Free radical scavenging behavior of folic acid: evidence for possible antioxidant activity

The free radical scavenging properties and possible antioxidant activity of folic acid are reported. Pulse radiolysis technique is employed to study the one-electron oxidation of folic acid in homogeneous aqueous solution. The radicals used for this study are CCl₃O₂^•, N₃^•, SO₄^•−, Br₂^•−, √OH, and O^•−. All these radicals react with folic acid under ambient condition at an almost diffusion-controlled rate producing two types of transients. The first transient absorption maximum is around 430 nm, which decays, and a simultaneous growth at around 390 nm is observed. Considering the chemical structure of folic acid, the absorption maximum at 430 nm has been assigned to a phenoxyl radical. The latter one is proposed to be a delocalized molecular radical. A permanent product has been observed in the oxidation of folic acid with CCl₃O₂^• and N₃^• radicals, with a broad absorption band around 370–400 nm. The bimolecular rate constants for all the radical-induced oxidation reactions of folic acid have been measured. Folic acid is seen to scavenge these radicals very efficiently. In the reaction of thiyl radicals with folic acid, it has been observed that folic acid can not only scavenge thiyl radicals but can also repair these thiols at physiological pH. While carrying out the lipid peroxidation study, in spite of the fact that folic acid is considerably soluble in water, we observed a significant inhibition property in microsomal lipid peroxidation. A suitable mechanism for oxidation of folic acid and repair of thiyl radicals by folic acid has been proposed.     

Trifluoroacetylated tyrosine-rich D-tetrapeptides have potent antioxidant activity

The term “oxidative stress” indicates a set of chemical reactions unleashed by a disparate number of events inducing DNA damage, lipid peroxidation, protein modification and other effects, which are responsible of altering the physiological status of cells or tissues. Excessive Reactive Oxygen Species (ROS) levels may accelerate ageing of tissues or induce damage of biomolecules thus promoting cell death or proliferation in dependence of cell status and of targeted molecules. In this context, new antioxidants preventing such effects may have a relevant role as modulators of cell homeostasis and as therapeutic agents. Following an approach of peptide libraries synthesis and screening by an ORAC_FL assay, we have isolated potent anti-oxidant compounds with well-defined structures. Most effective peptides are N-terminally trifluoroacetylated (CF₃) and have the sequence tyr-tyr-his-pro or tyr-tyr-pro-his. Slight changes in the sequence or removal of the CF₃ group strongly reduced antioxidant ability, suggesting an active role of both the fluorine atoms and of peptide structure. We have determined the NMR solution structures of the active peptides and found a common structural motif that could underpin the radical scavenging activity. The peptides protect keratinocytes from exogenous oxidation, thereby from potential external damaging cues, suggesting their use as skin ageing protectant and as cell surviving agents.     

Increase in heart glutathione redox ratio and total antioxidant capacity and decrease in lipid peroxidation after vitamin e dietary supplementation in guinea pigs

Dietary treatment with three diets differing in vitamin E, Low E (15 mg of vitamin E/kg diet), Medium E (150 mg/kg), or High E (1,500 mg/kg), resulted in guinea pigs with low (but nondeficient), intermediate, or high heart a-tocopherol concentration. Neither the antioxidant enzymes superoxide dismutase, catalase, glutathione peroxidase, and reductase, nor the nonenzymatic antioxidants, GSH, ascorbate, and uric acid were homeostatically depressed by increases in heart a-tocopherol. Protection from both enzymatic (NADPH dependent) and nonenzymatic (ascorbate-Fe²⁺) lipid peroxidation was strongly increased by vitamin E supplementation from Low to Medium E Whereas no additional gain was obtained from the Medium E to the High E group. The GSH/GSSG and GSH/total glutathione ratios increased as a function of the vitamin E dietary concentration closely resembling the shape of the dependence of heart a-tocopherol on dietary vitamin E. The results show the capacity of dietary vitamin E to increase the global antioxidant capacity of the heart and to improve the heart redox status in both the lipid and water-soluble compartments. This capacity occurred at levels six times higher than the minimum daily requirement of vitamin E, even in the presence of optimum dietary vitamin C concentrations and basal unstressed conditions. The need for vitamin E dietary supplementation seems specially important in this tissue due to the low constitutive levels of endogenous enzymatic and nonenzymatic antioxidants present of the mammalian heart in comparison with those of other internal organs.     

