Protective effect of whey protein hydrolysates on H2O2-induced PC12 cells oxidative stress via a mitochondria-mediated pathway

Whey protein hydrolysates (WPHs) were prepared with pepsin and trypsin. A PC12 cell model was built to observe the protective effect of WPHs against H₂O₂-induced oxidative stress. The results indicated that WPHs reduced apoptosis by 14% and increased antioxidant enzyme activities. Flow cytometry was used to assess the accumulation of reactive oxygen species (ROS), Ca²⁺ levels and the mitochondrial membrane potential (MMP). The results showed that WPHs suppressed ROS elevation and Ca²⁺ levels and stabilised MMP by 16%. The anti-apoptosis/pro-apoptosis proteins Bcl-2/Bax and poly (ADP-ribose) polymerase (PARP) were investigated by Western-blot analysis, which indicated that WPHs increased the expression of Bcl-2 while inhibiting the expression of Bax and the degradation of PARP. WPHs also blocked Caspase-3 activation by 62%. The results demonstrate that WPHs can significantly protect PC12 cells against oxidative stress via a mitochondria-mediated pathway. These findings indicate the potential benefits of WPHs as valuable food antioxidative additives.     

Caseinophosphopeptides exert partial and site-specific cytoprotection against H2O2-induced oxidative stress in Caco-2 cells

Caseinophosphopeptides can sequester prooxidant metals and scavenge free radicals, and may thus be used as functional food ingredients. The total antioxidant capacity (TEAC and ORAC) of two pools of caseinophosphopeptides (1–3 mg/ml), obtained from casein subjected to simulated gastrointestinal digestion (at two different pH values) and selective precipitation, was evaluated to determine dose–response activity. Pool B (which showed the highest antioxidant capacity due to the presence of more antioxidant amino acids) was used to test its cytoprotective effect against H₂O₂-induced oxidative stress in Caco-2 cells. Caseinophosphopeptides protected the cells against oxidative damage by preserving cell viability, increasing GSH content, inducing catalase enzyme activity, diminishing lipid peroxidation and maintaining a correct cell cycle progression. However, they failed to exert protection at a mitochondrial level (ROS and mitochondrial membrane potential), implying a partial and site-specific effect. Thus, their mechanism of action is not only related to free radical scavenging activity, but also to metal chelation and the modulation of intracellular signaling cascades.     

Free radical scavenging activity and comparative proteomic analysis of antioxidative protein against H2O2-induced oxidative stress in neuronal cells

Artemisia annua was enzymatically hydrolyzed by five proteases and seven carbohydrases. All enzymatic extracts scavenged DPPH, hydroxyl and alkyl radicals. Especially, the Protamex among the various proteases and Maltogenase among the various carbohydrases extracts exhibited the highest scavenging activity on hydroxyl radical. The extracts of A. annua clearly reduced neuronal cell death from H₂O₂-induced damage. In addition, a proteomic analysis, two-dimensional electrophoresis (2-DE) and matrix assisted laser desorption ionisation-time of flight/time of flight (MALDI-TOF/TOF) was used to identify the proteins of the neuronal cells whose expressions were or were not altered by the treatment of the Maltogenase extracts which showed the highest hydroxyl radical scavenging activity among all enzymatic extracts for 24 h. The protein characterisation revealed that translation elongation factor Tu (EF-Tu), Immunoglobulin E (IgE) and voltage-dependent anion channel 1 (VDAC-1) were involved in the cell survival effects against H₂O₂-induced apoptosis. These results suggest that EF-Tu, IgE and VDAC-1 have an important role in the reduction of neuronal apoptosis by oxidative stress, and the enzymatic extracts of A. annua shows potent antioxidative activities by regulating EF-Tu, IgE and VDAC-1.     

Protective effect of Centella asiatica extract and powder on oxidative stress in rats

The effect of Centella asiatica extract and powder in reducing oxidative stress in SpraqueDawley rats was evaluated. Lipid peroxidation was monitored by measuring malonaldehyde (MDA) level in blood. Activities of free radical-scavenging enzymes (superoxide dismutase and catalase) were determined using H₂O₂ decomposition and nitrobluetetrazolium reduction, respectively. Results showed that administration of H₂O₂ (0.1%) in drinking water of the rats, for 25 weeks, increased the malonaldehyde levels in erythrocytes of all the rats. However, rats receiving C. asiatica extract, powder and α-tocopherol had lower MDA levels than did the other rats, which indicates, decrease lipid peroxidation in these rats. Increase in catalase activity of the rats appears to be a response to H₂O₂ accumulation. The decrease in the activity of superoxide dismutase in C. asiatica- and α-tocopherol supplemented rats suggested a lower requirement for the enzyme and this indicates the protective effect of the plant in combating oxidative stress undergone by the rats. Results revealed that C. asiatica extract and powder may ameliorate H₂O₂-induced oxidative stress by decreasing lipid peroxidation via alteration of the antioxidant defence system of the rats.     

