HPLC–ESI-MS investigation of tyrosol and hydroxytyrosol oxidation products in virgin olive oil

The aim of this research is the characterisation of the oxidation products of several hydrophilic phenols of virgin olive oil (VOO), such as hydroxytyrosol (3,4-DHPEA) and tyrosol (p-HPEA) and, their identification in the product during storage. This work is aimed at finding analytical indicators that can be used both as molecular markers of VOO “freshness” and for the evaluation of the shelf life of the product itself. Two oxidation systems were examined for the oxidation of p-HPEA and 3,4-DHPEA: enzymatic oxidation and Fenton oxidation. Reaction products were different for the two systems and were identified as quinones, dimers, and acids. During the autoxidation process, Fenton reaction oxidation products, but not enzymatic oxidation products, were found in VOO.     

Production of hydrogen peroxide is responsible for the induction of apoptosis by hydroxytyrosol on HL60 cells

Hydroxytyrosol [3,4‐dihydroxyphenylethanol (3,4‐DHPEA)], a phenolic compound found exclusively in olive oil, exerts growth‐suppressive and pro‐apoptotic effects on different cancer cells. Although some molecular mechanisms involved in the pro‐apoptotic activity of 3,4‐DHPEA have been proposed, the initial stress signals responsible of this phenomenon are not known. Our aim was to assess the involvement of reactive oxygen species as mediators of apoptosis induced by 3,4‐DHPEA on HL60 cells. Apoptosis was determined by analyzing the nuclear fragmentation by both fluorescence microscopy and flow cytometry. The externalization of phosphatidylserine was evidenced using an Annexin V‐FITC kit. The concentration of H₂O₂ in the culture medium was measured by the ferrous ion oxidation‐xylenol orange method. The pro‐apoptotic effect of 3,4‐DHPEA (100 μM) was prevented by N‐acetyl‐cysteine, ascorbate, and α‐tocopherol. Catalase suppressed the 3,4‐DHPEA‐induced apoptosis, while the Fe(II)‐chelating reagent o‐phenantroline showed no effect, suggesting the involvement of H₂O₂ but not of OH^?. Indeed, 3,4‐DHPEA caused accumulation of H₂O₂ in the culture medium. Tyrosol (p‐hydroxyphenylethanol) and caffeic acid, compounds structurally similar to 3,4‐DHPEA but not able to generate H₂O₂, did not induce an appreciable apoptotic effect. This is the first study demonstrating that apoptosis induction by 3,4‐DHPEA is mediated by the extracellular production of H₂O₂.     

Olive Oil Phenolics Prevent Oxysterol‐Induced Proinflammatory Cytokine Secretion and Reactive Oxygen Species Production in Human Peripheral Blood Mononuclear Cells,Through Modulation of p38 and JNK Pathways

1 Scope

The aim of the present study was to investigate the ability of extra virgin olive oil (EVOO) polyphenols to counteract the proinflammatory effects induced by dietary and endogenous oxysterols in ex vivo immune cells.

2 Methods and results

Peripheral blood mononuclear cells (PBMCs), separated from the whole blood of healthy donors, were utilized and were stimulated with an oxysterols mixture, in the presence of physiologically relevant concentrations of the EVOO polyphenols, hydroxytyrosol, tyrosol, and homovanillic alcohol. Oxysterols significantly increased the production of proinflammatory cytokines, interleukin‐1β, regulated on activation, normal T‐cell expressed and secreted and macrophage migration inhibitory factor in ex vivo cultured PBMCs. Increased levels of reactive oxygen species (ROS) were also detected along with increased phosphorylation of the p38 and JNK. All phenolic compounds significantly reduced cytokine secretion induced by the oxysterols and inhibited ROS production and mitogen activated protein kinase phosphorylation.

3 Conclusions

These results suggest that extra virgin olive oil polyphenols modulate the immune response induced by dietary and endogenous cholesterol oxidation products in human immune cells and may hold benefit in controlling chronic immune and/or inflammatory processes.     

