Formation of alpha-tocopherol radical and recycling of alpha-tocopherol by ascorbate during peroxidation of phosphatidylcholine liposomes. An electron paramagnetic resonance study

The events accompanying the inhibitory effect of alpha-tocopherol and/or ascorbate on the peroxidation of soybean L-alpha-phosphatidylcholine liposomes, which are an accepted model of biological membranes, were investigated by electron paramagnetic resonance, optical and polarographic methods. The presence of alpha-tocopherol radical in the concentration range 10(-8)-10(-7) M was detected from its EPR spectrum during the peroxidation of liposomes, catalysed by the Fe3+-triethylenetatramine complex. The alpha-tocopherol radical, generated in the phosphatidylcholine bilayer, is accessible to ascorbic acid, present in the aqueous phase at physiological concentrations. Ascorbic acid regenerates from it the alpha-tocopherol itself. A kinetic rate constant of about 2 X 10(5) M-1 X s-1 was estimated from the reaction as it occurs under the adopted experimental conditions. The scavenging effect of alpha-tocopherol on lipid peroxidation is maintained as long a ascorbic acid is present.     

Location and recycling of mitochondrial alpha-tocopherol

Vitamin E in the form of alpha-tocopherol is crucial for mitochondrial integrity. We studied the distribution of alpha-tocopherol in rat muscle mitochondria in relation to the capacity of the electron transport chain to recycle the vitamin. Fractionation studies showed that almost 90% of the alpha-tocopherol in mitochondria is located in the outer membrane. This distribution was confirmed with the finding that ferricytochrome c, which does not penetrate the outer membrane, oxidized 70-80% of mitochondrial alpha-tocopherol in a time- and concentration-dependent manner. Despite the predominant outer membrane distribution of alpha-tocopherol, succinate and other mitochondrial respiratory substrates spared alpha-tocopherol from oxidative loss by both agents. Sparing of alpha-tocopherol by succinate was prevented by 2-thenoyltrifluoroacetone, but not by myxothiazol, which suggests that ubiquinol is the electron donor. Ferricytochrome c significantly increased total F2-isoprostanes, an effect that was prevented by succinate. Most alpha-tocopherol in muscle mitochondria is located in the outer membrane, where it is susceptible to oxidative loss. Nonetheless, alpha-tocopherol is partially spared by ubiquinol in the electron transport chain.     

Free radical scavenging properties of sulfinpyrazone

Sulfinpyrazone, a potent uricosuric drug, was tested in vitro for its scavenging action against oxygen free radicals. In this study, sulfinpyrazone was able to scavenge 1,1-diphenyl-2-picrylhydrazyl radical with IC 50 value of 29.82 μg/ml compared to butylated hydroxytoluene (BHT, IC 50 value=20.15 μg/ml) and Trolox (IC 50 value=16.01 μg/ml). It was able to scavenge superoxide anion with IC 50 value of 27.72 μg/ml compared to Trolox (IC 50 value=22.08 μg/ml) and ascorbic acid (IC 50 value=14.65 μg/ml). The hydroxyl radical scavenging activity of sulfinpyrazone is in a concentration-dependent fashion. In the range of concentrations used, sulfinpyrazone was not a scavenger toward H 2 O 2 . However, the intracellular H 2 O 2 -induced 2',7'-dichlorofluorescin diacetate (DCF-DA) fluorescence in HL-60 cells was significantly reduced by sulfinpyrazone during 30-60 min of incubation. Finally, phorbol-12-myristate-13-acetate induced-lucigenin chemiluminescence in whole blood was markedly inhibited by sulfinpyrazone. Our results suggest a new direction for the pharmacological actions of sulfinpyrazone in free radical scavenging properties.     