Biomarkers of diabetes-associated oxidative stress and antioxidant status in young diabetic patients with or without subclinical complications

The aims of the study were to ascertain the potential role of oxidative stress in the onset of disease-related pathophysiological complications in young type 1 diabetes patients. Indicative parameters of lipoperoxidation, protein oxidation, and changes in antioxidant defense system status were measured in blood samples from 26 young diabetic patients with recently diagnosed (< 6 months) microangiopathy (+DC), 28 diabetic patients without complications (−DC), and 40 healthy age-matched controls (CR). Both diabetic groups presented similar fructosamine and glycated hemoglobin (HbA1c) values. Results showed erythrocyte glutathione peroxidase activity, glutathione content, and plasma β-carotene to be significantly lower in diabetic patients compared with control subjects, but with no significant differences between −DC and +DC groups. Antioxidant enzyme superoxide dismutase activity was significantly higher in the erythrocytes of diabetic patients independently of the presence of microvascular complications. However, the plasma -tocopherol/total lipids ratio was significantly diminished in +DC group compared with −DC (p = .008). Lipid peroxidation indices measured in plasma included malondialdehyde, lipid hydroperoxides, and lipoperoxides, which were significantly elevated in our diabetic patients regardless of the presence of complications. Evidence of oxidative damage to proteins was shown both through the quantification of plasma protein carbonyl levels, which were significantly higher in −DC (0.61 ± 0.09 mmol/mg prot), and higher still in the +DC patients (0.75 ± 0.09 mmol/mg prot) compared with those of controls (0.32 ± 0.03 mmol/mg prot; p < .01) and immunoblot analysis of protein-bound carbonyls. Additionally, a marked increase in protein oxidation was observed in +DC patients through assessment of advanced oxidation protein products (AOPP) considered to be an oxidized albumin index; AOPP values were significantly higher in +DC than in −DC patients (p < .01) and CR (p < .0001). These results point to oxidatively modified proteins as a differential factor possibly related to the pathogenesis of diabetic complications.     

In vitro free radical scavenging activity of hepatic metallothionein induced in an Indian freshwater fish, Channa punctata Bloch

Mammalian metallothioneins (MT) have been reported to scavenge free radicals. There is no experimental evidence to show that fish MT has a similar property. In the present study cadmium-induced MT (Cd-MT) from the liver of an Indian freshwater fish Channa punctata Bloch was investigated for its free radical scavenging activity using three different in vitro assays. Exposure to cadmium chloride (0.2 mg/kg body weight; three doses on alternate days) resulted in a marked induction of Cd-MT in liver. Only a single isoform of Cd-MT was found to be induced. Molecular weight of Cd-MT was found to be 14 kDa as deduced by SDS-PAGE analysis. The purified Cd-MT effectively scavenged the following free radicals: superoxide radical (O2*-), 2,2'-azinobis 3-ethylbenzothiazoline-6-sulfonic acid (ABTS*+) and 1,1-diphenyl-picrylhydrazyl radical (DPPH*). The radical scavenging effect was found to be concentration-dependent. Also, the purified MT exhibited an inhibitory effect on ferric nitrilotriacetate (Fe-NTA) induced oxidative DNA damage in vitro. The cysteine residues of MT are proposed to be the main candidate for its radical scavenging activity. Findings of the present study strongly suggest a free radical scavenging role for fish MT. Present study adds to the little existing knowledge about fish MT and its possible biological functions.     

