Lipid hydroperoxide induced oxidative stress damage and antioxidant enzyme response in Caco-2 human colon cells

It has been demonstrated that reactive oxygen species, free radicals, and oxidative products, such as lipid hydroperoxides, participate in tissue injuries and in the onset and progression of degenerative diseases in humans. Studies were conducted using Caco-2 colon carcinoma cells to evaluate cellular damage caused by exposing cells for 30 min to oleic acid hydroperoxides (OAHPx) at concentrations varying from 0 to 25 microM. Cell membrane damage and DNA damage were significantly high even at the lowest concentration of 2.5 microM OAHPx compared to the control. Cell lipid peroxidation, indicated by conjugated diene concentration, increased exponentially with increasing OAHPx concentration. Antioxidant mechanisms in Caco-2 cells were evaluated by measuring catalase, superoxide dismutase (SOD), and glutathione peroxidase (GPx) activities. Cellular catalase and GPx activities were not significantly different from each other at 0 to 25 microM OAHPx concentrations. SOD activity decreased with increasing OAHPx concentration. These results show that existing enzymatic antioxidant mechanisms are not sufficient for complete detoxification of 5-25 microM lipid hydroperoxides.     

Hydrogen peroxide induced oxidative stress damage and antioxidant enzyme response in Caco-2 human colon cells

Studies were conducted to evaluate the cell damage caused by exposing human colon carcinoma cells, Caco-2, to hydrogen peroxide at concentrations varying from 0 to 250 microM for 30 min. Evaluation of cell viability, as measured by trypan blue dye exclusion test, showed that the loss of viability was < 5% at concentrations up to 250 microM hydrogen peroxide. Cell membrane damage and DNA damage as measured by the leakage of lactate dehydrogenase and the comet assay, respectively, were significantly high at concentrations >100 microM hydrogen peroxide compared to those of the control. Antioxidant mechanisms in Caco-2 cells were evaluated by measuring catalase, superoxide dismutase, and glutathione peroxidase activities. Catalase activities remained constant in cells treated with 50-250 microM hydrogen peroxide. Superoxide dismutase activity decreased, whereas glutathione peroxidase activity increased in cells treated with H(2)O(2) concentrations of >50 microM. This study showed that with increasing hydrogen peroxide concentration, cell membrane leakage and DNA damage increased, whereas the three antioxidant enzymes responded differently, as shown by mathematical models.     

Modulation of oxidative damage due to salt stress using salicylic acid in Hordeum vulgare

Oxidative status of salicylic acid (SA) treated barley (Hordeum vulgare) plants grown under saline conditions was examined in a two-year field study during 2012–2013 and 2013–2014 growing seasons. Salinity caused a marked oxidative stress which was manifested as increased concentration of hydrogen peroxide (H₂O₂) and reduced grain yield of barley. Barley plants induced antioxidant system to tolerate salt stress, so that activities of four antioxidant enzymes: peroxidase, catalase, superoxide dismutase and glutathione reductase were enhanced by 22%, 28%, 57% and 44% in the first and by 33%, 20%, 45% and 68% in the second year, respectively. Foliar application of SA in concentration over 0.5 mM enhanced the activities of all four antioxidant enzymes and reduced H₂O₂ content and so enhanced yield. However, higher concentrations of SA reduced grain yield in salt stressed plants in both years, which could be attributed to the negative interaction of antioxidant enzymes with higher concentrations of SA as a non-enzymatic antioxidant. Furthermore, SA is in benzoic acid group and at very higher concentrations can act as an herbicide. It was concluded that SA modulated the oxidative stress through enhanced activities of antioxidant enzymes and reduced the salt-induced adverse effect thereby improving grain yield.     

Boron stress,oxidative damage and antioxidant protection in potato cultivars (Solanum tuberosum L.)

