Protective effect of flavonoids on endothelial cells against linoleic acid hydroperoxide-induced toxicity

The protective effect of flavonoids against linoleic acid hydroperoxide (LOOH)-induced cytotoxicity was examined by using cultured endothelial cells. When the cells were incubated with both LOOH and flavonoids, most flavonols protected the cells from injury by LOOH. Flavones bearing an ortho-dihydroxy structure also showed a protective effect against the cytotoxicity of LOOH. However, flavanones had no effect. The structure-activity relationship revealed the presence of either the ortho-di-hydroxy structure in the B ring of the flavonoids or 3-hydroxyl and 4-oxo groups in the C ring to be important for the protective activities. The interaction between flavonoids and a-tocopherol was also examined in this system. Flavonoids that were protective against LOOH-induced cytotoxicity had at least an additive effect on the action of alpha-tocopherol against LOOH-induced damage.     

Inhibition of linoleic acid hydroperoxide-induced toxicity in cultured human fibroblasts by anthocyanidins

The protective effect of anthocyanidins against the toxicity induced by linoleic acid hydroperoxide (LOOH) was examined in cultured human fetal lung fibroblasts, TIG-7. Cyanidin was more effective than pelargonidin or delphinidin in inhibiting LOOH-induced cytotoxicity. The presence of a catechol moiety in the B ring is shown to be important for the protective activities against the cytotoxicity of LOOH.     

Protective effects of flavonoids on the cytotoxicity of linoleic acid hydroperoxide toward rat pheochromocytoma PC12 cells

The protective effects of nine flavonoids, including apigenin, eriodictyol, 3-hydroxyflavone, kaempherol, luteolin, quercetin, rutin, and taxifolin (Table 1), on the cytotoxicity of linoleic acid hydroperoxide (LOOH) toward rat pheochromocytoma PC12 cells were examined. The cytotoxicity was assessed by the trypan blue exclusion test and so-called MTT assay. When cells were preincubated with each flavonoid prior to LOOH exposure, quercetin, 3-hydroxyflavone, or luteolin decreased LOOH cytotoxicity toward undifferentiated cells, while only luteolin decreased efficiently LOOH cytotoxicity toward differentiated cells. On the other hand, when cells were coincubated with each flavonoid and LOOH, kaempherol, eriodictyol, quercetin, 3-hydroxyflavone, luteolin, or taxifolin decreased LOOH cytotoxicity toward undifferentiated and differentiated cells. On both preincubation prior to LOOH exposure and coincubation with LOOH, luteolin acted as the most efficiently protective agent against LOOH cytotoxicity. Further, these flavonoids showed protective effects on coincubation rather than preincubation. Flow cytometry using the fluorescence probe 2',7'-dichlorofluorescin diacetate revealed that LOOH increases the intracellular level of reactive oxygen species in undifferentiated cells in a dose-dependent manner, and that desferrioxamine mesylate suppresses the LOOH-induced increase in the level. These flavonoids suppress the LOOH-induced increase. Further, the protective effect of flavonoids on LOOH cytotoxicity correlates with the suppression of the LOOH-induced increase. These results suggest that such flavonoids are beneficial for neuronal cells under oxidative stress.     

Protection of coumarins against linoleic acid hydroperoxide-induced cytotoxicity

Coumarins comprise a group of natural phenolic compounds found in a variety of plant sources. Protective effects of coumarins against cytotoxicity induced by linoleic acid hydroperoxide were examined in cultured human umbilical vein endothelial cells. When the cells were incubated in medium supplemented with linoleic acid hydroperoxide and coumarins, esculetin (6,7-dihydroxycoumarin) and 4-methylesculetin protected cells from injury by linoleic acid hydroperoxide. Fraxetin and caffeic acid showed weak, but significant, protection. Esculin as well as esculetin and 4-methylesculetin were effective for protecting cells against linoleic acid hydroperoxide-induced cytotoxicity in the case of pretreatment for 24 h, however fraxetin and caffeic acid showed no protection. Since esculetin was detected after 24 h treatment with esculin, a sugar moiety in the esculin molecule appears to be hydrolyzed during pretreatment. Coumarins such as umbelliferone containing only one hydroxyl group showed no protective effect in pretreatment or concurrent treatment. Esculetin and 4-methylesculetin provided synergistic protection against cytotoxicity induced by linoleic acid hydroperoxide with alpha-tocopherol. Furthermore, the radical-scavenging ability of coumarins was examined in electron spin resonance spectrometry. Esucletin, 4-methylesculetin, fraxetin, and caffeic acid showed the quenching effect on the 1,1-diphenyl-2-picrylhydrazyl radical. These results indicate that the presence of an ortho catechol moiety in the coumarin molecules plays an important role in the protective activities against linoleic acid hydroperoixde-induced cytotoxicity.     