Antioxidant effects of water- and lipid-soluble nitroxide radicals in liposomes

Liposomes are today useful tools in different fields of science and technology. A lack of stability due to lipid peroxidation is the main problem in the extension of the use of these formulations. Recent investigative works have reported the protective effects of stable nitroxide radicals against oxidative processes in different media and under different stress conditions. Our group has focused its attention on the natural aging of liposomes and the protection provided by the water- and lipid-soluble nitroxide radicals 2,2,6,6-tetramethylpiperdine-1-oxyl (TEMPO) and doxylstearic acids (5-DSA, 12-DSA, and 16-DSA), respectively. Unilamellar liposomes were incubated under air atmosphere at 37°C, both in the absence and in the presence of these radicals. Conjugated dienes, lipid hydroperoxides, TBARS, membrane fluidity, and nitroxide ESR signal intensity were followed as a function of time. Our results demonstrated that doxylstearic acids were more efficient than TEMPO in retarding lipid peroxidation at all the concentrations tested. The inhibition percentages, depending on the total nitroxide concentration, were not proportional to the lipid–water partition coefficient. Furthermore, time-course ESR signals showed a slower decrease for doxylstearic acids than for TEMPO. No significant differences were found among 5-DSA, 12-DSA, and 16-DSA. We concluded that the nitroxide radical efficiency as antioxidant directly depends on both nitroxide concentration and lipophilicity.     

Hemoglobin components from trout (Salmo irideus): determination of their peroxidative activity

The peroxidative activity of trout hemoglobins, HbI and HbIV, which differ in their conformation, was compared with that of HbA. Artificial substrates (guaiacol and dopamine) and more physiological substrates such as model lipid membranes containing unsaturated fatty acids were used. The results indicate that all the hemoglobin molecules assayed show different levels of peroxidative activity. The capability to act as peroxidases is greater in HbIV than in HbI and HbA. In contrast, native globins did not show peroxidase activity. The different peroxidative activity of the Hbs is discussed in relation to stability both vs. protein oxidation and protein dissociation. The results confirm the view that hemoglobin may be of importance in establishing the life span of the erythrocyte itself.     

Antioxidants in the serum of children with insulin-dependent diabetes mellitus

To determine whether alteration in serum antioxidant status is related to the increased oxidative stress as a cause of diabetic angiopathy, we measured both the antioxidant activity (AOA) and total peroxyl radical-trapping antioxidant parameter (TRAP), and their component individual antioxidants in serum of children with insulin-dependent diabetes mellitus (IDDM). The AOA was measured as the ability to inhibit lipid autoxidation in brain homogenates. TRAP was assayed as the ability to delay lipid peroxidation induced by an azo initiator. Antioxidants measured were ceruloplasmin, transferrin, and albumin components of AOA; and ascorbic acid, uric acid, protein sulfhydryl, and alpha-tocopherol as components of TRAP. Serum AOA appeared to be decreased in the diabetics in relation to poor glycemic control, corresponding to the decrease in transferrin and albumin. Serum haptoglobin level was also decreased in the diabetics. Similarly, the directly measured TRAP value was decreased in the diabetic serum mainly due to the decreased contribution of unidentified chain-breaking antioxidants, despite the increase in ascorbic acid and alpha-tocopherol. The decrease in both types of antioxidant activity in the diabetic serum, as new findings, suggests that a defective serum antioxidant status contributes to the increased oxidative stress in IDDM.     

中药提取物对酵母菌抗真菌活性研究

目的探讨6味中药2种方法提取成分对酵母菌的抑菌和杀菌作用。方法采用药基琼脂稀释法,测定6味中药水提和醇提成分对白念珠菌和糠秕马拉色菌的MIC和MFC。结果对白念珠菌：水提黄连、醇提黄柏、醇提土槿皮MIC范围分别为0．625—1．25mg／mL、0．625～1．25mg/mL、0．313—0．625mg／mL;均值均为0．625mg／mL;对糠秕马拉色菌：水提和醇提黄连MIC范围分别为0．625～1．25mg／mL和1．25mg／mL,均值均为1．25mg／mL。对白念珠菌：醇提土槿皮MFC范围0．625～2．5mg/mL,均值0．625rag／mL。结论水提黄连、醇提黄柏和土槿皮对白念珠菌有较强抑菌作用,其中醇提土槿皮有较强杀菌作用。水提和醇提黄连对糠秕马拉色菌有较强抑菌作用。     