Protective effects of the crude extracts from yam (Dioscorea alata) peel on tert-butylhydroperoxide-induced oxidative stress in mouse liver cells

Researchers have shown that yam extracts contain antioxidative activity; however, there are few reports regarding the antioxidant activities of yam peel. The effects of water and 50% ethanolic extracts from Darsan yam (Dioscorea alata) peel on the oxidative status of tert-butylhydroperoxide (t-BHP)-treated mouse Hepa 1–6 and FL83B liver cell lines were investigated. The cytosols were analysed for H₂O₂ and malondialdehyde (MDA) levels and antioxidative enzymes activities, including superoxide dismutase, glutathione peroxidase (GPx) and catalase activities. Both water and 50% ethanolic extracts from yam peel did not affect cellular MDA level in t-BHP-treated cells, but they altered the level of H₂O₂. Water extract from yam peel amplified the t-BHP-induced cytotoxicity in Hepa 1–6 whilst the ethanolic extract showed protection in FL83B cells. GPx activity might play an important role in the protective effect associated with t-BHP-induced oxidative stress.     

Cytoprotective effect of fucoxanthin isolated from brown algae <Emphasis Type="Italic">Sargassum siliquastrum</Emphasis> against H<Subscript>2</Subscript>O<Subscript>2</Subscript>-induced cell damage

In this study, the cytoprotective effect of fucoxanthin, which was isolated from Sargassum siliquastrum, against oxidative stress induced DNA damage was investigated. Fucoxanthin, a kind of carotenoid, was pretreated to the medium and the protective effect was evaluated via 2′,7′-dichlorodihydrofluorescein diacetate, 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide, and comet assays. Intracellular reactive oxygen species were over produced by addition of hydrogen peroxide (H₂O₂), but the production was significantly reduced by the treatment with fucoxanthin. The fucoxanthin strongly enhanced cell viability against H₂O₂ induced oxidative damage and the inhibitory effect of cell damage was a dose-dependent manner. Furthermore, a protective effect against oxidative stress-induced cell apoptosis was also demonstrated via nuclear staining with Hoechst dye. These results clearly indicate that fucoxanthin isolated from S. siliquastrum possesses prominent antioxidant activity against H₂O₂-mediated cell damage and which might be a potential therapeutic agent for treating or preventing several diseases implicated with oxidative stress.     

Molecular mechanism underlying anti-apoptotic and anti-inflammatory effects of Mamao (Antidesma thwaitesianum Müll. Arg.) polyphenolics in human breast epithelial cells

There has been increasing interest in finding natural antioxidants to prevent free radical damage and retard the progress of chronic inflammatory diseases. Our previous data demonstrated the strong antioxidant properties of polyphenolics in Mamao seed (MS) and Mamao marc (MM) extracts. In this study we further investigated the effect of MS and MM polyphenolics on hydrogen peroxide (H₂O₂)-induced apoptosis and tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation, using human breast epithelial (MCF10A) cells. MS and MM extracts conferred dose-dependent protection against H₂O₂-induced apoptosis by inhibiting PARP/caspase-3 cleavage, inducing anti-apoptotic Bcl-2 expression, and down-regulating pro-apoptotic Bax. Moreover, MS and MM polyphenolics inhibited TPA-induced COX-2 and NF-κB activation by blocking the degradation of cytoplasmic IκBα, as well as subsequent nuclear translocation of p65 and attenuation of the activation of ERK, but not JNK and p38. These data establish the molecular mechanism for the anti-apoptotic and anti-inflammatory effects of MS and MM polyphenolics.     

Cytoprotective activity of extract of roasted coffee residue on mouse embryonic fibroblasts cells against apoptosis induced by oxidative stress

This study was conducted to evaluate the cytoprotective activity of roasted coffee residues (RCRs) extract on mouse embryonic fibroblast (MEF) cells. RCRs originated from Colombia and Honduras are relatively nontoxic to cell growth and even stimulate cell proliferation. Colombian RCRs showed most efficient protective effects on MEF cells against oxidative damage induced by H₂O₂ compared among the extracts prepared under the same roasting time. The most significant radical scavenging activity was measured in RCR with roasting time of 8.5 min. Phenolic and nonphenolic compounds in RCRs were chlorogenic acid, caffeine, caffeic acid, nicotinic acid, trigonelline, and 5-(hydroxymethyl)furfuralolehyde. Effect of Colombian RCRs on apoptosis occurred by oxidative damage was evaluated by morphological and flow cytometric analysis. Apoptotic cell accumulation was decreased by cotreatment of MEF cells with Colombian RCRs. These results suggested that antioxidant potency of RCRs suppresses the cytotoxicity which is induced by H₂O₂ and has a protective effect on MEF cell against oxidative stress.     

