槲皮素体外抗氧化及对小鼠血脂代谢作用的研究

本研究旨在探讨槲皮素体外抗氧化能力以及对高脂日粮小鼠血脂代谢的影响.体外分别测定了槲皮素对DPPH·,·OH和ABTS+·自由基的清除作用.动物实验:将昆明种雄性小鼠32只,随机分为4组,分别饲喂正常、高脂、高脂+0.05g/kg槲皮素、高脂+0.1g/kg槲皮素日粮.9周后测定小鼠肝脏活性氧(Reactive oxygen species,ROS)水平、丙二醛(Malondialdehyde,MDA)含量、抗氧化酶活力及血脂水平.结果表明:槲皮素对DPPH·,·OH和ABTS+·具有较强的清除作用,在一定范围内呈现出明显的剂量增加-效应增强的关系.0.05g/kg槲皮素能显著降低肝脏自由基水平及MDA含量(P＜0.05),增强抗氧化能力(P＜0.05),改善血脂水平(P＜0.05),而0.1g/kg槲皮素效果不显著.结论:0.05g/kg槲皮素可有效提高机体抗氧化能力,缓解高脂膳食造成的氧化应激,改善血脂代谢.     

Free radical scavenging and antioxidant activities of flavonoids extracted from the radix of Scutellaria baicalensis Georgi

Free radical scavenging and antioxidant activities of baicalein, baicalin, wogonin and wogonoside, the four major flavonoids in the radix of Scutellaria baicalensis Georgi, were examined in different systems. ESR results showed that baicalein and baicalin scavenged hydroxyl radical, DPPH radical and alkyl radical in a dose-dependent manner, while wogonin and wogonoside showed subtle or no effect on these radicals. Ten micromol/l of baicalein and baicalin effectively inhibited lipid peroxidation of rat brain cortex mitochondria induced by Fe(2+)-ascorbic acid, AAPH or NADPH, while wogonin and wogonoside showed significant effects only on NADPH-induced lipid peroxidation. In a study on cultured human neuroblastoma SH-SY5Y cells system, it was found that 10 micromol/l of baicalein and baicalin significantly protected cells against H(2)O(2)-induced injury. Baicalein was the most effective antioxidant among the four tested compounds in every system due to its o-tri-hydroxyl structure in the A ring. Compared with a well-known flavonoid, quercetin, the antioxidant activity of baicalein was lower in DPPH or AAPH system, but a little higher in those systems which might associate with iron ion. These results suggest that flavonoids in the radix of Scutellaria baicalensis with o-di-hydroxyl group in A the ring, such as baicalein and baicalin, could be good free radical scavengers and might be used to cure head injury associated with free radical assault.     

Phenolics as potential antioxidant therapeutic agents: mechanism and actions

Accumulating chemical, biochemical, clinical and epidemiological evidence supports the chemoprotective effects of phenolic antioxidants against oxidative stress-mediated disorders. The pharmacological actions of phenolic antioxidants stem mainly from their free radical scavenging and metal chelating properties as well as their effects on cell signaling pathways and on gene expression. The antioxidant capacities of phenolic compounds that are widely distributed in plant-based diets were assessed by the Trolox equivalent antioxidant capacity (TEAC), the ferric reducing antioxidant power (FRAP), the hypochlorite scavenging capacity, the deoxyribose method and the copper-phenanthroline-dependent DNA oxidation assays. Based on the TEAC, FRAP and hypochlorite scavenging data, the observed activity order was: procyanidin dimer > flavanol > flavonol > hydroxycinnamic acids > simple phenolic acids. Among the flavonol aglycones, the antioxidant propensities decrease in the order quercetin, myricetin and kaempferol. Gallic acid and rosmarinic acid were the most potent antioxidants among the simple phenolic and hydroxycinnamic acids, respectively. Ferulic acid displayed the highest inhibitory activity against deoxyribose degradation but no structure–activity relationship could be established for the activities of the phenolic compounds in the deoxyribose assay. The efficacies of the phenolic compounds differ depending on the mechanism of antioxidant action in the respective assay used, with procyanidin dimers and flavan-3-ols showing very potent activities in most of the systems tested. Compared to the physiologically active (glutathione, -tocopherol, ergothioneine) and synthetic (Trolox, BHA, BHT) antioxidants, these compounds exhibited much higher efficacy. Plant-derived phenolics represents good sources of natural antioxidants, however, further investigation on the molecular mechanism of action of these phytochemicals is crucial to the evaluation of their potential as prophylactic agents.     