Hyaluronic acid and chondroitin-4-sulphate treatment reduces damage in carbon tetrachloride-induced acute rat liver injury

Oxidative stress is involved in the pathogenesis of chemically mediated liver injury. Since glycosaminoglycans possess antioxidant activity, the aim of this work was to assess the protective effects of hyaluronic acid and chondroitin-4-sulphate treatment in a model of carbon tetrachloride-induced liver injury. Liver damage was induced in male rats by an intraperitoneal injection of carbon tetrachloride (1 ml/kg in vegetal oil). Serum alanine aminotransferase and aspartate aminotransferase, hepatic malondialdehyde, plasma TNF-alpha, hepatic reduced glutathione and catalase, and myeloperoxidase, an index of polymorphonuclear infiltration in the jeopardised hepatic tissue, were evaluated 24 h after carbon tetrachloride administration. Carbon tetrachloride produced a marked increase in serum alanine aminotransferase and aspartate aminotransferase activities, primed lipid peroxidation, enhanced plasma TNF-alpha levels, induced a severe depletion of reduced glutathione and catalase, and promoted neutrophil accumulation. Intraperitoneal treatment of rats with hyaluronic acid (25 mg/kg) or chondroitin-4-sulphate (25 mg/kg) failed to exert any effect in the considered parameter, while the combination treatment with both glycosaminoglycans (12,5 + 12,5 mg/kg) decreased the serum levels of alanine aminotransferase and aspartate aminotransferase, inhibited lipid peroxidation by reducing hepatic malondialdehyde, reduced plasma TNF-alpha, restored the endogenous antioxidants, and finally decreased myeloperoxidase activity. These results suggest that hyaluronic acid and chondroitin-4-sulphate possess a different antioxidant mechanism and consequently the combined administration of both glycosaminoglycans exerts a synergistic effect with respect to the single treatment.     

Evidence that free radical generation occurs during scorpion envenomation

Although it is well established that symptomatology, morbidity and death following scorpion envenomation are due to increases in neurotransmitter release secondary to toxins binding to voltage-sensitive sodium channels, the mechanism by which venom action is involved in damaging heart, liver, lungs and kidneys remains unclear. We hypothesized that scorpion toxins could induce the generation of high levels of free radicals responsible for membrane damage in organs targeted by venom action. We have investigated lipid peroxidation in different organs, through the evaluation of thiobarbituric acid reactive substances (TBARS), after experimental envenomation of rats by toxic fractions of Androctonus australis Hector venom. We have shown that scorpion toxins cause considerable lipid peroxidation in most vital organs. We also evaluated the protective effects of antioxidants in mice injected with lethal doses of toxins. Among the drugs tested, N-acetylcysteine (NAC) was effective in protecting the mice when injected prior to toxin application. However, the free radical scavenging properties of NAC seem less implicated in these protective effects than its ability to increase the fluidity of bronchial secretions. We therefore conclude that free radical generation only plays a minor role in the toxicity of scorpion venom.     

Trolox C, a lipid-soluble membrane protective agent, attenuates myocardial injury from ischemia and reperfusion

The lipophilic antioxidant Trolox C, a vitamin E analog, was administered to isolated, buffer-perfused rabbit hearts subjected to 25 min of global stop-flow ischemia and 30 min of reperfusion. In six hearts, Trolox C (200 microM) was infused for 15 min immediately prior to ischemia and for the first 15 min of reperfusion. Six control hearts received only vehicle. Gas chromatography analysis confirmed that effective myocardial levels of Trolox were attained. At 30 min reperfusion, the recovery of left ventricular developed pressure was 56 +/- 3% of baseline in control hearts versus 70 +/- 4% in Trolox-treated hearts (p < .01). There was also significant improvement in recovery of Trolox-treated hearts in diastolic pressure and both maximum and minimum values of the first derivative of left ventricular pressure (dP/dt). Creatine phosphokinase release into the coronary effluent at 30 min of reperfusion was 16.5 +/- 8.4 IU/min in untreated and 6.3 +/- 1.0 IU/min (p < .05) in Trolox-treated hearts. Thus Trolox C, a lipophilic antioxidant, attenuated myocardial injury during stop-flow ischemia and reperfusion.     