Direct analysis of glucuronidated metabolites of main olive oil phenols in human urine after dietary consumption of virgin olive oil

In the present study we report on a UPLC-MRM validated method for the simultaneous direct analysis of main glucuronidated metabolites of olive oil phenols: tyrosol, hydroxytyrosol and its O-methyl metabolite homovanillyl alcohol in human urine after dietary olive oil ingestion. The developed method was linear within the concentration range 20–2000 ng/mL with adequate recovery of analytes (>86%). Intra- and inter-day precision and accuracy were according to standard requirements for method validation criteria. Using the developed method, urinary concentrations and excretion rates of glucuronides of olive oil phenols were successfully estimated in an intervention study with 11 healthy volunteers supplemented with a dietary dose of virgin olive oil (VOO) (50 mL). Therefore, about 13% of the consumed olive oil polyphenols were recovered in 24-h urine, where 75% of them were in the form of glucuronides (3′- and 4′-O-hydroxytyrosol glucuronides, 4′-O-glucuronides of tyrosol and homovanillyl alcohol) and 25% as free compounds.     

Antioxidant activity of phenolic fractions in olive mill wastewater

Olive mill wastewater (OMW) contains a substantial amount of valuable antioxidant phenols that can be recovered for industrial application as food additives and pharmaceuticals. The present study was aimed at extracting different phenolic OMW fractions, and determining their antioxidant potential. Five different OMW fractions were obtained using fractionation techniques, their antioxidant potential determined by DPPH, ORAC and a β-carotene bleaching test. The total phenol level ranged between 115 and 170 mg/l. The phenolic compounds present in individual fractions were identified using the HPLC–PAD method, where the main compounds were hydroxytyrosol, tyrosol, caffeic acid, vanillic acid, verbascoside, oleuropein, ferulic acid, and p-coumaric acid. The five OMW fractions showed different antioxidant levels depending on the test used. DPPH test showed that the fraction of alkyl aromatic alcohols (AAAs) was the best with EC₅₀ of 20 mg/l and the pure hydroxytyrosol with 2 mg/l. ORAC test showed that AAA and semi hydrolysed total phenol (s-TP) fractions were significantly better than Trolox when compared to 20 mg/l of Trolox.     

Induction of apoptosis by diphlorethohydroxycarmalol isolated from brown alga,Ishige okamurae

The anti-cancer effect of diphlorethohydroxycarmalol (DC) isolated from Ishige okamurae, a brown alga, via the induction of apoptosis resulting from mitochondrial dysfunction was assessed in human promyelocytic leukemia (HL60) cells. The apoptotic mechanisms of DC induction on tumor cells were studied in this work for the first time. DCs were determined to evidence marked cytotoxicity on HL60 cells in a dose-dependent manner. The induction of apoptosis in HL60 cells by DC was evidenced by the accumulation of sub-G₁ cell population and nuclear condensation. Further investigations into the depletion of mitochondrial membrane potential (ΔΨ_m) determined that DC treatment induced apoptosis via the regulation of the expression of pro-survival and pro-apoptotic Bcl-2 family members. It was demonstrated that the anti-cancer activity of DC was mediated by apoptotic induction resulting from mitochondrial dysfunction. Our study results also indicated dysfunction and that DCs may constitute a new and promising agent for the treatment of human promyelocytic leukemia cells.     