Free radical scavenging and cytotoxic properties in the ellipticine series

In the series of cytotoxic intercalating compounds derived from ellipticine, we tried to correlate free radical scavenging properties with cytotoxic activities. Scavenging properties were determined in vitro on two experimental models: a) antioxidant activity of the drugs during the autoxidation of methyl linolenate induced by azo-bis-isobutyronitrile; this activity was measured either by the initial rate ratios in the presence and in the absence of the drug or by the length of the inhibition period of the reaction in the presence of the drug and b) ability to reduce DPPH free radicals. Cytotoxic properties were expressed by ID50 the dose which reduces by 50% the L 1210 cell growth rate as compared to controls after 48 h. It appears that antioxidant activity and reduction of DPPH both require the presence of a free OH group on the ellipticine ring. A good correlation is observed between cytotoxicity and antioxidant activity of the hydroxylated derivatives; minor structural modifications which result in a loss of cytotoxic activity also result in a loss of antioxidant properties. No such correlation is observed with DPPH reducing properties of ellipticine derivatives.     

Carotenoid radical chemistry and antioxidant/pro-oxidant properties

The purpose of this review is to summarise the current state of knowledge of (i) the kinetics and mechanisms of radical reactions with carotenoids, (ii) the properties of carotenoid radicals, and (iii) the antioxidant/pro-oxidant properties of carotenoids.     

Free radical tissue damage: protective role of antioxidant nutrients

Highly reactive molecules called free radicals can cause tissue damage by reacting with polyunsaturated fatty acids in cellular membranes, nucleotides in DNA, and critical sulfhydryl bonds in proteins. Free radicals can originate endogenously from normal metabolic reactions or exogenously as components of tobacco smoke and air pollutants and indirectly through the metabolism of certain solvents, drugs, and pesticides as well as through exposure to radiation. There is some evidence that free radical damage contributes to the etiology of many chronic health problems such as emphysema, cardiovascular and inflammatory diseases, cataracts, and cancer. Defenses against free radical damage include tocopherol (vitamin E), ascorbic acid (vitamin C), beta-carotene, glutathione, uric acid, bilirubin, and several metalloenzymes including glutathione peroxidase (selenium), catalase (iron), and superoxide dismutase (copper, zinc, manganese) and proteins such as ceruloplasmin (copper). The extent of tissue damage is the result of the balance between the free radicals generated and the antioxidant protective defense system. Several dietary micronutrients contribute greatly to the protective system. Based on the growing interest in free radical biology and the lack of effective therapies for many of the chronic diseases, the usefulness of essential, safe nutrients in protecting against the adverse effects of oxidative injury warrants further study.     

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.     

Free radical scavenging and antiproliferative properties of Biginelli adducts

A series of Biginelli adducts bearing different substituents at C-4 position were synthesized by using p-sulfonic acid calix[4]arene as a catalyst. The in vitro potential to scavenge reactive nitrogen/oxygen species (RNS and ROS) and the ability to inhibit cancer cells growth were then investigated. Four adducts were found to be potent scavengers of 2,2-diphenyl-1-picrylhydrazyl (RNS) and/or superoxide anion (ROS) radicals. The antiproliferative activity against cancer cells was disclosed for the first time for 16 monastrol analogs. The capacity of all compounds to inhibit cancer cells growth was dependent on the histological origin of cells, except for BA24, which was highly active against all cell lines. BA20 and BA33 were as potent as the reference drug doxorubicin against adriamycin-resistant ovarian and prostate cancer cells, respectively. These results highlight some monastrol analogs as lead compounds for the design of new free radical scavengers and anticancer agents.     

Free radical scavenging ability and antioxidant efficiency of curcumin and its substituted analogue

Free radical reactions of curcumin and its ethoxy substituted derivative (C1) 1,7-bis-(4-hydroxy-3-ethoxy phenyl)-1,6-heptadiene-3,5-dione have been studied using a pulse radiolysis technique in homogeneous aqueous-organic solutions like acetonitrile-water and isopropanol-water mixtures, as well as in neutral TX-100 and cationic CTAB micellar solutions. The phenoxyl radicals of curcumin or C1 were generated by one-electron transfer to several oxidants like N(3)(.), Br(2)(-.), CCl(3)O(2)(.), glutathione radicals which exhibit absorption from a 300-600-nm wavelength region with the maximum at 490-500 nm. Other important properties of the phenoxyl radicals such as extinction coefficient, radical lifetime and their formation and decay rate constants were also determined in these systems. The antioxidant property of curcumin and C1 were estimated in terms of their ability to inhibit the lipid peroxidation in liposomes and also in terms of trolox equivalent antioxidant capacity (TEAC). The results were compared with alpha-tocopherol.     