Ability of antioxidants to prevent oxidative mutations in Salmonella typhimurium TA102

An assay for the ability of antioxidants to prevent mutations induced by various oxidants in Salmonella typhimurium TA102 cells was developed. Protection against hydrogen-peroxide-induced mutagenicity was observed for quercetin, caffeic acid, ascorbic acid and dimethyl sulfoxide (used as a solvent for water-insoluble antioxidants). No protective effect was observed for green tea extract (weakly pro-oxidative), catechin, rutin, sinigrin, ferulic acid and alpha-tocopherol. Mutagenicity caused by tert-butyl hydroperoxide (tBOOH) was prevented most effectively by quercetin and ascorbic acid, whereas weaker effects were observed for green tea extract and for rutin, and no effect being observed for the other antioxidants tested. The results for hydrogen peroxide indicate iron chelation to be the most important protective mechanism. Radical scavenging appeared to be effective only with dimethyl sulfoxide and ascorbic acid, which are effective scavengers of hydroxyl radicals and were used here in high concentrations. It is proposed that the hydrogen-peroxide-induced mutations in the Salmonella cells are caused by hydroxyl radicals generated by iron ions closely associated with DNA. Protection against mutagenicity caused by tert-butyl hydroperoxide appears to occur mainly through the scavenging of alkoxyl and possibly of alkyl radicals.     

Antioxidant strategies in the treatment of stroke

Excessive production of free radicals is known to lead to cell injury in a variety of diseases, such as cerebral ischemia. In this review, we describe some of the numerous studies that have examined this oxidative stress and the efficiency of antioxidant strategies in focal cerebral ischemia. Besides using genetically modified mice, these strategies can be divided into three groups: (1) inhibition of free radical production, (2) scavenging of free radicals, and (3) increase of free radical degradation by using agents mimicking the enzymatic activity of endogenous antioxidants. Finally, the clinical trials that have tested or are currently testing the efficiency of antioxidants in patients suffering from stroke are reviewed. The results presented here lead us to consider that antioxidants are very promising drugs for the treatment of ischemic stroke.     

The correlation between active oxygens scavenging and antioxidative effects of flavonoids

The abilities of 15 flavonoids as a scavenger of active oxygens (hydroxyl radical and superoxide anion) were studied. Hydroxyl radical (^.OH) was generated by the Fenton system, and assayed by the determination of methanesulfinic acid (MSA) formed from the reaction of dimethyl sulfoxide (DMSO) with ^.OH. (+)-Catechin, (−)-epicatechin, 7,8-dihydroxy flavone, and rutin showed the ^.OH scavenging effect 100–300 times superior to that of mannitol, a typical ^.OH scavenger. The other flavonoids showed no ^.OH scavenging effect at their concentrations up to 50 μM. Baicalein, quercetin, morin, and myricetin unexpectedly increased the ^.OH production in the Fenton system. The flavonoids tested now, except monohydroxy flavones, were more or less inhibitive to the superoxide anion (O₂) generation in the hypoxanthine-xanthine oxidase system. A great part of this inhibitory effect was likely owing to suppression of xanthine oxidase activity by the flavonoids. The flavonoids, which scavenged ^.OH or O₂⁻, were necessarily antioxidants to the peroxidation of methyl linoleate. However, there was a type of flavonoid such as morin, which have neither ^.OH nor O₂⁻ scavenging effect, but was a strong antioxidant.     