Six potato cultivars grown in Turkey in boron-prone areas and differing in their tolerance towards high boron were studied to reveal whether boron causes oxidative stress. To assess stress level, chlorophyll fluorescence and growth parameters were measured. Oxidative damage was assessed as malondialdehyde level, and antioxidant protection was evaluated as ascorbate (AA), dehydroascorbate, reduced glutathione (GSH) and oxidized glutathione amounts and superoxide dismutase, catalase, ascorbate peroxidase (APX) and glutathione reductase (GR) activities. High boron stress affected photosynthesis negatively in a threshold-dependent manner and inhibited growth. No pronounced changes in oxidation of lipids occurred in any cultivar. Activation of APX suggested the involvement of an ascorbic acid–reduced glutathione cycle in the protection against oxidative stress caused by high boron. Efficient work of this antioxidant system was probably hindered by boron complexation with NAD(P)⁺/NAD(P)H and resulted in the inhibition of GR and a decrease in AA and GSH. Hence, oxidative stress associated with high boron is a secondary component of boron toxicity which arises from metabolic changes caused by boron interference with major metabolites. Potato cultivars tolerate excess boron stress well and show damage only in very high boron concentrations. The potato cvs best suited for high boron soils/breeding purposes are cvs Van Gogh and Agria.

Abbreviations: AA: ascorbic acid; APX: ascorbate peroxidase; CAT: catalase; DHA: dehydroascorbic acid; DHAR: dehydroascorbate reductase; DTNB: 5; 5′-dithiobis-2-nitrobenzoic acid; DTT: dithiotreitol; F_v/F_m: photosynthetic efficiency at the dark-adapted state; GR: glutathione reductase; GSH: reduced glutathione; GSSG: oxidized glutathione; MDA: malondialdehyde; ROS: reactive oxygen species; SOD: superoxide dismutase; TCA: trichloroacetic acid     

Modulation of the ROS‐scavenging system in salt‐stressed wheat plants inoculated with arbuscular mycorrhizal fungi

Although there is evidence for a positive involvement of the antioxidant defense system in plant response to salt stress, there is poor information regarding the influence of mycorrhizal symbiosis on enzymatic and nonenzymatic antioxidant defense in wheat under saline conditions. The present article focuses on the contribution of mycorrhizae to antioxidant defense in salt‐stressed wheat plants. Two wheat (Triticum aestivum L.) cultivars, Sids 1 and Giza 168, were grown under nonsaline or two saline conditions (4.7 and 9.4 dS m^–1) with and without arbuscular mycorrhizal fungi (AMF) inoculation. Salt stress considerably decreased root colonization and plant productivity, particularly in Giza 168. Interestingly, mycorrhizal colonization alleviated the adverse effect of salt stress and significantly enhanced plant productivity, especially in Sids 1. The concentration of glycinebetaine, the activities of antioxidative enzymes (superoxide dismutase, peroxidase, catalase, and glutathione reductase) and the concentrations of antioxidant molecules (glutathione and ascorbate) were increased under saline conditions; these increases were more significant in salt‐stressed mycorrhizal plants, especially in Sids 1. Salt stress induced oxidative damage through increased lipid peroxidation, electrolyte leakage, and hydrogen peroxide concentration, particularly in Giza 168. Mycorrhizal colonization altered plant physiology and significantly reduced oxidative damage. Elimination of reactive oxygen species (ROS) can be one of the mechanisms how AMF improve wheat adaptation to saline soils and increase its productivity.     

Chemical characterization of the avenanthramide-rich extract from oat and its effect on D-galactose-induced oxidative stress in mice

The present study was to characterize the avenanthramide-rich extract (ARE) from oat bran and assess its effect on activity and gene expression of antioxidant enzymes in D-galactose-induced oxidative-stressed mice. High-performance liquid chromatography (HPLC) analysis found that ARE had 6.07% N-(3',4'-dihydroxycinnamoyl)-5-hydroxyanthranilic acid (Bc), 4.37% N-(4'-hydroxycinnamoyl)-5-hydroxyanthranilic acid (Bp), and 5.36% N-(4'-hydroxy-3'-methoxycinnamoyl)-5-hydroxyanthranilic acid (Bf). In addition, ARE was also rich in vanillic acid (0.60%), caffeic acid (0.50%), syringic acid (0.54%), p-coumaric acid (0.16%), ferulic acid (0.08%), and sinapic acid (0.03%). Administration of D-galactose markedly lowered not only the activity of superoxide dismutase (SOD) and glutathione peroxidase (GPx) but also the gene expression of manganese superoxide dismutase (SOD), copper-zinc SOD, glutathione peroxidase (GPx), and lipoprotein lipase (LPL) mRNA in mice. Administration of ARE significantly reversed the D-galactose-induced oxidative stress by increasing the activity of the antioxidant enzymes and upregulating their gene expression. This was accompanied by a significant decrease in the malondialdehyde (MDA) level in mice given ARE compared to the control. The results demonstrated that ARE possessed the antioxidant activity and was effective against D-galactose-induced oxidative stress.     

