Inhibitory action of Oren-gedoku-to extract on enzymatic lipid peroxidation in rat liver microsomes

We examined the inhibitory action of the extract of Oren-gedoku-to, a traditional herbal medicine known to act as an antioxidant, on enzymatic lipid peroxidation in rat liver microsomes. Simultaneous addition of a spray-dried preparation of Oren-gedoku-to extract (Tsumura TJ-15) inhibited enzymatic lipid peroxidation induced by reduced beta-nicotinamide adenine dinucleotide phosphate (NADPH) and ADP/Fe3+ complex in liver microsomes in a dose-dependent manner. When the inhibition by TJ-15 of enzymatic lipid peroxidation in liver microsomes was kinetically analyzed, this medicine showed a competitive inhibition against NADPH or ADP/Fe3+ complex. TJ-15 inhibited the NADPH-driven enzymatic reduction of ADP/Fe3+ complex or cytochrome c in liver microsomes competitively. TJ-15 enhanced NADPH consumption by liver microsomes with ADP/Fe3+ complex. Treatment with TJ-15 after the onset of enzymatic lipid peroxidation in liver microsomes inhibited the progression of lipid peroxidation in a dose-dependent manner. The present results indicate that Oren-gedoku-to extract inhibits enzymatic lipid peroxidation in rat liver microsomes in the initiation and propagation steps in a dose-dependent manner. These results also suggest that Oren-gedoku-to extract inhibits enzymatic lipid peroxidation in rat liver microsomes not only through its antioxidant action but also through reduction of the supply of electrons derived from NADPH to ADP/Fe3+ complex in liver microsomes both in a competitive manner and through stimulation of NADPH oxidation.     

Antioxidant properties of Thonningianin A,isolated from the African medicinal herb,Thonningia sanguinea

The antioxidant properties of Thonningianin A (Th A), an ellagitannin, isolated from the methanolic extract of the African medicinal herb, Thonningia sanguinea were studied using the NADPH and Fe2+/ascorbate-induced lipid peroxidation (LPO), electron spin resonance spectrometer and the deoxyribose assay. Th A at 10 microM inhibited both the NADPH and Fe2+/ascorbate-induced LPO in rat liver microsomes by 60% without inhibitory effects on cytochrome P450 activity. Th A was similar to the synthetic antioxidant, tannic acid, as an inhibitor of both the NADPH and Fe2+/ascorbate-induced LPO but potent than gallic acid, vitamin C and vitamin E. While Th A poorly scavenged the hydroxyl radical generated by the Fenton reaction it dose-dependently scavenged 1,1-diphenyl-2-picrylhydrazyl, superoxide anion and peroxyl radicals with IC50 of 7.5, 10 and 30 microM, respectively. Furthermore, Th A showed inhibitory effects on the activity of xanthine oxidase with an IC50 of 30 microM. In the deoxyribose assay both T. sanguinea and its methanolic component Th A showed only site-specific (Fe3+ + H2O2) but not non-site-specific (Fe3+ + EDTA + H2O2) hydroxyl radical scavenging suggesting chelating ability for iron ions. Spectroscopic studies showed that Th A enhanced absorbance in the visible region in the presence of Fe2+ ions. These results indicate that the antioxidant properties of Th A involve radical scavenging, anti-superoxide formation and metal chelation.     

Influence of prenylated and non-prenylated flavonoids on liver microsomal lipid peroxidation and oxidative injury in rat hepatocytes.