An essential difference between the flavonoids monoHER and quercetin in their interplay with the endogenous antioxidant network

Antioxidants can scavenge highly reactive radicals. As a result the antioxidants are converted into oxidation products that might cause damage to vital cellular components. To prevent this damage, the human body possesses an intricate network of antioxidants that pass over the reactivity from one antioxidant to another in a controlled way. The aim of the present study was to investigate how the semi-synthetic flavonoid 7-mono-O-(β-hydroxyethyl)-rutoside (monoHER), a potential protective agent against doxorubicin-induced cardiotoxicity, fits into this antioxidant network. This position was compared with that of the well-known flavonoid quercetin. The present study shows that the oxidation products of both monoHER and quercetin are reactive towards thiol groups of both GSH and proteins. However, in human blood plasma, oxidized quercetin easily reacts with protein thiols, whereas oxidized monoHER does not react with plasma protein thiols. Our results indicate that this can be explained by the presence of ascorbate in plasma; ascorbate is able to reduce oxidized monoHER to the parent compound monoHER before oxidized monoHER can react with thiols. This is a major difference with oxidized quercetin that preferentially reacts with thiols rather than ascorbate. The difference in selectivity between monoHER and quercetin originates from an intrinsic difference in the chemical nature of their oxidation products, which was corroborated by molecular quantum chemical calculations. These findings point towards an essential difference between structurally closely related flavonoids in their interplay with the endogenous antioxidant network. The advantage of monoHER is that it can safely channel the reactivity of radicals into the antioxidant network where the reactivity is completely neutralized.     

Pitfalls in a Method for Assessment of Total Antioxidant Capacity

A relatively simple and widely applied method for quantitating the total antioxidant capacity of body fluids and drug solutions based on the absorbance of the ABTS radical cation was evaluated. In this assay, the end-point is an antioxidant-induced decrease in absorbance at a fixed time. This decrease is used as an index of total antioxidant capacity. It is shown that Trolox, potassium cyanide and quercetin all decrease the absorbance of ABTS radical cations at a fixed time, but by different mechanisms. Trolox scavenges the ABTS radical, potassium cyanide inhibits radical formation, while quercetin acts by both mechanisms. Using this method antioxidant capacity may be overestimated, due to both a scavenger effect and an effect on the rate of ABTS oxidation. To distinguish between these effects, a post-addition assay was used in which the sample is added when the formation of radicals is stable. Using post- addition assay conditions enables discrimination between effects on radical scavenging and on the radical formation, two major mechanisms for antioxidant action. In extrapolating the results to an ill vivo situation it should be questioned: (i) whether the peroxidase process does indeed mimic the process of radical formation in vivo, and (ii) whether the ABTS radicals do resemble the radical species involved in an in vivo situation. Results obtained in the ABTS radical-based methods should therefore be reviewed critically before the antioxidant capacity can be assessed.     

In vitro antioxidant activity of Hedyotis corymbosa (L.) Lam. aerial parts

The methanolic extract of the aerial part of Hedyotis corymbosa (L.) Lam. (Rubiaceae) was screened for antioxidant activity using 1,1-diphenyl-2-picryl hydroxyl (DPPH) quenching assay, 2,2'-azinobis-3-ethylbenzothiozoline-6-sulfonic acid (ABTS) cation decolorization test, ferric reducing power (FRP), scavenging capacity towards hydroxyl ion (OH*) radicals and nitric oxide (NO) radical inhibition activity using established assay procedures. Total phenolics and total flavonoid contents were, also determined. The plant yielded 210 mg gallic acid equivalent/100 g phenolic content and 55 mg quercetin equivalent/100 g flavonoid content. The extract exhibited high antiradical activity against DPPH, ABTS, nitric oxide and hydroxyl radicals with EC50 value of 82, 150, 130, and 170 microg/ml, respectively. The FRP increased with increasing concentration of the sample. The antioxidant activity of the extract was comparable with that of the standard butylated hydroxyl toluene (BHT). High correlation between total phenolic/flavonoid contents and scavenging potential of different reactive oxygen species (R2 = 0.785-0.998) indicated the polyphenols as the main antioxidants.     