Comparative study of antioxidant properties and cytoprotective activity of flavonoids

Antioxidant properties and cytoprotective activity of flavonoids (rutin, dihydroquercetin, quercetin, epigallocatechin gallate (EGCG), epicatechin gallate (ECG)) were studied. All these compounds inhibited both NADPH- and CCl₄-dependent microsomal lipid peroxidation, and the catechins were the most effective antioxidants. The I ₅₀ values calculated for these compounds by regression analysis were close to the I ₅₀ value of the standard synthetic antioxidant ionol (2,6-di-tert-butyl-4-methylphenol). The antiradical activity of flavonoids to O ₂ was studied in a model photochemical system. Rate constants of the second order reaction obtained by competitive kinetics suggested flavonoids to be more effective scavengers of oxygen anion-radicals than ascorbic acid. By competitive replacement all flavonoids studied were shown to be chelating agents capable of producing stable complexes with transition metal ions (Fe²⁺, Fe³⁺, Cu²⁺). The flavonoids protected macrophages from asbestos-induced damage, and the protective effect increased in the following series: rutin < dihydroquercetin < quercetin < ECG < EGCG. The cytoprotective effect of flavonoids was in strong positive correlation with their antiradical activity to O ₂ .     

Effects of Dietary Antioxidants on Human DNA Ex Vivo

The protective effect of fruits and vegetables against cancer is well established. It is believed that this effect is mediated by antioxidants and decreased oxidative damage to DNA. However, the identity of the antioxidant(s) responsible is not clear. Moreover, a potentially damaging pro-oxidant effect of some antioxidants has been reported. In this study the ex vivo effects of several dietary antioxidants, including quercetin, various catechins, ascorbic acid and &#102 -tocopherol, were investigated, at concentrations up to 200 &#117 &#119 M, using the single cell gel electrophoresis (comet) assay for DNA damage. Lymphocytes from three healthy subjects were pre-incubated with these antioxidants, and the comet assay was performed on treated, untreated, challenged and unchallenged cells in parallel, oxidant challenge being induced by 5 &#117 min exposure to hydrogen peroxide (final concentrations H 2 O 2 : 30, 45, or 60 &#117 &#119 M). Results using this ex vivo cellular assay showed protection by some antioxidants (quercetin, caffeic acid), no effect by some (catechin, epicatechin, catechin gallate, epicatechin gallate) and an apparently damaging effect by others (epigallocatechin, epigallocatechin gallate). Damage may have been caused by production of H 2 O 2 from these polyphenolics. Neither ascorbic acid nor &#102 -tocopherol protected or damaged DNA. Further study of the role of quercetin and caffeic acid in DNA protection is needed.     

Effects of dietary antioxidants on human DNA ex vivo

The protective effect of fruits and vegetables against cancer is well established. It is believed that this effect is mediated by antioxidants and decreased oxidative damage to DNA. However, the identity of the antioxidant(s) responsible is not clear. Moreover, a potentially damaging pro-oxidant effect of some antioxidants has been reported. In this study the ex vivo effects of several dietary antioxidants, including quercetin, various catechins, ascorbic acid and alpha-tocopherol, were investigated, at concentrations up to 200 microM, using the single cell gel electrophoresis (comet) assay for DNA damage. Lymphocytes from three healthy subjects were pre-incubated with these antioxidants, and the comet assay was performed on treated, untreated, challenged and unchallenged cells in parallel, oxidant challenge being induced by 5 min exposure to hydrogen peroxide (final concentrations H2O2: 30, 45, or 60 microM). Results using this ex vivo cellular assay showed protection by some antioxidants (quercetin, caffeic acid), no effect by some (catechin, epicatechin, catechin gallate, epicatechin gallate) and an apparently damaging effect by others (epigallocatechin, epigallocatechin gallate). Damage may have been caused by production of H2O2 from these polyphenolics. Neither ascorbic acid nor alpha-tocopherol protected or damaged DNA. Further study of the role of quercetin and caffeic acid in DNA protection is needed.     