Prenylated chalcones from hops and beer were compared with non-prenylated flavonoids [chalconaringenin (CN), naringenin (NG), genistein (GS) and quercetin (QC)] for their ability to inhibit lipid peroxidation in rat liver microsomes. Chalcones with prenyl- or geranyl-groups (5 and 25 microM) were more effective inhibitors of microsomal lipid peroxidation than CN, NG or GS induced by Fe(2+)/ascorbate. Prenylated chalcones were effective inhibitors of microsomal lipid peroxidation induced by Fe(3+)-ADP/NADPH and by tert-butyl hydroperoxide (TBH) but to a lesser extent compared to the Fe(2+)/ascorbate system. An increase of prenyl substituents decreased antioxidant activity in the lipid peroxidation systems. Certain flavonoids behaved as prooxidants in the iron-dependent lipid peroxidation systems. For example, at 5 microM, NG enhanced iron/ascorbate-induced lipid peroxidation whereas CN, diprenylxanthohumol and tetrahydroxanthohumol enhanced Fe(3+)-ADP/NADPH-induced lipid peroxidation. None of the flavonoids (25 microM), except QC, inhibited NADPH cytochrome P450-reductase activity of rat liver microsomes, suggesting that the mechanism of inhibition of lipid peroxidation induced by Fe(3+)-ADP/NADPH is not due to inhibition of the reductase enzyme. Chalcones exhibiting antioxidant activity against TBH-induced lipid peroxidation such as xanthohumol and 5'-prenylxanthohumol, and NG, with no antioxidant property at 5 microM concentration protected cultured rat hepatocytes from TBH toxicity. Other antioxidants (desmethylxanthohumol and CN) in the TBH system were not cytoprotective. These results demonstrate the importance of prenyl groups in the antioxidant activity of hop chalcones in the various in vitro systems of lipid peroxidation. Furthermore, the antioxidant activity of the flavonoids has little or no bearing on their ability to protect rat hepatocytes from the toxic effects of TBH.     

Iron complexing activity of mangiferin, a naturally occurring glucosylxanthone, inhibits mitochondrial lipid peroxidation induced by Fe2+-citrate

Mangiferin, a naturally occurring glucosylxanthone, has been described as having antidiabetic, antiproliferative, immunomodulatory and antioxidant activities. In this study we report for the first time the iron-complexing ability of mangiferin as a primary mechanism for protection of rat liver mitochondria against Fe(2+)-citrate induced lipid peroxidation. Thiobarbituric acid reactive substances and antimycin A-insensitive oxygen consumption were used as quantitative measures of lipid peroxidation. Mangiferin at 10 microM induced near-full protection against 50 microM Fe(2+)-citrate-induced mitochondrial swelling and loss of mitochondrial transmembrane potential (DeltaPsi). The IC(50) value for mangiferin protection against Fe(2+)-citrate-induced mitochondrial thiobarbituric acid reactive substance formation (9.02+/-1.12 microM) was around 10 times lower than that for tert-butylhydroperoxide mitochondrial induction of thiobarbituric acid reactive substance formation. The xanthone derivative also inhibited the iron citrate induction of mitochondrial antimycin A-insensitive oxygen consumption, stimulated oxygen consumption due to Fe(2+) autoxidation and prevented Fe(3+) ascorbate reduction. Absorption spectra of mangiferin-Fe(2+)/Fe(3+) complexes also suggest the formation of a transient charge transfer complex between Fe(2+) and mangiferin, accelerating Fe(2+) oxidation and the formation of a more stable Fe(3+)-mangiferin complex unable to participate in Fenton-type reaction and lipid peroxidation propagation phase. In conclusion, these results show that in vitro antioxidant activity of mangiferin is related to its iron-chelating properties and not merely due to the scavenging activity of free radicals. These results are of pharmacological relevance since mangiferin and its naturally contained extracts could be potential candidates for chelation therapy in diseases related to abnormal intracellular iron distribution or iron overload.     

Synthesis and in vitro studies of pyrone derivatives as scavengers of active oxygen species.

The antioxidant properties of eleven alpha-pyrones and four gamma-pyrones were evaluated by means of three different tests: reduction of the stable free radical, 1,1-diphenyl-2-picrylhydrazyl (DPPH), superoxide anion scavenging assay and lipid peroxidation assay. In the DPPH test, 6-aryl-5,6-dihydro-4-hydroxypyran-2-ones (3) and 4-hydroxypyran-2-one (5f) were the most active derivatives with IC50 values ranging from 36.7 to 394 mumol/l. Potent superoxide anion scavenging properties appeared in derivatives possessing phenol moieties. Thus phenolic pyrones 5e and 5f exhibited a noteworthy activity (IC50 = 0.180 and 0.488 mmol/l, respectively) when reference compound, ascorbic acid, demonstrated only 24% inhibition at a concentration of 1 mg/ml. In addition derivative 5f significantly inhibited the Fe2+/ADP/ascorbate-induced lipid peroxidation of rat liver microsomes with an IC50 value of 0.069 mmol/l. Due to its multiple mechanism of protective action, compound 5f may be useful for the treatment of oxidative tissue injury in human disease.     