Toxic reactivity of wheat (Triticum aestivum) plants to herbicide isoproturon

The herbicide isoproturon is widely used for controlling weed/grass in agricultural practice. However, the side effect of isoproturon as contaminants on crops is unknown. In this study, we investigated isoproturon-induced oxidative stress in wheat ( Triticum aestivum). The plants were grown in soils with isoproturon at 0-20 mg/kg and showed negative biological responses. The growth of wheat seedlings with isoproturon was inhibited. Chlorophyll content significantly decreased at the low concentration of isoproturon (2 mg/kg), suggesting that chlorophyll was rather sensitive to isoproturon exposure. The level of thiobarbituric acid reactive substances (TBARS), an indicator of cellular peroxidation, showed an increase, indicating oxidative damage to plants. The isoproturon-induced oxidative stress resulted in a substantial change in activities of the majority of antioxidant enzymes, including superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX). Activities of the antioxidant enzymes showed a general increase at low isoproturon concentrations and a decrease at high isoproturon concentrations. Activities of CAT in leaves showed progressive suppression under the isoproturon exposure. Analysis of nondenaturing polyacrylamide gel electrophoresis (PAGE) confirmed these results. We also tested the activity of glutathione S-transferase (GST) and observed the activity stimulated by isoproturon at 2-10 mg/kg.     

Soy isoflavones protect the intestine from lipid hydroperoxide mediated oxidative damage

The effects of 24 h supplementation of human colon carcinoma cells (Caco-2) with isoflavones, genistein, and daidzein and their activities against oleic acid hydroperoxide mediated oxidative stress were investigated. Genistein, at 25, 50, and 100 microM, and daidzein, at 25 and 50 microM, did not induce cell injury to Caco-2 cells. Both compounds reduced cell injury and DNA damage mediated by 5 microM oleic acid hydroperoxides in Caco-2 cells. The effects of genistein and daidzein on antioxidant enzymes were dependent upon the compound and its concentration.     

Salicylic acid improves the antioxidant ability against arsenic-induced oxidative stress in sunflower (Helianthus annuus) seedling

The present study investigated the potential role of external salicylic acid (SA) in alleviating Arsenic (As) toxicity in sunflower leaves. The exposure of plants to 10 µM As inhibited biomass production and intensively increased the accumulation of As in both roots and leaves. The levels of some important parameters associated with oxidative stress, namely lipid peroxidation, electrolyte leakage, and hydrogen peroxide (H₂O₂) production were increased. SA application alleviated the negative effect of As on growth and led to decrease in oxidative injuries. Furthermore, SA application led to higher activity of catalase, ascorbate peroxidase, and glutathione peroxidase, and concomitantly decreased superoxide dismutase and guaiacol peroxidase activities. As important antioxidants, ascorbate and glutathione contents in sunflower leaves exposed to As were significantly decreased by SA treatment. These results reveal that SA is more effective in alleviating As toxicity at higher concentrations than that at lower concentrations.     