Free radical scavenging reactions and antioxidant activity of embelin: biochemical and pulse radiolytic studies

Embelin (from Embelia ribes) is a component of herbal drugs and possess wide range of medicinal properties. These properties may be, in part, due to scavenging of oxidizing free radicals. In this context, free radical scavenging reactions and antioxidant activity of embelin (2,5-dihydroxy-3-undecyl-1,4-benzoquinone) have been studied. It has been found to scavenge DPPH radical and inhibit hydroxyl radical induced deoxyribose degradation. It has been also found to inhibit lipid peroxidation and restore impaired Mn-superoxide dismutase in rat liver mitochondria. Further, kinetics and mechanism of the reactions of embelin with hydroxyl, one-electron oxidizing, organo-haloperoxyl and thiyl radicals have been studied using nanosecond pulse radiolysis technique. Its redox potential has been also evaluated with cyclic voltammetry. These studies suggest that embelin can act as a competitive antioxidant in physiological conditions.     

The role of oxygen radicals in experimental acute pancreatitis

Oxygen-derived free radicals mediate an important step in the initiation of experimental acute pancreatitis. Thereby, it seems that these reactive oxygen metabolites are generated at an early stage of disease. The source of the enhanced production of oxygen radicals still remains unclear. Experimentally, the efficiency of scavenger treatment varied between different models, whereby these differences depended on the experimental model and not on the form of pancreatitis which was induced. Most studies pretreated the experimental animals before inducing acute pancreatitis. This does not mirror the clinical reality, since patients are admitted to the hospital after onset of the disease. It was shown in Cerulein pancreatitis, however, that scavenger treatment also mitigated the pancreatic tissue damages after induction of acute pancreatitis. Moreover, antioxidant treatment also attenuated the extrapancreatic complications, thus improving the final outcome of the disease. The first indirect observations also suggest that in human acute recurrent and chronic pancreatitis, oxygen free radicals are generated and add to the damages seen. Therefore, well-defined controlled clinical studies with patients suffering from acute pancreatitis are needed to validate the role of oxygen radicals in this disease.     

β-Amyloid Neurotoxicity In Vitro: Evidence of Oxidative Stress but Not Protection by Antioxidants

Abstract: Recent data from several groups suggest that the primary mechanism of β-amyloid neurotoxicity may be mediated by reactive oxygen species. To evaluate this hypothesis, we first compared the efficacy of antioxidant agents in preventing toxicity caused by oxidative insults (iron, hydrogen peroxide, and tert -butyl hydroperoxide) and β-amyloid peptides in cultured rat hippocampal neurons. Tested antioxidants (propyl gallate, Trolox, probucol, and promethazine) generally provided significant protection against oxidative insults but not β-amyloid peptides. Next, we examined whether β-amyloid causes oxidative stress, by comparing levels of lipid peroxidation after exposure to either iron or β-amyloid. In a cell-free system, iron but not β-amyloid generated lipid peroxidation. In culture, both insults caused rapid increases in lipid peroxidation, with iron inducing higher levels at later time points. Pretreatment with the antioxidant probucol significantly reduced lipid peroxidation caused by both insults but only attenuated iron toxicity, suggesting that lipid peroxidation does not contribute directly to cell death induced by β-amyloid. Finally, we observed that increasing basal levels of oxidative stress by pretreating cultures with subtoxic doses of iron significantly increased neuronal vulnerability to β-amyloid. The ability of β-amyloid to induce oxidative stress and the demonstration that oxidative stress potentiates β-amyloid toxicity support the clinical use of antioxidants for AD. However, these data do not support the theory that the primary mechanism of β-amyloid toxicity involves oxidative pathways, indicating a continued need to identify additional cellular responses to β-amyloid that underlie its neurodegenerative actions.     