Inhibition of xanthine oxidase by phenolic conjugates of methylated quinic acid

The caffeoyl conjugates of prenylhydroquinone glucoside and of quinic acid, either in the carboxyl-free or carboxymethyl forms, isolated from Phagnalon rupestre (Asteraceae), showed inhibitory activity on lipid peroxidation induced by Fe 2+/ascorbate and by CCl4/NADPH in rat liver microsomes, with IC50 values ranging from 3 to 11 microM. After having demonstrated their effect on the xanthine oxidase-regulated superoxide production, the active compounds were tested for the direct inhibition of this enzyme. Methylated dicaffeoylquinic conjugates competitively inhibited the enzyme and the highest potency was obtained for the 4,5-diester, with an IC50 value of 3.6 microM, nearly ten times lower than that of the 3,5-analogue. In conclusion, the presence of the caffeoyl moiety is essential for both the antiperoxidative and radical scavenging activities, and the methylation of the quinic carboxyl group enhances the potency on xanthine oxidase inhibitory activity.     

Inhibition of heart mitochondrial lipid peroxidation by non-toxic concentrations of carvedilol and its analog BM-910228

Carvedilol, a non-selective beta-adrenoreceptor blocker, has been shown to possess a high degree of cardioprotection in experimental models of myocardial damage. Reactive oxygen species have been proposed to be implicated in such situations, and antioxidants have been demonstrated to provide partial protection to the reported damage. The purpose of our study was to investigate the antioxidant effect of carvedilol and its metabolite BM-910228 by measuring the extent of lipid peroxidation in a model of severe oxidative damage induced by ADP/FeSO(4) in isolated rat heart mitochondria. Carvedilol and BM-910228 inhibited the thiobarbituric acid-reactive substance formation and oxygen consumption associated with lipid peroxidation with IC(50) values of 6 and 0.22 microM, respectively. Under the same conditions, the IC(50) values of alpha-tocopheryl succinate and Trolox were 125 and 31 microM, respectively. As expected, the presence of carvedilol and BM-910228 preserved the structural and functional integrity of mitochondria under oxidative stress conditions for the same concentration range shown to inhibit lipid peroxidation, since they prevented the collapse of the mitochondrial membrane potential (DeltaPsi) induced by ADP/FeSO(4) in respiring mitochondria. It should be stressed that neither carvedilol nor BM-910228 induced any toxic effect on mitochondrial function in the concentration range of the compounds that inhibits the peroxidation of mitochondrial membranes. In conclusion, the antioxidant properties of carvedilol may contribute to the cardioprotective effects of the compound, namely through the preservation of mitochondrial functions whose importance in myocardial dysfunction is clearly documented. Additionally, its hydroxylated analog BM-910220, with its notably superior antioxidant activity, may significantly contribute to the therapeutic effects of carvedilol.     

Effect of the flavanolignans of Silybum marianum L. on lipid peroxidation in rat liver microsomes and freshly isolated hepatocytes.

The effect of several flavanolignans (silicristin, silidianin, silybin and isosilybin) present in silymarin, the extract of Silybum marianum fruits, was tested on lipid peroxidation in rat liver microsomes and freshly isolated hepatocytes. In microsomes lipid peroxidation was generated by ADP/Fe2+ and NADPH. All flavanolignans inhibited peroxidation in a concentration dependent manner. In hepatocytes lipid peroxidation was induced by ADP/Fe3+ complex and cell damage was evaluated as LDH activity released in the medium. The inhibition of the peroxidative process by flavanolignans was also evident in this model, even if with a potency order different from that found in microsomes. In contrast, the effect on LDH release was significant only for silybin and isosilybin, the other compounds being inactive on this parameter.     