Excess nickel–induced changes in antioxidative processes in maize leaves

Maize (Zea mays L. cv. 777) plants grown in hydroponic culture were treated with 100 µM NiSO₄ (moderate nickel (Ni) excess). In addition to growth parameters, metabolic parameters representative of antioxidant responses in leaves were assessed 24 h and 3, 7, and 14 d after initiating the Ni treatment. Extent of oxidative damage was measured as accumulation of malondialdehyde and hydrogen peroxide in leaves 7 and 14 d after treatment initiation. Apart from increasing membrane‐lipid peroxidation and H₂O₂ accumulation, excess supply of Ni suppressed plant growth and dry mass of shoots but increased dry mass of roots and decreased the concentrations of chloroplastic pigments. Excess supply of Ni, though inhibited the catalase (EC 1.11.1.6) activity, increased peroxidase (EC 1.11.1.7), ascorbate peroxidase (EC 1.11.1.11), and superoxide dismutase (EC 1.15.1.1) activities. Localization of isoforms of these enzymes (peroxidase, ascorbate peroxidase, and superoxide dismutase) on native gels also revealed increases in the intensities of pre‐existing bands. Enhanced activities of peroxidase, ascorbate peroxidase, and superoxide dismutase, however, did not appear to be sufficient to ameliorate the effects of excessively generated reactive oxygen species due to excess supply of Ni.     

Characterization of two unknown compounds in methanol extracts of rosemary oil

In this study, two unknown compounds in rosemary oil, containing 3% carnosic acid and 0.3% carnosol, were identified and characterized. After methanol extraction, purification, and analysis by reversed-phase HPLC and LC-MS, a recovery of 92% (+/-8%) of carnosic acid was obtained, but no carnosol was found. However, two unknown compounds with a molecular weight of 330.2 and 302.2 were consistently detected. From additional LC-MS-MS, (1)H NMR, and elemental analyses, it became clear that the first compound (M(w) = 330.2) could not be carnosol. It was hypothesized that it originated from the breakdown of the intramolecular bond of carnosol, followed by the addition of a water molecule. Possibly, an unsaturated double bond was formed after dehydration. Assuming that this compound was an intermediate in the conversion to rosmanol, the second unknown compound (M(w) = 302.2) may have resulted from the breakdown of the intramolecular bond of rosmanol. Similarly, an unsaturated double bond may have been formed. After splitting off carbon oxide, a detectable molecule with a molecular weight of 302.2 was observed.     

Enhanced carnosic acid levels in two rosemary accessions exposed to cold stress conditions

Two rosemary accessions were subjected to chilling temperatures in control environmental cabins analyzing their variations in rosmarinic and carnosic acids together with their adaptability to these stress conditions. Cold stressed plants of both accessions showed increases in caffeic acid and carnosic acid concentration levels, while other secondary metabolites such as rosmarinic acid, naringin, cirsimaritin, hispidulin, and carnosol showed different responses in both accessions. In addition, cold stressed plants exhibited significant reductions in chlorophylls, beta-carotene, and violaxanthin levels as well as the maximum quantum yield of PSII in both accessions. Hydrogen peroxide and lipid peroxidation levels showed similar responses in both accessions, which were positively and negatively correlated with rosmarinic and carnosic acids. From these results it is therefore suggested that carnosic acid biosynthesis in rosemary plants is induced by chilling periods. On the other hand, we demonstrate that not all rosemary accessions are equally well adapted to chilling temperatures. In fact, for (one) accession cold treated plants severe losses in chlorophyll, beta-carotene, and even xanthophylls (including zeaxanthin and antheraxanthin) were observed, despite no visual symptoms of leaf injury. More research is needed to understand rosmarinic acid variations in rosemary plants under stress conditions.     