α-tocopherol,ascorbic acid,and rutin inhibit synergistically the copper-promoted LDL oxidation and the cytotoxicity of oxidized LDL to cultured endothelial cells

Low-density lipoproteins (LDL) mildly oxidized by copper ions or UV radiations exhibit a cytotoxic effect to cultured endothelial cells. Rutin, a polyphenolic flavonoid, ascorbic acid, and α-tocopherol were able to inhibit the peroxidation of LDL and their subsequent cytotoxicity. The mixture of the three compounds (rutin/ascorbic acid/α-tocopherol, 4/4/1) exhibited a supra-additive antioxidant effect. The inhibition of the cytotoxic effect was well correlated with that of TBARS formation. Another important conclusion is that these antioxidants were able to prevent directly at the cellular level the cytotoxic effect of oxidized LDL, since cells preincubated with them were protected against the cytotoxic effect of previously oxidized LDL. The protective effect of antioxidants was limited because of their own toxicity. The antioxidant mixture permitted a maximal cytoprotective effect with relatively lower concentrations to be obtained and the cytotoxicity of high concentrations to be avoided. In conclusion, rutin, ascorbic acid, and α-tocopherol constitute two lines of defense in protecting cells against injury owing to oxidation of LDL (1) at the LDL level, by inhibiting the LDL oxidation and the subsequent cytotoxicity, and (2) at the cellular level, by protecting the cells directly, i.e., by increasing their resistance against the cytotoxic effect of oxidized LDL.     

Protective effect of phenolic antioxidants on the cytotoxicity induced by phosphatidylcholine hydroperoxide

Phosphatidylcholine hydroperoxides show weak but distinct toxicity toward cultured human umbilical vein endothelial cells. The protective effect of phenolic antioxidants against the cytotoxicity of phosphatidylcholine hydroperoxides was examined. Probucol depressed the toxicity most effectively among the antioxidants studied under both pretreatment and concurrent treatment conditions. alpha-Tocopherol showed a protective effect in the case of concurrent treatment. Protection by phenolic antioxidants against the cytotoxicity of phosphatidylcholine hydroperoxides seems to depend on their incorporation rate into cells, their affinity for phospholipids, their antioxidative activity, and their orientation in membranes.     

Protective Effect of Phenolic Antioxidants on the Cytotoxicity Induced by Phosphatidylcholine Hydroperoxide

Phosphatidylcholine hydroperoxides show weak but distinct toxicity toward cultured human umbilical vein endothelial cells. The protective effect of phenolic antioxidants against the cytotoxicity of phosphatidylcholine hydroperoxides was examined. Probucol depressed the toxicity most effectively among the antioxidants studied under both pretreatment and concurrent treatment conditions. α-Tocopherol showed a protective effect in the case of concurrent treatment. Protection by phenolic antioxidants against the cytotoxicity of phosphatidylcholine hydroperoxides seems to depend on their incorporation rate into cells, their affinity for phospholipids, their antioxidative activity, and their orientation in membranes.     

Methylated polyphenols are poor "chemical" antioxidants but can still effectively protect cells from hydrogen peroxide-induced cytotoxicity

Several polyphenolic compounds, including flavonoids and phenolic acids, were compared with their per-methylated forms in both chemical and cell-based assays for antioxidant capacity. Methylation largely eliminated "chemical" antioxidant capacity, according to ferric reducing antioxidant power and oxygen radical absorbance capacity assays. Methylation, however, only moderately reduced protection of human Jurkat cells in culture, from hydrogen peroxide-mediated cytotoxicity, at physiologically relevant concentrations. Neither methylated nor un-methylated compounds were detectably metabolized by the cells. It appears that the protective mechanism of polyphenolic antioxidants against high concentrations of hydrogen peroxide in human cells may be largely unrelated to chemical antioxidant capacity.     

Antioxidants and herbal extracts protect HT-4 neuronal cells against glutamate-induced cytotoxicity

Antioxidant therapy has been shown to be beneficial in neurological disorders including Alzheimer's disease and cerebral ischemia. Glutamate-induced cytotoxicity in HT-4 neuronal cells has been previously demonstrated to be due to oxidative stress caused by depletion of cellular glutathione (GSH). The present study demonstrates that a wide variety of antioxidants inhibit glutamate-induced cytotoxicity in HT-4 neuronal cells. Low concentrations of α-tocopherol and its analogs were highly effective in protecting neuronal cells against cytotoxicity. Purified flavonoids and herbal extracts of Gingko biloba (EGb 761) and French maritime pine bark (Pycnogenol®) were also effective. We have previously shown that pro-glutathione agents can spare GSH and protect cells from glutamate insult in a C6 glial cell model. The protective effects of nonthiol-based antioxidants tested in the HT-4 line were not mediated via GSH level modulation. In contrast, protective effects of thiol-based pro-glutathione agents α-lipoic acid (LA) and N-acetyl cysteine (NAC) corresponded with a sparing effect on GSH levels in glutamate-treated HT-4 cells. Glutamate-induced cytotoxicity in HT-4 cells is a useful model system for testing compounds or mixtures for antioxidant activity.     