Intermembrane transport and antioxidant action of alpha-tocopherol in liposomes

Studies were made of the ability of alpha-tocopherol, incorporated into unilamellar liposomes from saturated or unsaturated phospholipids (donor liposomes) to inhibit the accumulation of lipid peroxidation (LPO) products in unilamellar liposomes from rat cerebral cortex lipids (acceptor liposomes) in the presence of LPO inducer (Fe + ascorbate). With the molar alpha-tocopherol: phospholipids rations from 1:1000 to 1:100 in donor liposomes, obtained through sonication of lipid dispersions, alpha-tocopherol was incorporated into both monolayers of liposomes and was distributed in monomeric form without forming clusters. Based on the dependencies of LPO inhibition on the alpha-tocopherol concentrations, we chose the ones that completely prevented the accumulation of LPO products in donor liposomes. Under these conditions LPO inhibition in mixtures of donor and acceptors liposomes was fully determined by the antioxidant effect of alpha-tocopherol in acceptor liposomes due to its intermembrane transfer. The efficiency of the "intermembrane" antioxidant action of alpha-tocopherol increased in the course of preincubation of donor and acceptor liposomes (up to 60 min) and this increase was more pronounced when the donor liposomes contained unsaturated phospholipids. Evidence was obtained that the intermembrane transfer of alpha-tocopherol did not result from the fusion of donor and acceptor liposomes during preincubation.     

Free radical damage in neonatal rat cardiac myocyte cultures: effects of alpha-tocopherol, Trolox, and phytol

A number of investigations have implicated free radicals in the progression of ischemic/reperfusion injury. alpha-Tocopherol has been found to attenuate alterations due to ischemia and reperfusion in an isolated heart model. The present study was intended to directly examine neonatal rat cardiac ventricular cell cultures exposed to a free radical generating system catalyzed by xanthine oxidase. The effectiveness of alpha-tocopherol in the attenuation of the resultant changes and the mechanism by which the effects of alpha-tocopherol may be exerted were evaluated. Cultures were either nontreated or pretreated for 18 h with 20 microM alpha-tocopherol or the subcomponents of the alpha-tocopherol molecule, phytol and Trolox. Exposure of cell cultures to free radicals resulted in significant increases in lipid peroxidation products, release of both lactate dehydrogenase and 3H-arachidonate, and structural alterations. Pretreatment with alpha-tocopherol showed significant attenuation of the changes associated with exposure to free radicals. Trolox and phytol at equal molar doses were not as effective as alpha-tocopherol in protecting the myocytes against injury. Thus, alpha-tocopherol seems beneficial in its ability to reduce free radical-mediated changes by functioning as a lipophilic antioxidant. Additionally, the intact, native alpha-tocopherol molecule exceeded the protective capabilities of either of its subcomponents.     

Free radical generation and antioxidant content in chloroplasts from soybean leaves exposed to ultraviolet-B

The aim of this work was to study the effect of ultraviolet-B (UV-B) exposure on oxidative status in chloroplasts isolated from soybean ( Glycine max cv . Hood). Chloroplasts were isolated from soybean leaves excised from either control seedlings or those exposed to 30 and 60 kJ m⁻² day⁻¹ of UV-B radiation for 4 days. Chloroplastic oxidative conditions were assessed as carbon-centered radical, carbonyl groups and ascorbyl radical content. Treatment with UV-B increased the carbon-centered radical-dependent EPR signal significantly by 55 and 100% in chloroplasts from leaves exposed to 30 and 60 kJ m⁻² day⁻¹ UV-B, respectively, compared to radical content in chloroplasts from control leaves. The content of carbonyl groups increased by 37 and 62% in chloroplasts isolated from soybean leaves irradiated for 4 days with 30 and 60 kJ m⁻² day⁻¹ UV-B, respectively. The content of soluble metabolites in isolated chloroplasts should not be taken as absolute in vivo values; however, these data are valuable for comparative studies. UV-B exposure did not significantly affect ascorbyl radical content compared to controls. The content of ascorbic acid and thiols in chloroplasts isolated from leaves exposed to 60 kJ m⁻² day⁻¹ UV-B was increased by 117 and 20.8%, respectively, compared to controls. Neither the content of total carotene nor that of β -carotene or α -tocopherol was affected by the irradiation. The results presented here suggest that the increased content of lipid radicals and oxidized proteins in the chloroplasts isolated from leaves exposed to UV-B could be ascribed to both the lack of antioxidant response in the lipid soluble fraction and the modest increase in the soluble antioxidant content.     