Assessment of the anti-inflammatory activity and free radical scavenger activity of tiliroside

Three flavonoids, gnaphaliin, pinocembrin and tiliroside, isolated from Helichrysum italicum, were studied in vitro for their antioxidant and/or scavenger properties and in vivo in different models of inflammation. In vitro tests included lipid peroxidation in rat liver microsomes, superoxide radical generation in the xanthine/xanthine oxidase system and the reduction of the stable radical 1,1-diphenyl-2-pycryl-hydrazyl (DPPH). Acute inflammation was induced by application of 12-O-tetradecanoylphorbol 13-acetate (TPA) to the mouse ear or by subcutaneous injection of phospholipase A(2) or serotonin in the mouse paw. Eczema provoked on the mouse ear by repeated administration of TPA was selected as a model of chronic inflammation. The flavonoids were assayed against sheep red blood cell-induced mouse paw oedema as a model of delayed-type hypersensitivity reaction. The most active compound, both in vitro and in vivo, was tiliroside. It significantly inhibited enzymatic and non-enzymatic lipid peroxidation (IC(50)=12.6 and 28 microM, respectively). It had scavenger properties (IC(50)=21.3 microM) and very potent antioxidant activity in the DPPH test (IC(50)=6 microM). In vivo, tiliroside significantly inhibited the mouse paw oedema induced by phospholipase A(2)(ED(50)=35.6 mg/kg) and the mouse ear inflammation induced by TPA (ED(50)=357 microg/ear). Pinocembrin was the only flavonoid that exhibited anti-inflammatory activity in the sheep red blood cell-induced delayed-type hypersensitivity reaction. However, only tiliroside significantly reduced the oedema and leukocyte infiltration induced by TPA. As in the case of other flavonoids, the anti-inflammatory activity of tiliroside could be based on its antioxidant properties, although other mechanisms are probably involved.     

The correlation of the oxidation potentials of structurally related dibenzo[1,4]dichalcogenines to their antioxidance capacity in biological systems undergoing free radical-induced lipid peroxidation

A series of structurally related dibenzo[1,4]dichalcogenines possessing similar lipid solubilities but greatly differing oxidation potentials were tested for their ability to inhibit stimulated lipid peroxidation in ADP/Fe2+/ascorbate-treated liver microsomes and in hepatocytes treated with either t-butylhydroperoxide or diquat. In general, there was a close correlation between the half-wave oxidation potential of a particular compound and its antioxidant activity in the microsome and cell systems, with compounds possessing the lowest potential being the most potent antioxidants and vice versa. The Te/O and Te/S and S/O-substituted compounds, with oxidation potentials between 0.65 and 0.87 V, demonstrated most potent activity. Above this potential the antioxidant activity of the structure declined rapidly. The Te/O and Te/S compounds are among the most potent synthetic antioxidants described possessing IC50 values in the microsome system lower than 0.5 microM. This study clarifies the critical role of redox potential of an antioxidant site on a particular molecule without the complication of variable lipid solubility and may allow the definition of an optimal potential for antioxidant activity in biological systems.     

内南五味子木脂素戈米辛J对肝线粒体膜脂质过氧化和超氧阴离子自由基的作用

目的　研究来自内南五味子的戈米辛J对脂质过氧化的影响和清除超氧阴离子自由基 (O·2 )的能力。方法　采用离体大鼠肝线粒体膜的脂质过氧化模型和黄嘌呤氧化酶 鲁米诺化学发光法。结果　戈米辛J象VitE一样能剂量依赖性抑制Fe2 + VitC和ADP/NADPH所致的脂质过氧化 ;戈米辛J的IC50 分别为 5 75 ( 95 %可信限 :5 42～ 6 11)和0 95 ( 0 14～ 6 5 4) μmol·L-1。VitE的IC50 分别为 74 8( 30 2～ 185 3)和 6 5 1( 0 13～ 319) μmol·L-1。戈米辛J抑制Fe2 + VitC和ADP/NADPH所致的脂质过氧化的作用比VitE的作用分别强 13倍和 6 8倍。戈米辛J也剂量依赖地抑制黄嘌呤 黄嘌呤氧化酶 鲁米诺化学发光 ,其抑制发光强度 5 0 %的浓度 (IC50 )为 2 18 2 2 μmol·L-1。结论　戈米辛J具有抑制OH·诱导的脂质过氧化和清除O·2 的作用     