Effect of salicylic acid and salt on wheat seed germination

Abstract

The effects of pretreatment with salicylic acid on wheat seed germination (Triticum aestivum L. cv. Roshan), lipid peroxidation, and superoxide dismutase, catalase, polyphenol oxidase, and peroxidase activity were studied under conditions of salt stress. Seeds treated with different concentrations of salicylic acid were used for measuring germination traits. Salt stress was induced by sodium chloride solution. Seeds were soaked in salicylic acid solution for 24 h, dried with sterile paper, transferred to sterile Petri dishes, and treated with 10 ml NaCl solution at different concentrations. After 1 week, the number of germinated seeds, root length, seedling length, and dry weight were recorded. Antioxidant enzyme activity and lipid peroxidation were also assayed. Salinity decreased seed germination. Thus, a high concentration of NaCl (200 mM) decreased germination by 17.6% compared with control treatment. Salicylic acid significantly increased germination in stressed and control seeds. Salicylic acid increased the level of cell division of seedlings and roots, which increased plant growth. Salt stress significantly increased the activity of the antioxidative enzymes catalase, superoxide dismutase, peroxidase, and polyphenol oxidase in wheat seedlings, and salicylic acid reduced the activity of antioxidant enzymes as stress signal molecules. Our results indicated that scavenging of reactive oxygen species was effective, especially by salicylic acid, and that membrane damage was limited. The aim of the present work was to study the character of changes in enzymatic systems induced by NaCl and salicylic acid in wheat seedlings under conditions of salt stress. In brief, salicylic acid treatment reduced the damaging action of salinity on embryo growth and accelerated a restoration of growth processes; thereupon it may be effective for the improvement of seed germination in arid and semi-arid regions.     

Variation of Antioxidant Activity in Dreissena polymorpha Specimens Exposed to 2,2′,4,4′,5,6′-Hexa BDE (BDE-154)

We evaluated the imbalance of the oxidative status in zebra mussel (Dreissena polymorpha) specimens exposed for 96?h to environmentally relevant concentrations (0.1, 0.5, and 1???g/L) of the 2,2??,4,4??,5,6??-hexa BDE (BDE-154). The activities of three antioxidant enzymes, catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), and the phase II detoxifying enzyme glutathione S-transferase (GST), were measured in the cytosolic fraction from a pool of zebra mussels. Significant variations in the activity of each single enzyme were noticed at each treatment, indicating that exposure to BDE-154 was able to impair the oxidative status of treated bivalves through the increase of reactive oxygen species. In detail, SOD and GPx were significantly induced, while CAT and GST were depressed with respect to the baseline levels. These data have confirmed that the raise of oxidative stress is the main cause of the BDE-154-induced genetic damage observed in a previous study on the zebra mussel.     

Active oxygen detoxifying enzymes and phenylalanine ammonia-lyase in the ethylene-induced chilling tolerance in citrus fruit

The effects of applying ethylene (2 microL x L(-)(1)) during cold storage of Fortune mandarins on the development of chilling-induced peel damage and on changes in the activities of the enzymes of the antioxidant system, superoxide dismutase, catalase (CAT), ascorbate peroxidase, guaiacol peroxidase, and glutathione reductase, and on phenylalanine ammonia-lyase (PAL) have been investigated. Chilling damage was reduced by applying ethylene during fruit storage at 1.5 degrees C. PAL activity increased in response to cold stress and was higher in fruit held under ethylene than under air during the whole storage period, whereas CAT was temporarily higher in ethylene-treated fruit. In contrast, the activities of the other enzymes were not increased by ethylene. The global results suggest that the ethylene-induced chilling tolerance in Fortune mandarins might be due to increased PAL and CAT activities.     

Melatonin alleviates cold‐induced oxidative damage in maize seedlings by up‐regulating mineral elements and enhancing antioxidant activity

Melatonin, known as an animal hormone and an antioxidant with a low molecular weight, is one of the most commonly used substances to improve plant resistance against various environmental stresses. However, there are no studies explaining the effects of melatonin on the relationship between defense system and mineral composition of plants under stressed and unstressed‐conditions. The present study was conducted to investigate whether the mitigating effect of melatonin is associated with its modulating influence on the mineral elements of cold‐stressed maize seedlings. The seedlings were treated with melatonin (1 mM) and cold stress (10/7°C) for 3 d separately and in combination. After 3 d, the seedlings were harvested to determine several physiological, biochemical, and molecular parameters. Melatonin application effectively mitigated the damages from cold stress, as demonstrated by higher relative water concentration, chlorophyll concentration and antioxidant enzyme activities (superoxide dismutase, guaiacol peroxidase, catalase, ascorbate peroxidase, and glutathione reductase), as well as lower superoxide, hydrogen peroxide, and malondialdehyde concentrations. Similarly, melatonin significantly ameliorated cold‐induced reductions in the concentrations of potassium, phosphorus, sulfur, magnesium, iron, copper, manganese, and zinc. Besides, it further increased calcium and boron concentrations compared to cold stress alone. Our results reveal that melatonin has an important modulating influence on the mineral element composition of plants and mitigates cold stress through up‐regulation of these elements and simultaneously enhanced antioxidant activity.     