Curcumin protects mouse neuroblastoma Neuro-2A cells against hydrogen-peroxide-induced oxidative stress

Curcumin has been traditionally used in China and India for food and medicinal purposes. It has been shown to possess potent antioxidative activity both in vitro and in vivo. In the present study, the neuroprotective effects and the potential mechanisms of curcumin against H₂O₂-induced oxidative stress in mouse neuroblastoma Neuro-2A cells were investigated. Treatment with curcumin at 20 and 25 μg/mL for 1 h prior to H₂O₂ exposure significantly attenuated cell viability loss, reduced apoptosis, suppressed the elevation of intracellular reactive oxygen species (ROS) and calcium levels, and stabilised mitochondrial membrane potential. Furthermore, curcumin could block H₂O₂-mediated degradation of the protein IκBα and subsequent activation of nuclear factor κB, thus inhibiting the expression of its target gene cyclooxygenase 2. These results indicate that curcumin has potential protective effects against H₂O₂-induced oxidative stress in neuron cells, which might make curcumin a suitable therapeutic agent for prevention and treatment of neurodegenerative diseases associated with oxidative stress.     

Evidence for protective effects of coffees on oxidative stressinduced apoptosis through antioxidant capacity of phenolics

This study evaluated total phenolics, total flavonoids, and antioxidant capacity of randomly selected regular and decaffeinated coffees commercially available in Korea and their protective effects in human hepatic epithelial HepG2 cell line against oxidative stress. All coffees tested exhibited potent antioxidant capacity in chemical systems and, consequently, significant protection of cells from oxidative stress in vitro in a dose-dependent manner. In particular, H₂O₂-induced apoptosis as evaluated by annexin V staining and flow cytometry was prevented by coffee extracts, resulting in the enhanced cell viability. Of interest, the content of total phenolics and flavonoids in coffees demonstrated a positive correlation with antioxidant capacity, indicating that the antioxidant capacity of coffees may be attributed to those phytochemicals. In accordance with previous studies, caffeoylquinic acid (CQA) and its derivatives including 3-CQA, 4-CQA, 5-CQA, 3,4-diCQA, 3,5-diCQA, and 4,5-diCQA were identified as phenolic phytochemicals by a reversed-phase HPLC, with 5-CQA being a major component. Taken together, the present study demonstrated protective effects of regular and decaffeinated coffees on cells in vitro against overwhelming oxidative stress due to richness in phenolics, especially CQA and its derivatives. Coffees, regular or decaffeinated, may serve as a good source of health-beneficial phytochemicals in diet.     

Comparison between conjugated linoleic acid and essential fatty acids in preventing oxidative stress in bovine mammary epithelial cells

Some in vitro and in vivo studies have demonstrated protective effects of conjugated linoleic acid (CLA) isomers against oxidative stress and lipid peroxidation. However, only a few and conflicting studies have been conducted showing the antioxidant potential of essential fatty acids. The objectives of the study were to compare the effects of CLA to other essential fatty acids on the thiol redox status of bovine mammary epithelia cells (BME-UV1) and their protective role against oxidative damage on the mammary gland by an in vitro study. The BME-UV1 cells were treated with complete medium containing 50 μM of cis-9,trans-11 CLA, trans-10,cis-12 CLA, α-linolenic acid, γ-linolenic acid, and linoleic acid. To assess the cellular antioxidant response, glutathione, NADPH, and γ-glutamyl-cysteine ligase activity were measured 48 h after addition of fatty acids (FA). Intracellular reactive oxygen species and malondialdehyde production were also assessed in cells supplemented with FA. Reactive oxygen species production after 3 h of H₂O₂ exposure was assessed to evaluate and to compare the potential protection of different FA against H₂O₂-induced oxidative stress. All FA treatments induced an intracellular GSH increase, matched by high concentrations of NADPH and an increase of γ-glutamyl-cysteine ligase activity. Cells supplemented with FA showed a reduction in intracellular malondialdehyde levels. In particular, CLA isomers and linoleic acid supplementation showed a better antioxidant cellular response against oxidative damage induced by H₂O₂ compared with other FA.     