Quality attributes and their relations in fresh black ripe ‘Kalamon’ olives (Olea europaea L.) for table use – phenolic compounds and total antioxidant capacity

Quality attributes were investigated in fresh Greek black table ‘Kalamon’ olives prior to processing. Fruit weight, dimensions, respiration and ethylene production rates, firmness, peel colour, moisture, oil content, total antioxidant capacity (TAC), the concentration of total phenolics (TP) and phenolic compounds (hydroxytyrosol, oleuropein, tyrosol, verbascoside, luteolin‐7‐O‐glucoside, luteolin, rutin) were determined in olives from different orchards. There was a significant effect of orchard on most attributes, but not on fruit firmness. Verbascoside, oleuropein and hydroxytyrosol were the major phenolics, and the presence of verbascoside in ‘Kalamon’ olives is revealed for the first time. Positive correlations were found among fruit weight, dimensions, respiration and ethylene. TAC was positively related mainly to TP, hydroxytyrosol, verbascoside and rutin, but inversely to oleuropein. Luteolin was inversely related to luteolin‐7‐O‐glucoside. Colour darkening was directly related to TAC, while colour parameters were positively and moderately affected by oil and moisture.     

The use of polyphenolic extract,purified hydroxytyrosol and 3,4-dihydroxyphenyl acetic acid from olive mill wastewater for the stabilization of refined oils: a potential alternative to synthetic antioxidants

Ethyl acetate extracts of olive mill waste water (OMWW) were prepared, under in optimal conditions, using a continuous counter-current extraction unit. The antiradical and antioxidant activities of the OMWW extract as well as pure phenolic compounds identified in this extract were evaluated. Results showed that pure hydroxytyrosol and 3,4-dihydroxyphenyl acetic acid had the highest radical-scavenging effects on 1,1-diphenyl-2-picrylhydrazyl radical and the highest antioxidant activities using the β-carotene linoleate model system.The effect of addition of individual phenolic compounds and OMWW extract to refined olive and husk oils was compared with that of control, BHA and BHT at 50 °C. Unexpectedly, 3,4-dihydroxyphenyl acetic acid had the highest protective effect against oil oxidation. Oils with added 3,4-dihydroxyphenyl acetic acid had lower PV than oils with added hydroxytyrosol, the most studied powerful antioxidant. Moreover, the addition of OMWW extract, at 500 ppm, resulted in lower PV values than BHA, p-hydroxyphenylacetic acid, tyrosol on the control.The results suggested that 3,4-dihydroxyphenyl acetic acid, hydroxytyrosol and OMWW extract possess useful antioxidant properties and may be used as alternatives in the search for natural replacement of synthetic antioxidant food additives.     

Nuclear factor erythroid 2-related factor 2 antioxidant response element pathways protect bovine mammary epithelial cells against H2O2-induced oxidative damage in vitro

The experiment was conducted to determine the role of nuclear factor (erythroid-derived 2)-like factor 2 (NFE2L2, formerly Nrf2) antioxidant response element (ARE) pathway in protecting bovine mammary epithelial cells (BMEC) against H₂O₂-induced oxidative stress injury. An NFE2L2 small interfering RNA (siRNA) interference or a pCMV6-XL5-NFE2L2 plasmid fragment was transfected to independently downregulate or upregulate expression of NFE2L2. Isolated BMEC in triplicate were exposed to H₂O₂ (600 μM) for 6 h to induce oxidative stress before transient transfection with scrambled siRNA, NFE2L2-siRNA, pCMV6-XL5, and pCMV6-XL5-NFE2L2. Cell proliferation, apoptosis and necrosis rates, antioxidant enzyme activities, reactive oxygen species (ROS) and malondialdehyde (MDA) production, protein and mRNA expression of NFE2L2 and downstream target genes, and fluorescence activity of ARE were measured. The results revealed that compared with the control, BMEC transfected with NFE2L2-siRNA3 had proliferation rates that were 9 or 65% lower without or with H₂O₂, respectively. These cells also had apoptosis and necrosis rates that were 27 and 3.5 times greater with H₂O₂ compared with the control group, respectively. In contrast, transfected pCMV6-XL5-NFE2L2 had proliferation rates that were 64.3% greater or 17% lower without or with H₂O₂ compared with the control group, respectively. Apoptosis rates were 1.8 times lower with H₂O₂ compared with the control. In addition, compared with the control, production of ROS and MDA and activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), and glutathione-S-transferase (GST) increased markedly in cells transfected with pCMV6-XL5-NFE2L2 and without H₂O₂. However, compared with the control, production of ROS and MDA and activity of CAT and GSH-Px increased markedly, whereas activities of SOD and GST decreased in cells transfected with pCMV6-XL5-NFE2L2 and incubated with H₂O₂. Compared with the control, cells transfected with NFE2L2-siRNA3 with or without H₂O₂ had lower production of ROS and MDA and activity of SOD, CAT, GSH-Px, and GST. Cells transfected with pCMV6-XL5-NFE2L2 with or without H₂O₂ had markedly higher protein and mRNA expression of NFE2L2, heme oxygenase-1 (HMOX-1), NADH quinone oxidoreductase 1, glutamate cysteine ligase catalytic subunit, and glutamyl cystine ligase modulatory subunit compared with the control incubations. Cells transfected with NFE2L2-siRNA3 without or with H₂O₂ had markedly lower protein and mRNA expression of NFE2L2, HMOX-1, NADH quinone oxidoreductase 1, glutamyl cystine ligase modulatory subunit, and glutamate-cysteine ligase catalytic subunit compared with the control incubations. In addition, expression of HMOX-1 was 5.3-fold greater with H₂O₂ compared with the control. Overall, results indicate that NFE2L2 plays an important role in the NFE2L2-ARE pathway via the control of HMOX-1. The relevant mechanisms in vivo merit further study.     

