT, alpha-tocopherol and trolox were used as reference antioxidants and radical scavenger compounds. Moreover, this study will bring an innovation for further studies related to antioxidant properties of L-adrenaline. According to present study, L-adrenaline had effective in vitro antioxidant and radical scavenging activity.     

Antioxidant effect of manganese.

The antioxidant effects of manganese and other transition metals were studied as the inhibition of microsomal lipid peroxidation and crocin bleaching by peroxyl radicals. The peroxyl radical scavenging capacity was measured by competition kinetics analysis. While Zn(II), Ni(II), and Fe(II) were almost completely ineffective, Mn(II) and Co(II) showed a free radical scavenging capacity, exhibiting relative rate constant ratios respectively of 0.513 and 0.287. This indicates that Mn(II) is by far the most active. Therefore, the chain-breaking antioxidant capacity of Mn(II) seems to be related to the rapid quenching of peroxyl radicals according to the reaction R-OO. + Mn(II) + H(+)-->ROOH+Mn(III). The antioxidant mechanism is discussed considering the different reduction potentials of the examined cations.     

Antioxidant effect of anthocyanin on enzymatic and non-enzymatic lipid peroxidation.

In vitro enzymatic and non-enzymatic polyunsaturated fatty acid peroxidation was significantly inhibited in a dose dependent manner by purified anthocyanin, a deep-red colour pigment from carrot cell culture. The kinetics showed that anthocyanin is a non-competitive inhibitor of lipid peroxidation. Anthocyanin has been found to be a potent antioxidant compared to classical antioxidants such as butylated hydroxy anisole (BHA), butylated hydroxy toulene (BHT) and alpha tocopherol. This natural agent, in addition to imparting colour to the food, might prevent autooxidation of lipids as well as lipid peroxidation in biological systems.     

Diphenylacetaldehyde-generated excited states promote damage to isolated rat liver mitochondrial DNA, phospholipids, and proteins.

This work studies damage to rat liver mitochondrial protein, lipid, and DNA caused by electronically excited states generated by cytochrome c-catalyzed diphenylacetaldehyde enol oxidation to triplet benzophenone. The extension of lipid peroxidation was estimated by production of thiobarbituric acid-reactive substances and by formation of Schiff bases with membrane proteins, evaluated by SDS-polyacrylamide gel electrophoresis. Concomitant with DPAA-driven mitochondrial permeabilization, extensive mtDNA fragmentation occurred and DNA adducts with aldehydes-products of fatty acid oxidation-were observed. The degree of lipid peroxidation and mtDNA alterations were significantly decreased by butylated hydroxytoluene, a potent peroxidation chain breaker. The lipid peroxidation process was also partially inhibited by the bioflavonoid rutin and urate totally prevented the mitochondrial transmembrane potential collapse. In all cases, the mitochondrial damage was dependent on the presence of phosphate ions, a putative bifunctional catalyst of carbonyl enolization. These data are consistent with the notion that triplet ketones may act like alkoxyl radicals as deleterious reactive oxygen species on biologic structures. Involvement of singlet dioxygen formed by triplet-triplet energy transfer from benzophenone in the model reaction with DPAA/cytochrome c in the presence of DCP liposomes was suggested by quenching of the accompanying chemiluminescence upon addition of histidine and lycopene.