Dietary antioxidants provide differential subcellular protection in epithelial cells

Abstract

This study aimed to evaluate the organelle-specific antioxidant/pro-oxidant actions of clinically important dietary antioxidants against oxidative stress. An in vitro cellular model was employed to investigate the antioxidant/pro-oxidant effects of various concentrations (1, 10 and 100 μM) of ascorbic acid, α-tocopherol and β-carotene during H₂O₂-induced oxidative stress. Damage to nuclear and mitochondrial genomes was analyzed by quantitative polymerase chain reaction and oxidation of membrane lipids was measured via colorimetric assays. The key findings were: (i) dietary antioxidants conferred a dose-dependent protective effect (with a pro-oxidant shift at higher concentrations); (ii) the protection conferred to different sub-cellular organelles is highly specific to the dietary antioxidant; (iii) the mtDNA is highly sensitive to oxidative attack compared to nDNA (P < 0.05); and (iv) mtDNA protection conferred by dietary antioxidants was required to improve protection against oxidative-induced cell death. This study shows that antioxidant-induced protection of mtDNA is an important target for future oxidative stress therapies.     

Natural antioxidants of plant origin against ozone damage of sensitive crops

The increasing concentrations of ambient ozone observed during recent decades in many industrial and rural regions of the world present hazard for vegetation and human health. The problem of protection of sensitive vegetation from ozone damage could be ameliorated by replacement of sensitive biotypes with more tolerant ones as well as by application of chemical protectants. However, application of synthetic protectants will pollute the environment and agricultural production and may also have dose-dependent toxicity to vegetation. Therefore, it is urgent to develop alternative, environmentally antiozonants, for example, compounds based on natural plant antioxidants. In this article the literature has been reviewed in search of works relating to the potential of natural plant antioxidants that might serve to protect sensitive vegetation from ozone damage. The following groups of antioxidants have been discussed: (i) ascorbic acid and its derivatives, (ii) phytohormones, (iii) flavonoids, and (iv) polyamines. The physiological aspects of their protective effect on ozone-sensitive crops have been considered. Possible phytotoxicity resulting from their application in the field has been discussed. The issues needing further studies have been outlined.     

Flora of the forest-steppe zone of West Siberia as a source of biologically active compounds

Plants of 76 species from 25 families growing in the forest-steppe zone of West Siberia are analyzed for the content of low-molecular biologically active compounds, such as flavonoids, tannins, catechins, coumarins, saponins, and alkaloids. It is established that 33 species hold promise as a source of flavonoids, 29 species tannins, 21 coumarins, 13 saponins, 13 alkaloids, and 1 catechins.     

Catechins delay lipid oxidation and alpha-tocopherol and beta-carotene depletion following ascorbate depletion in human plasma

Blood plasma was incubated with 50 mM AAPH [2, 2'-azobis-(2-amidinopropane) hydrochloride] in the absence or presence of catechins (5-100 microM). Lipid oxidation was evaluated by measuring the formation of 2-thiobarbituric acid reactive substances (TBARS). The concentration of alpha-tocopherol (AT), beta-carotene (BC), ascorbic acid (AA), and catechins was determined by reverse phase high performance liquid chromatography (HPLC) with electrochemical detection. All the assayed catechins inhibited plasma TBARS formation. Based on the calculated IC50, the order of effectiveness was: epicatechin gallate (ECG) > epigallocatechin gallate (EGCG) > epigallocatechin (EGC) > epicatechin (EC) > catechin (C). Catechins protected plasma AT and BC from AAPH-mediated oxidation. The order of effectiveness for AT protection was ECG > EGCG > EC = C > EGC; and for BC protection, the order was EGCG > ECG > EGC > > EC > C. The addition of catechins modified the kinetics of TBARS formation and AT depletion, but the rate of AA depletion was not affected. Catechin oxidation did not start until the complete depletion of AA, and it preceded AT depletion. These results indicate that catechins are effective antioxidants in human blood plasma, delaying the lipid oxidation and depletion of endogenous lipid-soluble antioxidants (AT and BC).     