Toxicological effects of microcystin-LR on earthworm (<Emphasis Type="Italic">Eisenia fetida</Emphasis>) in soil

Microcystin-LR (MC-LR) is a cyclic heptapeptide toxin produced by cyanobacteria in eutrophic water. It can be transferred into soil–crop systems via irrigation and cyanobacterial paste fertilization. No studies have examined the potential toxicity of MC-LR to soil animals. Therefore, in the present study, the toxicological effects of MC-LR on earthworm (Eisenia fetida), including survival, growth, reproduction, oxidative stress, and cell viability, were investigated. The LC₅₀ of MC-LR was 0.149 μg cm^?2 at 72 h based on a filter paper test and 0.460 mg kg^?1 at 14 days based on an acute soil test. MC-LR seriously affected the reproduction of earthworms. Based on hatchability, the EC₅₀ of MC-LR was 0.268 mg kg^?1, similar to environmentally relevant concentrations of microcystins. The changes in activities of superoxide dismutase, guaiacol peroxidase, catalase, and glutathione peroxidase, together with the levels of glutathione and malondialdehyde, indicated that oxidative damage and lipid peroxidation played significant roles in MC-LR toxicity. In addition, the toxicity of MC-LR in earthworms increased despite degradation of MC-LR in soil over time, possibly due to the formation of toxic metabolites of MC-LR or the bioaccumulation of MC-LR in earthworms. A reduction in the neutral red retention time along with an increase in coelomocyte apoptosis with increasing MC-LR concentrations indicated a severe damage to viability. These results suggest that environmentally relevant MC-LR concentrations in agricultural soil may cause reproductive, biochemical, and cellular toxicity to Eisenia fetida. This information can be used in ecological risk assessments on MC-LR in soil.     

Identification and antioxidant activity of novel chlorogenic acid derivatives from bamboo (Phyllostachys edulis)

One known and two novel antioxidant compounds have been isolated from bamboo (Phyllostachys edulis). The butanol-soluble extract of the bamboo leaves was found to have a significant antioxidant activity, as measured by scavenging the stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical and the superoxide anion radical (O(2)(-)) in the xanthine/xanthine oxidase assay system. Antioxidant activity-directed fractionation of the extract led to the isolation and characterization of three structural isomeric chlorogenic acid derivatives: 3-O-(3'-methylcaffeoyl)quinic acid (1), 5-O-caffeoyl-4-methylquinic acid (2), and 3-O-caffeoyl-1-methylquinic acid (3). Compounds 2 and 3 were isolated and characterized for the first time from the natural products. In the DPPH scavenging assay as well as in the iron-induced rat microsomal lipid peroxidation system, compounds 2 (IC(50) = 8.8 and 19.2 microM) and 3 (IC(50) = 6.9 and 14.6 microM) showed approximately 2-4 times higher antioxidant activity than did chlorogenic acid (IC(50) = 12.3 and 28.3 microM) and other related hydroxycinnamates such as caffeic acid (IC(50) =13.7 and 25.5 microM) and ferulic acid (IC(50) = 36.5 and 56.9 microM). Among the three compounds, compound 1 yielded the weakest antioxidant activity, and the DPPH scavenging and lipid peroxidation inhibitory activity (IC(50) = 16.0 and 29.8 microM) was lower than those of chlorogenic and caffeic acids. All three compounds exhibited both superoxide scavenging activities and inhibitory effects on xanthine oxidase. Their superoxide anion (O(2)(-)) scavenging activities (IC(50) = 1, 4.3 microM; 2, 2.8 microM; and 3, 1.2 microM) were markedly stronger than those of ascorbic acid (IC(50) = 56.0 microM), alpha-tocopherol (IC(50) > 100 microM), and other test compounds, although their inhibition effects on xanthine oxidase may contribute to the potent scavenging activity. alpha-Tocopherol exerted a significant inhibitory effect (65.5% of the control) on superoxide generation in 12-O-tetradecanoylphorbol-13-acetate-induced human promyelocytic leukemia HL-60 cells, and compound 3 showed moderate activity (36.0%). On the other hand, other compounds including 1, 2, chlorogenic acid, and other antioxidants were weakly active (24.8-10.1%) in the suppression of superoxide generation.     