Adriamycin-induced lipid peroxidation in mitochondria and microsomes

The effect of the anti-neoplastic agent adriamycin on the peroxidation of lipids from rat liver and heart mitochondria and rat liver microsomes was investigated. The extent of total lipid peroxidation was determined by assaying for malondialdehyde (MDA), while the degradation of unsaturated fatty acids was monitored using gas chromatography. For liver mitochondria and microsomes, the formation of MDA was dependent on the concentrations of adriamycin, Fe3+, and protein, as well as time. In the presence of 50 microM adriamycin and saturating amounts of NADH, 1.5 +/- 0.2 nmol MDA/mg protein/60 min was produced with liver mitochondria. Upon addition of 25 microM Fe3+, the amount of MDA generated was increased to 6.5 +/- 0.1 nmol/mg protein/60 min. Liver microsomes produced amounts which were approximately 2-fold higher under all conditions. No MDA formation could be detected in rat heart mitochondria. The addition of 50 microM chlorpromazine completely inhibited peroxidation, whereas 0.5 to 1.0 mM p-bromophenacyl bromide blocked MDA formation by 50%. Analysis of fatty acids by gas chromatography showed that there was about a 50% decrease in arachidonic and docosahexaenoic acids in liver mitochondria and microsomes, but no change in the fatty acid content of heart mitochondria when incubated with both 50 microM adriamycin and 25 microM Fe3+ for 1 hr. These results suggest that (1) therapeutic concentrations of adriamycin enhance the peroxidation of lipids in liver mitochondria and microsomes through an enzymatic mechanism, especially in the presence of Fe3+; and (2) toxicity of this drug may be related to the degradation of membrane lipids.     

The pyrimido-pyrimidine derivative RA-642: a potent inhibitor of ferrous-induced lipid peroxidation in cell membranes

The effects of three pyrimido-pyrimidine derivatives (RA-642, dipyridamole and mopidamol) on hydroxyl anion-induced lipid peroxidation in cell membranes from liver, brain, kidney, lung and heart rat tissue were studied using d-alpha-tocopherol as standard for lipid peroxidation. Ferrous sulfate and ascorbic acid (FeAs) were used to induce lipid peroxidation via the formation of hydroxyl anions. The products resulting from the reaction with thiobarbituric acid were taken to be indicators of lipid peroxidation. Thiobarbituric acid reactive substances (TBARS) were produced by different rat tissues in the following sequence: brain greater than liver greater than kidney greater than heart greater than lung. Dose-response and time-response curves were plotted for all compounds. Inhibiting concentrations, 50% (IC50), ranged from 0.3-1.4 microM for RA-642, and 2.5 and 4.6 microM for dipyridamole. In liver mitochondrial membranes, IC50s of these compounds were 0.4 +/- 0.2 and 5.8 +/- 1.2 microM, respectively. At 15 min after beginning TBARS production, dipyridamole and RA-642 did not exert any inhibitory effect.     

Phytochemical characterization and radical scavenger activity of flavonoids from Helichrysum italicum G. Don (Compositae)

The glycosidic fraction of the flavonoids extracted from the flowering tops of the Helichrysum italicum G. Don was isolated, purified and characterized. This fraction was constituted by three compounds, which were assigned the structure of 4,2',4',6'-tetrahydroxychalcone-2'-glucoside, kaempferol-3-glucoside and naringenin-glycoside. Radical scavenger properties of the single glycosyl-flavonoids and of the in toto glycosidic fraction were tested with in vitro systems where different reactive oxygen species are generated (superoxide ions, hydroxyl radicals) and on lipid peroxidation induced by ADP/Fe2+ and NADPH or CCl4 in rat liver microsomes. The formation of reactive oxygen species was detected by cytochrome c reduction, salicylic acid hydroxylation and hyaluronic acid depolymerization. The action of the glycosidic fraction on the release of TXB2 and 12-HETE in human platelets, after collagen stimulation, was also evaluated. The glycosidic fraction inhibited in a dose dependent fashion lipid peroxidation in rat liver microsomes treated with ADP/Fe2+ or CCl4. This effect is due to the ability of flavonoids to scavenge free radicals at different stages of the process (superoxide ions, hydroxyl and lipid peroxide radicals). The single glycosyl-flavonoids exhibited a different scavenger activity, depending on the oxygen species and the chemical structure of the compounds. No effect of the fraction was observed on TXB2 and 12-HETE formation at 100 microns concentration.     