Allylmagnolol,a novel magnolol derivative as potent antioxidant

We reported the discovery of potent antioxidants based on magnolol, a naturally occurring biphenolic obtained from the bark of Magnolia officinalis. The allylmagnolols 3a,b were synthesized via O-alkylation of the biphenols followed by Claisen rearrangement. In-vitro using enhanced chemiluminescence (CL) and flow cytometric assays in whole cells revealed that both 3a and 3b displayed promising free radical scavenging effects in PMA- and LPS-stimulated models as compared with magnolol. Further DNA labeling analysis for cytotoxicity indicated that these analogues show no cytotoxic effects for the scavenging of the oxygen-derived free radicals under PMA-stimulated concentrations. The results from 3,3'-bisallylmagnolol (3b) suggested that the naturally occurring constituent was suitable to be a lead compound for the development of potential antioxidants for certain diseases.     

Direct evidence for recycling of myeloperoxidase-catalyzed phenoxyl radicals of a vitamin E homologue, 2,2,5,7,8-pentamethyl-6-hydroxy chromane,by ascorbate/dihydrolipoate in living HL-60 cells

Myeloperoxidase (MPO)-catalyzed one-electron oxidation of endogenous phenolic constituents (e.g., antioxidants, hydroxylated metabolites) and exogenous compounds (e.g., drugs, environmental chemicals) generates free radical intermediates: phenoxyl radicals. Reduction of these intermediates by endogenous reductants, i.e. recycling, may enhance their antioxidant potential and/or prevent their potential cytotoxic and genotoxic effects. The goal of this work was to determine whether generation and recycling of MPO-catalyzed phenoxyl radicals of a vitamin E homologue, 2,2,5,7,8-pentamethyl-6-hydroxychromane (PMC), by physiologically relevant intracellular reductants such as ascorbate/lipoate could be demonstrated in intact MPO-rich human leukemia HL-60 cells. A model system was developed to show that MPO/H(2)O(2)-catalyzed PMC phenoxyl radicals (PMC*) could be recycled by ascorbate or ascorbate/dihydrolipoic acid (DHLA) to regenerate the parent compound. Absorbance measurements demonstrated that ascorbate prevents net oxidation of PMC by recycling the phenoxyl radical back to the parent compound. The presence of DHLA in the reaction mixture containing ascorbate extended the recycling reaction through regeneration of ascorbate. DHLA alone was unable to prevent PMC oxidation. These conclusions were confirmed by direct detection of PMC* and ascorbate radicals formed during the time course of the reactions by EPR spectroscopy. Based on results in the model system, PMC* and ascorbate radicals were identified by EPR spectroscopy in ascorbate-loaded HL-60 cells after addition of H(2)O(2) and the inhibitor of catalase, 3-aminotriazole (3-AT). The time course of PMC* and ascorbate radicals was found to follow the same reaction sequence as during their recycling in the model system. Recycling of PMC by ascorbate was also confirmed by HPLC assays in HL-60 cells. Pre-loading of HL-60 cells with lipoic acid regenerated ascorbate and thus increased the efficiency of ascorbate in recycling PMC*. Lipoic acid had no effect on PMC oxidation in the absence of ascorbate. Thus PMC phenoxyl radical does not directly oxidize thiols but can be recycled by dihydrolipoate in the presence of ascorbate. The role of phenoxyl radical recycling in maintaining antioxidant defense and protecting against cytotoxic and genotoxic phenolics is discussed.     