The response of phenylalanine ammonia‐lyase,polyphenol oxidase and phenols to cold stress in the olive tree (Olea europaea L. cv. Picual)

BACKGROUND: In this study the activities of phenylalanine ammonia‐lyase (PAL) and polyphenol oxidase (PPO) and the concentrations of hydroxytyrosol, tyrosol and oleuropein in olive tree (Olea europaea L. cv. Picual) leaves were investigated before and after cold stress by freezing. The air temperature fell to below ?7 °C and, according to the specific field conditions, four categories of orchard were selected: not cold stressed (NS), lightly cold stressed (LS), moderately cold stressed (MS) and heavily cold stressed (HS). RESULTS: In LS and MS samples the PAL specific activity at saturated substrate concentration rose 4.8‐ and 1.9‐fold respectively compared with NS samples. In HS samples the PAL activity declined by 47% compared with NS samples. A low level of PAL protein was detected in all samples affected by cold stress. In LS, MS and HS samples the PPO specific activity at saturated substrate concentration was 1.9‐, 4.4‐ and 9.8‐fold higher respectively than in NS samples. K_m values also increased after cold stress. In MS and HS samples the concentration of oleuropein was 69 and 82% higher respectively than in NS samples, while the concentrations of hydroxytyrosol and tyrosol decreased. CONCLUSION: The response of PAL activity may be part of a recovery process of the olive leaf against cold stress, while the response of PPO and oleuropein may be part of an antioxidant protection mechanism. Copyright © 2009 Society of Chemical Industry     

Antioxidative and anti-hydrogen peroxide activities of various herbal teas

Herbal teas, i.e., extracts of herbs, are popular because of their fragrance and antioxidative activity. Since the antioxidative activity comes mainly from polyphenols, total polyphenol concentrations and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activities in herbal teas were measured and compared. Levels of H₂O₂ in the teas were also examined, since the production of H₂O₂ in beverages such as coffee and green tea, has been reported. Only a small amount of H₂O₂ was detected in the herbal teas just after their preparation with hot water. However, H₂O₂ was gradually produced during incubation at 25 °C after extraction with hot water, especially when the teas were incubated in phosphate buffer at pH 7.4. To examine the anti-H₂O₂ activity of herbal teas, various teas were added to a catechin-enriched green tea, which produce much H₂O₂, and they were incubated at 25 °C for one day. Addition of hibiscus and thorn apple tea decreased the production of H₂O₂ in the catechin-enriched green tea, possibly because of a lowering of the pH of the mixture.     