Unraveling a mechanism of honey antibacterial action: Polyphenol/H2O2-induced oxidative effect on bacterial cell growth and on DNA degradation

Several compounds with antibacterial activities were identified in honey however, a mechanism by which they lead to bacterial growth inhibition and bacterial death remains still unknown. We recently found that honeys possess DNA degrading activity mediated by honey hydrogen peroxide and an unknown honey component(s). Here we provide evidence that active honeys (MIC₉₀ of 6.25–12.5% v/v) possessed significantly higher levels of phenolics (p < 0.02) of higher radical scavenging activities (p < 0.005) than honeys of average activity. Removal of H₂O₂ by catalase eliminated bacteriostatic activities caused by both phenolics and H₂O₂ suggesting that the growth inhibition resulted from the coupling chemistry between these compounds. Both phenolics and H₂O₂ were involved in DNA degradation by honeys. Treatment of plasmid DNA with H₂O₂ alone did not affect the DNA integrity but H₂O₂ removal from honey by catalase prevented DNA degradation. Polyphenols extracted from honeys degraded plasmid DNA in the presence of H₂O₂ and Cu(II) in the Fenton-type reaction. The extent of DNA degradation was inversely related to the polyphenol concentration in this system as well as in honeys. At low content, honey polyphenols exerted pro-oxidant activity damaging to DNA. In conclusion, honey phenolics with pro-oxidant activities were necessary intermediates that conferred oxidative action of H₂O₂. Phenolic/H₂O₂-induced oxidative stress constituted the mechanism of honey bacteriostatic and DNA damaging activities.     

Effect of polyphenolic compounds from Coriandrum sativum on H2O2-induced oxidative stress in human lymphocytes

Polyphenolic compounds are widely distributed in plants and known to be excellent antioxidants in vitro. They have the capacity to reduce free-radical formation by scavenging free radicals and protecting antioxidant defences. The present study evaluated the antioxidant potencies of polyphenolic compounds from a spice, Coriandrum sativum against hydrogen peroxide-induced oxidative damage in human lymphocytes. Pretreatment with polyphenolic rich fractions protected human lymphocytes against H₂O₂-induced oxidative damage. H₂O₂ treatment significantly decreased the activities of antioxidant enzymes, such as superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, glutathione-S-transferase and caused decreased glutathione content and increased thiobarbituric acid-reacting substances (TBARS). Treatment with polyphenolic fractions (50 μg/ml) increased the activities of antioxidant enzymes and glutathione content and reduced the levels of TBARS significantly. Observed reduction in the level of lipid peroxides showed a decreased tendency of peroxidative damage. We conclude that, under these experimental conditions, polyphenolic compounds effectively suppress hydrogen peroxide-induced oxidative stress.     

Essential rosemary oil protects testicular cells against DNA-damaging effects of H2O2 and DMNQ

Rosemary oil (RO) is popular in the Mediterranean region as a culinary additive which has the ability to protect delicate organs such as liver, brain and heart. We examined the effect of RO consumption on resistance of rat testicular cells (TCs) against DNA-damaging effects of the oxidative agents H₂O₂ and DMNQ and on the activity of the antioxidant enzymes glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD). DNA lesions were detected by conventional and modified comet assay and the activities of GSH-Px and SOD were measured spectrophotometrically. Since TCs represent a mixture of haploid, diploid and tetraploid cells, we used flow cytometry for their differentiation and calculation of DNA-damaging effects of H₂O₂ and DMNQ in cells of different ploidy. The results showed that the oxidative DNA lesions were significantly reduced in TCs from rats administered RO; however, the activity of antioxidant enzymes did not differ in TCs from control and RO-supplemented rats.     

ANTIOXIDANT ENZYME RESPONSE STUDIES IN H2O2-STRESSED PORCINE MUSCLE TISSUE FOLLOWING TREATMENT WITH FAVA BEAN SPROUT EXTRACT AND L-DOPA

Readily absorbed fava bean sprout extract (FSE), rich in levo‐dihydroxy phenylalanine (L‐DOPA) and a high total phenolic content, as well as synthetic L‐DOPA countered oxidative stress caused by H₂O₂ through stimulation of an antioxidant enzyme response in porcine muscle tissue. Evidence suggests that the phenolic treatments such as FSE had a protective effect on the stressed porcine muscle by maintaining the cellular redox homeostasis, through modulation of the cellular antioxidant enzymes such as superoxide dismutase, catalase and total peroxidase via enhanced pentose phosphate pathway (PPP) through stimulation of glucose‐6‐phosphate dehydrogenase (G6PDH) resulting in higher NADPH (reduced nicotinamide adenine dinucleotide phosphate). A marked increase in proline content was observed in the case of FSE and L‐DOPA treatments both under stressed and unstressed conditions indicating an interface to proline‐linked PPP. The major implication from this study is the evidence that FSE and L‐DOPA have direct roles as quenchers of free radicals and also as potential modulators of the endogenous cellular antioxidant enzyme response with L‐DOPA in a natural fava bean background being more protective and stable.     