Synthesis and antioxidant evaluation of novel silybin analogues

In this work, we evaluated the antioxidant properties of the eight novel silybin analogues for their capacity to scavenge free radicals including superoxide anion radicals and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals in vitro. Compound 7d demonstrated an excellent antioxidant effect in scavenging superoxide anion free radical with an IC50 value of 26.5 microM, while the IC50 of quercetin (the reference compound) was 38.1 microM. Compounds 7b, 7e, 7h showed certain scavenging activities for both types of free radicals.     

Hemin/nitrite/H2O2 induces brain homogenate oxidation and nitration: effects of some flavonoids

Oxidative injury has been implicated in the pathogenesis of numerous neurodegenerative diseases. Recently, it has been found that with the existence of hydrogen peroxide and nitrite, hemin catalyzes protein nitration. We hypothesize under certain pathological conditions, hemin catalyzed protein nitration may happen in the brain. In this paper, the effects of three flavonoids, i.e. quercetin, catachin and baicalein on hemin/nitrite/H2O2 induced brain homogenate oxidation and nitration were studied. The results showed that hemin/nitrite/H2O2 system could effectively induce brain homogenate protein oxidation and nitration. Quercetin, catachin and baicalein dose-dependently inhibited hemin/nitrite/H2O2 system-induced protein nitration in a dose-dependent manner, the inhibition of protein nitration was in the order of quercetin>catachin>baicalein. These compounds also inhibited hemin/H2O2 system-induced lipid peroxidation, the inhibition order was baicalein >quercetin>catachin. However, these flavonoids showed marginal effect on hemin/nitrite/H2O2 system caused protein oxidation and thiol oxidation. The inhibition activities of flavonoids on hemin/nitrite/H2O2 system-induced protein nitration may closely relate to their radical scavenging activities, since the inhibition order of protein nitration is the same as the radical scavenging order. These results indicate hemin/nitrite/H2O2 system induces different types of oxidative assault on bio-molecules. Flavonoids could act as antioxidants inhibiting ROS and RNS caused brain damage.     

Mechanism of ROS scavenging and antioxidant signalling by redox metallic and fullerene nanomaterials: Potential implications in ROS associated degenerative disorders

BackgroundThe balance between oxidation and anti-oxidation is believed to be critical in maintaining healthy biological systems. However, our endogenous antioxidant defense systems are incomplete without exogenous antioxidants and, therefore, there is a continuous demand for exogenous antioxidants to prevent stress and ageing associated disorders. Nanotechnology has yielded enormous variety of nanomaterials (NMs) of which metallic and carbonic (mainly fullerenes) NMs, with redox property, have been found to be strong scavengers of ROS and antioxidants in preclinical in vitro and in vivo models.Scope of reviewRedox activity of metal based NMs and membrane translocation time of fullerene NMs seem to be the major determinants in ROS scavenging potential exhibited by these NMs. A comprehensive knowledge about the effects of ROS scavenging NMs in cellular antioxidant signalling is largely lacking. This review compiles the mechanisms of ROS scavenging as well as antioxidant signalling of the aforementioned metallic and fullerene NMs.Major conclusionsDirect interaction between NMs and proteins does greatly affect the corona/adsorption formation dynamics but such interaction does not provide the explanation behind diverse biological outcomes induced by NMs. Indirect interaction, however, that could occur via NMs uptake and dissolution, NMs ROS induction and ROS scavenging property, and NMs membrane translocation time seem to work as a central mode of interaction.General significanceThe usage of potential antioxidant NMs in biological systems would greatly impact the field of nanomedicine. ROS scavenging NMs hold great promise in the future treatment of ROS related degenerative disorders.     