The influence of antioxidants in the thiyl radical induced lipid peroxidation and geometrical isomerization in micelles of linoleic acid

Abstract

The biomimetic model of micelles of linoleic acid containing 2-mercaptoethanol and the antioxidant was examined under gamma irradiation up to 400?Gy in aerobic or deoxygenated conditions where thiyl radicals are the main reactive species. Lipid peroxidation was retarded by ascorbic acid and α-tocopherol, whereas this process was strongly inhibited by resveratrol as effectively as the ascorbic acid/α-tocopherol mixture. Furthermore, antioxidants have a much stronger inhibitory effect on the peroxidation in the presence of 2-mercaptoethanol, and at the same time show protective properties of the double bond, decreasing the cis–trans isomerization. Under anaerobic conditions, cis–trans isomerization occurred and antioxidants efficiency increased along the series: resveratrol < α-tocopherol?<?ascorbic acid. This result is explained taking into account the double bond localization in the hydrophobic core of the micelle and the need of co-localization of the antioxidant in order to get an anti-isomerizing activity and protection of the natural lipid geometry.     

Inhibitory effect of flavonoids on low-density lipoprotein peroxidation catalyzed by mammalian 15-lipoxygenase

Lipoxygenase-dependent low-density lipoprotein (LDL) oxidation is believed to be involved in atherogenesis. Inhibition of lipoxygenase-induced lipid peroxidation might, therefore, be an important mode to suppress the development of atherosclerosis. Because dietary antioxidants inhibit LDL oxidation in vitro and their intake is inversely associated with coronary heart diseases, we compared the inhibitory effect of three typical flavonoids-quercetin, epicatechin, and flavone-with alpha-tocopherol and ascorbic acid against human LDL oxidation catalyzed by mammalian 15-lipoxygenase. The oxidative modification of LDL was monitored by measurement of cholesteryl ester hydroperoxide (CE-OOH) formation and consumption of antioxidants by using HLPC. Quercetin and epicatechin were the strongest inhibitors of LDL oxidation catalyzed by 15-lipoxygenase; ascorbic acid was an effective inhibitor in the first 3 h of oxidation; and fivefold alpha-tocopherol-enriched LDL showed a partial inhibition of CE-OOH formation only after 4-6 h of incubation. Flavone had no effect. Quercetin, ascorbic acid, and alpha-tocopherol were consumed in the first 3 h of incubation. Consumption of LDL alpha-tocopherol was partially inhibited by ascorbic acid and quercetin, whereas epicatechin and flavone were without effect. These results emphasize the inhibitory effect of the flavonoids quercetin and epicatechin on 15-lipoxygenase-mediated LDL lipid peroxidation. At similar concentrations, they are stronger antioxidants than ascorbic acid, alpha-tocopherol, and flavone.     

Inhibition of the cytotoxicity of protein toxins by a novel plant metabolite,Mansonone-D

We have studied the effect of several structurally related mansonones on the cytotoxicity of plant and bacterial toxins in Vero and BER-40, a brefeldin A-resistant mutant of Vero cells. Mansonone-D (MD), a sesquiterpenoid ortho-naphthoquinone, inhibited the cytotoxicity of ricin, modeccin, Pseudomonas toxin, and diphtheria toxin in Vero cells to different extents. The inhibition of ricin cytotoxicity was dose dependent and reversed upon removal of the drug. Protection of ricin cytotoxicity was also observed in the presence of cycloheximide, indicating that de novo protein synthesis is not required for the protective effect. Although MD inhibited the degradation and excretion of ricin, the binding and internalization of ricin was not affected. In contrast, MD strongly reduced the specific binding of diphtheria toxin in Vero cells. Fluorescence microscopic studies show that MD treatment dramatically alters the morphology of the Golgi apparatus in Vero cells. The kinetic studies reveal that the protection of ricin cytotoxicity is the consequence of decreased toxin translocation to the cytosol in MD-treated cells. The reactive ortho-quinone moiety of MD is important for the protective effect as thespesone, a para-naphthoquinone with a heterocyclic ring structure identical to that of MD, did not inhibit the cytotoxicity of toxins. Thespone, a dehydromansonone-D, lacking two hydrogens from the heterocyclic dihydrofuran ring of MD, inhibited the cytotoxicity of ricin, but was albeit less potent than MD. Neither mansonone-E nor mansonone-H with reactive ortho-quinone moiety, but with a different heterocyclic structure, had any effect on the cytotoxicity of ricin indicating that the protective effect of MD is specifically related to the overall structure of the metabolite. J. Cell. Physiol. 176:40–49, 1998. Published 1998 Wiley-Liss, Inc.

1 This article was prepared by a group of United States government employees and non-United States government employees, and as such is subject to 17 U.S.C. Sec. 105.