Comparative study on the action of tocopherols and tocotrienols as antioxidant: chemical and physical effects

alpha-Tocopherol is known as the most abundant and active form of vitamin E homologues in vivo, but recently the role of other forms of vitamin E has received renewed attention. The antioxidant properties were compared for alpha-, beta-, gamma- and delta-tocopherols and tocotrienols. The following results were obtained: (1). the corresponding tocopherols and tocotrienols exerted the same reactivities toward radicals and the same antioxidant activities against lipid peroxidation in solution and liposomal membranes; (2). tocopherols gave more significant physical effect than tocotrienols on the increase in rigidity at the membrane interior; (3). tocopherols and tocotrienols showed similar mobilities within the membranes, but tocotrienols were more readily transferred between the membranes and incorporated into the membranes than tocopherols; (4). alpha-tocopherol and alpha-tocotrienol, but not the other forms, reduced Cu(II) to give Cu(I) together with alpha-tocopheryl and alpha-tocotrienyl quinones, respectively and exerted prooxidant effect in the oxidation of methyl linoleate in SDS micelles.     

The effect of rebamipide on gastric xanthine oxidase activity and type conversion in ethanol-treated rats

Rebamipide, a novel antipeptic ulcer drug, 2-(4-chlorobenzoylamino)-3-[2(1H)-quinolinone-4-yl]-propionic acid, was studied for its inhibitory effect on gastric xanthine oxidase activity and type conversion of the enzyme that has a profound role in free radical generation. Intraperitoneal administration of rebamipide at 60 mg/kg body weight reduced gastric mucosal hemorrhagic lesions and lipid peroxidation, which was proportional to the inhibitory effect of rebamipide on alcohol-induced xanthine oxidase-type conversion and enzyme activity. It was also observed that the activity of xanthine oxidase was significantly inhibited by administration of rebamipide at 60 mg/kg body weight, leading to a significant reduction of lipid peroxide content in alcohol-treated rats. The results suggest that alcohol-induced gastric mucosal lesions might be, in part, due to the increased activity of xanthine oxidase and type conversion rate of the enzyme and the protective effect of rebamipide on gastric mucosal lesions would result from its ability to protect against oxidative stress on gastric mucosal lesions of alcohol-treated rats.     

Antioxidant functions of carotenoids

Carotenoid pigments, including hydrocarbons such as β-carotene or xanthophylls such as lutein and zeaxanthin, are very widely distributed in nature, where they play an important role in protecting cells and organisms against the harmful effects of light, air, and sensitizer pigments. This process has been demonstrated in bacteria, algae, plants, animals, and even in humans in the light-sensitive disease, erythropoietic protoporphyria. The primary mechanism of action of this phenomenon appears to be the ability of carotenoids to quench excited sensitizer molecules as well as quench ¹O₂.

In addition to this protection, and potentially of even greater biological importance, is the fact that carotenoids can also serve as antioxidants under conditions other than photosensitization. This review presents the data available indicating the extent of this important function. Antioxidant action can be documented in both enzymic and nonenzymic systems, and has been reported in subcellular, cellular, and animal studies. In fact, the many reports indicating that carotenoids may possess some anticarcinogenic properties may well be related to their ability to interact with and quench various radical species that can be generated within cells.     

Kinetic evaluation of lipophilic inhibitors of lipid peroxidation in DLPC liposomes

The authors have developed a kinetic method that allows one to obtain relative reactivity constants for lipophilic antioxidants in free radical systems. Two experimental model systems were developed: (a) a methanolic solution using AMVN as the free radical initiator and linoleic acid as the substrate, and (b) a multilamellar vesicle system composed of dilinoleoylphosphatidylcholine and AAPH as the substrate and the initiator, respectively. The use of these two systems allows researchers not only to determine the intrinsic reactivity of a potential antioxidant, but also to evaluate its potency in a membranous system where the contribution of the physical properties of the antioxidant to the inhibition of lipid peroxidation is important. These results show that all antioxidants tested acted in these systems as free radical scavengers, and they validate the synergism between intrinsic scavenging ability and membrane affinity and/or membrane-modifying physical properties in the inhibition of lipid peroxidation.