Effect of natural exogenous antioxidants on aging and on neurodegenerative diseases

Abstract

Aging and neurodegenerative diseases share oxidative stress cell damage and depletion of endogenous antioxidants as mechanisms of injury, phenomena that are occurring at different rates in each process. Nevertheless, as the central nervous system (CNS) consists largely of lipids and has a poor catalase activity, a low amount of superoxide dismutase and is rich in iron, its cellular components are damaged easily by overproduction of free radicals in any of these physiological or pathological conditions. Thus, antioxidants are needed to prevent the formation and to oppose the free radicals damage to DNA, lipids, proteins, and other biomolecules. Due to endogenous antioxidant defenses are inadequate to prevent damage completely, different efforts have been undertaken in order to increase the use of natural antioxidants and to develop antioxidants that might ameliorate neural injury by oxidative stress. In this context, natural antioxidants like flavonoids (quercetin, curcumin, luteolin and catechins), magnolol and honokiol are showing to be the efficient inhibitors of the oxidative process and seem to be a better therapeutic option than the traditional ones (vitamins C and E, and β-carotene) in various models of aging and injury in vitro and in vivo conditions. Thus, the goal of the present review is to discuss the molecular basis, mechanisms of action, functions, and targets of flavonoids, magnolol, honokiol and traditional antioxidants with the aim of obtaining better results when they are prescribed on aging and neurodegenerative diseases.     

Evaluation of antioxidant activity of <Emphasis Type="Italic">Melothria maderaspatana in vitro</Emphasis>

In this study, an aqueous extract of leaves from Melothria maderaspatana was tested for in vitro antioxidant activity. Free radical scavenging assays, such as hydroxyl radical, hydrogen peroxide, superoxide anion radical and 2,2-diphenyl-1-picryl hydrazyl (DPPH), 2,2’-azinobis-(3-ethyl-enzothiazoline-6-sulfonic acid) (ABTS) radical scavenging, and reducing power assay, were studied. The extract effectively scavenged hydroxyl radical, hydrogen peroxide and superoxide anion radicals. It also scavenged DPPH and ABTS radicals. Furthermore, it was found to have reducing power. All concentrations of leaf extract exhibited free radical scavenging and antioxidant power, and the preventive effects were in a dose-dependent manner. The antioxidant activities of the above were compared to standard antioxidants such as butylated hydroxytoluene (BHT), ascorbic acid, and α-tocopherol. The results obtained in the present study indicate that the M. maderaspatana extract could be considered a potential source of natural antioxidant.     

Free radical scavenging and antioxidant potential of mangrove plants: a review

Free radicals derived from reactive oxygen species and reactive nitrogen species are generated in our body by normal cellular metabolism which is enhanced under stress conditions. The most vulnerable biological targets of free radicals are cell structures including proteins, lipids and nucleic acids. Since antioxidants synthesized in the body are not sufficient under oxidative stress, their exogenous supply is important to prevent the body from free radical-induced injury. Recent researches have shown that antioxidants of plant origin with free radical scavenging property could have great importance as therapeutic agents in management of oxidative stress. Mangrove plants growing in inhospitable environment of the intertidal regions of land and sea in tropics and sub-tropics are equipped with very efficient free radical scavenging system to withstand the variety of stress conditions. These mangrove plants possess variety of phytochemical and are rich in phenolic compounds such as flavonoids, isoflavones, flavones, anthocyanins, coumarins, lignans, catechins, isocatechins, etc., which served as source of antioxidants. Isolation and identification of these antioxidant compounds offer great potential for their pharmaceutical exploitations. However, no comprehensive literature is available on antioxidants’ studies in mangrove plants in particular. Hence, the present review discusses the antioxidant potential of mangrove plants with its specific role under salt stress as well as the progress made so far in evaluation of antioxidant activities of different mangrove species.     