Characterization of the potent neuroprotective properties of the natural vitamin E alpha-tocotrienol

The natural vitamin E tocotrienols possess properties not shared by tocopherols. Nanomolar alpha-tocotrienol, not alpha-tocopherol, is potently neuroprotective. On a concentration basis, this finding represents the most potent of all biological functions exhibited by any natural vitamin E molecule. We sought to dissect the antioxidant-independent and -dependent neuroprotective properties of alpha-tocotrienol by using two different triggers of neurotoxicity, homocysteic acid (HCA) and linoleic acid. Both HCA and linoleic acid caused neurotoxicity with comparable features, such as increased ratio of oxidized to reduced glutathione GSSG/GSH, raised intracellular calcium concentration and compromised mitochondrial membrane potential. Mechanisms underlying HCA-induced neurodegeneration were comparable to those in the path implicated in glutamate-induced neurotoxicity. Inducible activation of c-Src and 12-lipoxygenase (12-Lox) represented early events in that pathway. Overexpression of active c-Src or 12-Lox sensitized cells to HCA-induced death. Nanomolar alpha-tocotrienol was protective. Knock-down of c-Src or 12-Lox attenuated HCA-induced neurotoxicity. Oxidative stress represented a late event in HCA-induced death. The observation that micromolar, but not nanomolar, alpha-tocotrienol functions as an antioxidant was verified in a model involving linoleic acid-induced oxidative stress and cell death. Oral supplementation of alpha-tocotrienol to humans results in a peak plasma concentration of 3 microm. Thus, oral alpha-tocotrienol may be neuroprotective by antioxidant-independent as well as antioxidant-dependent mechanisms.     

Free radical scavenging activity, metal chelation and antioxidant power of some of the Indian spices

Food constituents are the major source of various phytochemicals and micronutrients. The importance of these dietary constituents has been stressed in recent years due to their antioxidant and anticarcinogenic potential. Spices used in Indian foods such as cloves (Syzygium aromaticum), licorice (Glycyrrhiza glabra), mace (aril of Myristica fragans), and greater cardamom (Amomum subulatum) were tested for their antioxidant properties in vitro. The metal chelating activity, bleomycin dependent DNA oxidation, diphenyl-p-picryl hydrazyl (DPPH) radical scavenging activity and the ferric reducing /antioxidant power (FRAP) were measured in rat liver homogenate in presence of spices. Metal chelating activity was significantly high with all the spice extracts except mace. The spices due to higher reducing potential (in presence of bleomycin-FeCl_{3}) showed increased DNA oxidation. Cloves showed the highest DPPH radical scavenging activity, followed by licorice, mace and cardamom. FRAP values for cloves were also the highest, while other spices showed comparatively lesser FRAP values. The results show that the spices tested are strong antioxidants and may have beneficial effects on human health.     

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.     

Free radical theory of aging.

Free radical reactions are ubiquitous in living things. Studies on the origin and evolution of life provide a reasonable explanation for the prominent presence of this unruly class of chemical reactions. These reactions have been implicated in aging. This phenomenon is the accumulation of changes responsible for the sequential alterations that accompany advancing age and the associated progressive increases in the chance of disease and death. Aging changes are attributed to the environment and disease, and to an inborn process, the aging process. The latter produces aging changes at an exponentially increasing rate with advancing age. Past improvements in general living conditions have decreased the chances for death so that they are now near limiting values in the developed countries. In these countries the intrinsic aging process is the major cause of disease and death after about age 28. The free radical theory of aging postulates that aging changes are caused by free radical reactions. The data supporting this theory indicate that average life expectancy at birth may be increased by 5 or more years, by nutritious low caloric diets supplemented with one or more free radical reaction inhibitors.