Neuroprotective effects of the citrus flavanones against H2O2-induced cytotoxicity in PC12 cells

The citrus flavanones hesperidin, hesperetin, and neohesperidin are known to exhibit antioxidant activities and could traverse the blood-brain barrier. H2O2 formation induces cellular oxidative stress associated with neurodegenerative diseases. In this study, protective effects of pretreatments (6 h) with hesperidin, hesperetin, and neohesperidin (0.8, 4, 20, and 50 microM) on H2O2-induced (400 microM, 16 h) neurotoxicity in PC12 cells were evaluated. The results showed that hesperetin, hesperidin, and neohesperidin, at all test concentrations, significantly ( p < 0.05) inhibited the decrease of cell viability (MTT reduction), prevented membrane damage (LDH release), scavenged ROS formation, increased catalase activity, and attenuated the elevation of intracellular free Ca2+, the decrease of mitochondrial membrane potential (except those of 0.8 microM neohesperidin-treated cells) and the increase of caspase-3 activity in H2O2-induced PC12 cells. Meanwhile, hesperidin and hesperetin attenuated decreases of glutathione peroxidase and glutathione reductase activities and decreased DNA damage in H2O2-induced PC12 cells. These results first demonstrate that the citrus flavanones hesperidin, hesperetin, and neohesperidin, even at physiological concentrations, have neuroprotective effects against H2O2-induced cytotoxicity in PC12 cells. These dietary antioxidants are potential candidates for use in the intervention for neurodegenerative diseases.     

Morphology and physiology of zinc‐stressed mulberry plants

The aim of this study was to induce symptoms of zinc deficiency and Zn excess and to relate the generation of reactive oxygen species (ROS) and the altered cellular redox environment to the effects of Zn stress in mulberry (Morus alba L.) cv. Kanva‐2 plants. The antioxidative responses of Zn‐stressed mulberry plants were studied by determining malondialdehyde content (MDA, a measure of lipid peroxidation) as indicator of oxidative damage and the ratio of dehydroascorbate (DHA) to ascorbic acid (AsA) as an index of the cellular redox state. The Zn‐deficiency effects appeared as faint paling and upward cupping of the young emerging leaves. The paling intensified with time, and affected leaves finally developed necrotic spots. At advanced stage of Zn deficiency, newly emerged leaves were spindle‐shaped, pale, and small in size. Apart from their stunted appearance, the plants supplied excess Zn did not show any specific visible symptom. Leaf water status of mulberry plant was affected in Zn‐stressed plants. Deficient leaves had decreased water potential (Ψ) and specific water content (SWC), contained less tissue Zn, less chloroplastic pigments, and high tissue Fe and Mn concentrations. However, excess supply of Zn was found to increase Ψ and decrease tissue Fe. Both hydrogen peroxide and MDA accumulated in leaves of Zn‐stressed plants. While the concentration of DHA did not vary in Zn‐deficient leaves, it was increased in leaves of plants supplied excess Zn. The ratio of the redox couple (DHA to AsA) was increased both in Zn‐deficient or Zn‐excess plants. The activities of superoxide dismutase (EC 1.15.1.1), catalase (EC 1.11.1.6), peroxidase (EC 1.11.1.7), and ascorbate peroxidase (EC 1.11.1.11) increased in Zn‐stressed plants. The results suggest that deficiency or excess of Zn aggravates oxidative stress through enhanced generation of ROS and a disturbed redox homeostasis in mulberry plants.