Dihydroquercetin (taxifolin) and other flavonoids as inhibitors of free radical formation at key stages of apoptosis

Formation of free radicals in mitochondria plays a key role in the development of apoptosis, which includes formation of superoxide by the respiratory chain, formation of radicals by cytochrome c-cardiolipin complex in the presence of hydrogen peroxide or lipids, and chain lipid peroxidation resulting in cytochrome c release from mitochondria and initiation of the apoptotic cascade. In this work the effect of taxifolin (dihydroquercetin) and some other antioxidants on these three radical-producing reactions was studied. Peroxidase activity of the complex of cytochrome c with dioleyl cardiolipin estimated by chemiluminescence with luminol decreased by 50% with quercetin, taxifolin, rutin, Trolox, and ionol at concentrations 0.7, 0.7, 0.8, 3, and 10 μM, respectively. The lipid radical production detected by coumarin C-525-activated chemiluminescence decreased under the action of rutin and taxifolin in a dose-dependent manner, so that a 50% inhibition of chemiluminescence was observed at the antioxidant concentrations of 3.7 and 10 μM, respectively. Thus, these two radical-producing reactions responsible for apoptosis onset are inhibited by antioxidants at rather low concentrations. Experiments performed on liver slices and mash showed that taxifolin, quercetin, naringenin, and Trolox have low inhibitory effect on the lucigenin-dependent chemiluminescence in the tissue only at concentrations higher than 100 μM.     

Antioxidative properties of ascorbigen in using multiple antioxidant assays

The antioxidative properties of ascorbigen, one of the major indole-derived compounds of Brassica vegetables, were systematically evaluated using multiple assay systems with comparison to the well-known antioxidants ascorbic acid and Trolox. We first performed assays using model radicals, DPPH radical, galvinoxyl radical, and ABTS radical cation (ABTS^?+). Ascorbigen showed stronger activity than that of ascorbic acid in the ABTS^?+-scavenging assay but showed no activity in the DPPH radical- and galvinoxyl radical-scavenging assays. In the ABTS^?+-scavenging assay, the indole moiety of ascorbigen contributed to scavenging of the radicals to produce indole-3-aldehyde as one of the final reaction products. The activity of ascorbigen was then evaluated by an oxygen radical absorbance capacity assay and an oxidative hemolysis inhibition assay using physiologically relevant peroxyl radicals, AAPH-derived radicals. Ascorbigen showed much stronger antioxidant activity than did ascorbic acid and Trolox. Therefore, antioxidant activity of ascorbigen might be more beneficial than has been thought for daily health care.     

Synergistic interaction between the probucol phenoxyl radical and ascorbic acid in inhibiting the oxidation of low density lipoprotein.

Chain-breaking antioxidants such as butylated hydroxytoluene, alpha-tocopherol, and probucol have been shown to decrease markedly the oxidative modification of low density lipoprotein (LDL). Their mechanism of action appears to involve scavenging of LDL-lipid peroxyl radicals. The purpose of this study was to investigate the occurrence of radical reactions produced during oxidation of LDL and LDL-containing probucol initiated by lipoxygenase or copper. In addition, we have investigated the possibility of a synergistic interaction between ascorbate and probucol in inhibiting the oxidation of LDL. Incubation of LDL-containing probucol and lipoxygenase produced a composite electron spin resonance (ESR) spectrum due to the endogenous alpha-tocopheroxyl radical and probucol-derived phenoxyl radical. The spectral assignment was further verified by chemical oxidation of alpha-tocopherol and probucol. In the presence of ascorbic acid, these radicals in the LDL particle were reduced to their parent compounds with concomitant formation of the ascorbate radical. In both the peroxidation of linoleic acid and the copper-initiated peroxidation of LDL, the antioxidant activity of probucol was significantly increased by low (3-6 microM) concentrations of ascorbate. The probucol-dependent inhibition of LDL oxidation was enhanced in the presence of ascorbic acid. We conclude that the reaction between the phenoxyl radical of probucol and ascorbate results in a synergistic enhancement of the antioxidant capacity of these two compounds and speculate that such reactions could play a role in maintaining the antioxidant status of LDL during oxidative stress in vivo.