Dopaminergic pharmacology and antioxidant properties of pukateine, a natural product lead for the design of agents increasing dopamine neurotransmission

The dopaminergic and antioxidant properties of pukateine [(R)-11-hydroxy-1,2-methylenedioxyaporphine, PUK], a natural aporphine derivative, were analyzed in the rat central nervous system. At dopamine (DA) D1 ([3H]-SCH 23390) and D2 ([3H]-raclopride) binding sites, PUK showed IC50 values in the submicromolar range (0.4 and 0.6 microM, respectively). When the uptake of tritiated dopamine was assayed by using a synaptosomal preparation, PUK showed an IC50 = 46 microM. In 6-hydroxydopamine unilaterally denervated rats, PUK (8 mg/kg but not 4 mg/kg) elicited a significant contralateral circling, a behavior classically associated with a dopaminergic agonist action. When perfused through a microdialysis probe inserted into the striatum, PUK (340 microM) induced a significant increase in dopamine levels. In vitro experiments with a crude rat brain mitochondrial suspension showed that PUK did not affect monoamine oxidase activities, at concentrations as high as 100 microM. PUK potently (IC50 = 15 microM) and dose-dependently inhibited the basal lipid peroxidation of a rat brain membrane preparation. As a whole, PUK showed a unique profile of action, comprising an increase in extracellular DA, an agonist-like interaction with DA receptors, and antioxidant activity. Thus, PUK may be taken as a lead compound for the development of novel therapeutic strategies for Parkinson disease.     

Tungtungmadic acid, a novel antioxidant, fromSalicornia herbacea

Tungtungmadic acid (3-caffeoyl-4-dihydrocaffeoyl quinic acid) is a new chlorogenic acid derivative that was isolated from the Salicornia herbacea. The structure of tungtungmadic acid was determined using chemical and spectral analysis. The antioxidant activity of tungtungmadic acid was evaluated using various antioxidant assays, including free radical scavenging, lipid peroxidation and hydroxyl radical-induced DNA strand breaks assays. Tungtungmadic acid (IC50 = 5.1 microM and 9.3 microM) was found to have higher antioxidant activity in the DPPH scavenging assay as well as in the iron-induced liver microsomal lipid peroxidation system. In addition, the tungtungmadic acid was also effective in protecting the plasmid DNA against strand breakage induced by hydroxyl radicals.     

Flavin-dependent antioxidant properties of a new series of meso-N,N'-dialkyl-imidazolium substituted manganese(III) porphyrins

A number of synthetic manganese complexes exhibit both in vitro and in vivo catalytic antioxidant activities. This study reports that the antioxidant potencies of a new series of meso-N,N'-dialkyl-imidazolium substituted manganese(III) porphyrins are dependent, in part, on their ability to redox cycle with endogenous flavin-dependent oxidoreductases. Inhibition of lipid peroxidation activities of these novel cationic porphyrins was compared using rat brain homogenate as a source of lipids and endogenous oxidoreductases. Iron and ascorbate was used as initiators of lipid peroxidation, and two indices of lipid peroxidation (thiobarbituric acid reactive species (TBARS) and F(2)-isoprostanes) were determined. All meso-N,N'-dialkyl-imidazolium substituted porphyrins tested were potent inhibitors of lipid peroxidation with IC(50) ranging from 0.1 to 34 microM with a metal-dependent potency of Mn(III)>Co(III)>Zn(II). A flavin-dependent oxidoreductase antioxidant process was supported by the ability of the diphenyleneiodonium chloride (DPI, a flavoenzyme inhibitor) to decrease the potency of Mn-porphyrins in the lipid peroxidation model and that Mn-porphyrins stimulate NADPH oxidation in rat brain homogenates. These data suggest that metalloporphyrins may have differential antioxidant effects in tissues due to the presence or absence of flavin-dependent oxidoreductases.     

Antioxidant effect of calcium antagonists on microsomal membranes isolated from different brain areas.