Antioxidant effect derived from bioaccessible fractions of fruit beverages against H2O2-induced oxidative stress in Caco-2 cells

This work evaluates the effect of bioaccessible fractions from fruit beverages against oxidative stress (OS) in Caco-2 cells. A fruit beverage (grape + orange + apricot) (with/without milk and/or iron/zinc) was subjected to in vitro gastrointestinal digestion, and bioaccessible fractions were incubated with Caco-2 cell cultures. Following preincubation, OS was induced with 5 mM H₂O₂. Intracellular reactive oxygen species (ROS), mitochondrial potential (Δψ_m), mitochondrial metabolism (MTT test), intracellular reduced glutathione (GSH) and superoxide dismutase activity (SOD) were measured. The data evidenced viable cultures with increased mitochondrial metabolism and GSH-Rd activities, without alteration in SOD activity. Accordingly, more preserved mitochondrial integrity was also evidenced, allowing the action of antioxidant systems in preincubated cultures. Based on these data, we can conclude that a cytoprotective effect is derived from bioaccessible fractions of fruit beverages, though this effect failed to prevent intracellular ROS accumulation in Caco-2 cell cultures exposed to 5 mM H₂O₂.     

In vitro antioxidant synergism and antagonism between food extracts can lead to similar activities in H2O2‐induced cell death,caspase‐3 and MMP‐2 activities in H9c2 cells

BACKGROUND: The cardio‐health‐promoting activity of some foods may be due to their specific antioxidant content. The antioxidant activity of a mixture of plant extracts has been shown to differ from the activity of the individual extracts. As a result, the activity of the mixture can be described as synergistic, antagonistic or additive. This in vitro study evaluated the relationship between the in vitro antioxidant capacity of mixtures and their bioactivity when cardiomyocytes (H9c2) were challenged with H₂O₂. RESULTS: A mixture of raspberry and adzuki bean extracts produced a synergistic response and a mixture of broccoli and soybean extracts produced an antagonistic response in chemical‐based antioxidant assays. When these extracts were tested in cell cultures, individually and in mixtures, the mixture of raspberry and adzuki bean protected the cardiomyocytes from H₂O₂‐induced cell damage significantly better than the individual extracts. Conversely, the mixture of broccoli and soybean extracts was less effective in protecting H9c2 cells. The synergistic and antagonistic effects of the mixtures in protecting cell damage were brought about by enhanced or reduced ability in attenuating caspase‐3 and matrix metalloproteinase‐2 activities elevated by H₂O₂. CONCLUSION: Food mixtures with synergistic antioxidant activity and protective property against reactive oxygen species‐induced cell death can potentially be incorporated into novel functional foods or beverages with optimum health benefit. The antagonistic effect of food mixtures can be a health concern and thus should be avoided. Copyright © 2012 Society of Chemical Industry     

Antioxidant activity of Fragilariopsis pseudonana and protective effect against hydrogen peroxide-induced inhibition of gap junctional intercellular communication

This study was aimed to evaluate antioxidant activities of a methanol extract from the polar microalgae Fragilariopsis pseudonana (FP) and to investigate the protective effect for gap junctional intercellular communication (GJIC) in WB-F344 normal rat liver epithelial cells. The results indicated that the methanol extract of FP (FPM) scavenged ABTS free radicals and inhibited copper-induced low density lipoprotein oxidation in a dose-dependent manner. Exposing HepG2 cells to FeSO₄ increased intracellular reactive oxygen species formation and lipid peroxides, which was significantly reduced by treatment with FPM. Furthermore, FPM increased the activities of superoxide dismutase and glutathione peroxidase in cells. FPM recovered the hydrogen peroxide (H₂O₂)-induced inhibition of GJIC and attenuated the phosphorylation of connexin 43 (Cx43) and extracellular signal-regulated kinase1/2 (ERK1/2). Taken together, these results demonstrated that FPM has antioxidant activity and protects against H₂O₂-induced inhibition of GJIC by blocking phosphorylation of Cx43 via activation of ERK1/2.