The antioxidant effect of Ca2+ antagonists on sheep brain microsomal membranes and on liposomes prepared with total lipids extracted from the membranes was studied. Microsomal membranes were isolated from three brain areas: frontal cortex, hippocampus and caudate nucleus. Lipid peroxidation was induced by ascorbic acid and measured by malondialdehyde formation. Seven Ca2+ antagonists representative of the major chemical classes (dihydropyridines, benzothiazepines, phenylalkylamines, alkylamines, diphenylpiperazines) were tested for their antioxidant activity over a wide range of concentrations (0-500 microM). The order of antioxidant activity on frontal cortex membrane phospholipids, expressed as 50% inhibition of peroxidation (antioxidant IC50), was: nifedipine (IC50 = 4 microM) greater than flunarizine (IC50 = 48 microM) greater than bepridil (IC50 = 50 microM) greater than verapamil (IC50 = 74 microM). The dihydropyridines, nitrendipine and nimodipine, and the benzothiazepine, diltiazem, did not affect peroxidation even at a concentration of 500 microM. Membrane phospholipids are the substrate for free radical-induced damage since the extent of peroxidation in brain microsomal membranes was equal to that produced in liposomes prepared from membrane lipids. Although the lipophilicity of certain Ca2+ channel antagonists can enhance their antioxidant activity, our data suggest that Ca2+ antagonists inhibit peroxidation of the membrane lipid bilayer by a free radical scavenger effect that may be related to their chemical structure.     

Membrane antiperoxidative activities of D-propranolol, L-propranolol and dimethyl quaternary propranolol (UM-272).

With the isolated sarcolemmal membrane model, the antiperoxidative activities of D-propranolol, L-propranolol and the non-beta-blocker analogue UM-272 (dimethyl quaternary propranolol) were compared. Membrane lipid peroxidation induced by the Fe-catalysed, hydroxyl radical producing system (dihydroxyfumarate + Fe3+) was assessed by MDA formation. In the range of 10-200 microM, both D- and L-propranolol exhibited identical concentration-dependent inhibitory effects (20-70%), whereas UM-272 was about 2/3 as active as propranolol. In contrast, lignocaine (lidocaine), a well known membrane stabilizer, did not produce any effect. When purified rat hepatic microsomes were peroxidized by NADPH + Fe3+, comparable antiperoxidative effects were produced by the propranolol enantiomers and the dimethyl analogue. Thus, the antioxidant activities of these agents are related to their intrinsic chemical properties which might contribute to the reported beneficial effects of propranolol and UM-272 in models of ischaemia/reperfusion injury.     

Different antioxidant effects of polyphenols on lipid peroxidation and hydroxyl radicals in the NADPH-, Fe-ascorbate- and Fe-microsomal systems

Antioxidant and pro-oxidant effects of 14 naturally occurring polyphenols (PP) on rat liver microsomal lipid peroxidation (LP) and hydroxyl radical (*OH) production were studied in NADPH-dependent, 50 microM Fe(2+)-500 microM ascorbate (Fe-AA) or 50 microM Fe(2+) system, respectively. LP determined by the thiobarbituric acid method was inhibited in the NADPH system by flavonols and trans-resveratrol that were more effective than other flavonoids and derivatives of benzoic and cinnamic acid and were mostly more efficient than in the Fe-AA system. Inhibition of LP in the Fe system was higher by one order of magnitude than in the Fe-AA system. *OH production in the NADPH system, measured by formaldehyde production, was decreased by myricetin, fisetin and quercetin, but increased by kaempferol, morin and trans-resveratrol, indicating that z.rad;OH played a minor role in LP, which all of these PP inhibited. None of these PP at up to 40 microM concentration quenched *OH in the Fe-AA system. All tested PP, except trans-resveratrol and gentisic acid, spectrally interacted with Fe(2+) or Fe(3+), indicating formation of complexes or oxidation of PP. In contrast to the NADPH system we found no correlation between Fe(2+) chelation and inhibition of Fe-AA- or Fe-dependent LP indicating that iron chelation did not play a significant role in the two latter systems. It is concluded that the inhibition of LP by PP was apparently due to their hydrogen donating properties rather than chelation of iron.