Acetylation of hydroxytyrosol enhances its transport across differentiated Caco-2 cell monolayers

Hydroxytyrosol and hydroxytyrosyl acetate are two well-known phenolic compounds with antioxidant properties that are present in virgin olive oil. Since the in vivo biological activity of polyphenols is dependent on their intestinal absorption and metabolism, the absorption of hydroxytyrosol and hydroxytyrosyl acetate and the extent to which they are conjugated and metabolised during transfer across intestinal Caco-2/TC7 cell monolayers, was investigated. LC-DAD and LC-MS were used for the quantification and identification of metabolites. Further evidence was obtained by observing metabolite susceptibility to β-glucuronidase treatment and by comparison of products of in vitro conjugation reactions of authentic phenolics with those produced by the CaCo-2 cells. Homovanillyl alcohol was the only conjugate detected as a result of hydroxytyrosol metabolism, and accounted for 20% of the total metabolites detected in the basolateral compartment after 2 h of incubation. Hydroxytyrosyl acetate was largely converted into free hydroxytyrosol (38.4%) and subsequently metabolised into homovanillyl alcohol (6.7%). In addition, hydroxytyrosyl acetate glucuronide (17.4%) together with non-metabolised hydroxytyrosyl acetate (37.5%) were also detected. Both hydroxytyrosyl acetate and hydroxytyrosol were transferred across human Caco-2/TC7 cell monolayers, but the acetylated compound exhibited an apparent permeability (Papp_AP→BL/Papp _BL→AP) 2.1-fold higher than free hydroxytyrosol. For both compounds, all conjugates were preferentially transported to the basolateral side. These results show that the acetylation of hydroxytyrosol significantly increases its transport across the small intestinal epithelial cell barrier, and supports further research into hydroxytyrosyl acetate as a hydroxytyrosol prodrug offering enhanced bioavailability.     

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.     

Selenomethionine increases proliferation and reduces apoptosis in bovine mammary epithelial cells under oxidative stress

The decline in mammary epithelial cell number as lactation progresses may be due, in part, to oxidative stress. Selenium is an integral component of several antioxidant enzymes. The present study was conducted to examine the effect of oxidative stress and selenomethionine (SeMet) on morphology, viability, apoptosis, and proliferation of bovine mammary epithelial cells (BMEC) in primary culture. Cells were isolated from mammary glands of lactating dairy cows and grown for 3 d in a low-serum gel system containing lactogenic hormones and 0 or 100 μM H₂O₂ with 0, 10, 20, or 50 nM SeMet. Hydrogen peroxide stress increased intracellular H₂O₂ to 3 times control concentrations and induced a loss of cuboidal morphology, cell-cell contact, and viability of BMEC by 25%. Apoptotic cell number more than doubled during oxidative stress, but proliferating cell number was not affected. Supplementation with SeMet increased glutathione peroxidase activity 2-fold and restored intracellular H₂O₂ to control levels with a concomitant return of morphology and viability to normal. Apoptotic BMEC number was decreased 76% below control levels by SeMet and proliferating cell number was increased 4.2-fold. These findings suggest that SeMet modulated apoptosis and proliferation independently of a selenoprotein-mediated reduction of H₂O₂. In conclusion, SeMet supplementation protects BMEC from H₂O₂-induced apoptosis and increased proliferation and cell viability under conditions of oxidative stress.     

Anti-hydrogen peroxide activity of fish and soy sauce

Shoyu is the Japanese name for soy sauce and the most popular liquid condiment (seasoning) used in Japanese cuisine as well as in cuisines of other oriental countries. Shoyu is prepared by digesting mold-cultured soybeans and wheat seeded with an aspergillus (koji in Japanese) in the presence of sodium chloride. Gyoshoyu is produced when the soybeans and wheat are replaced with fish. Both Shoyu and Gyoshoyu have high level of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity. Hydrogen peroxide is produced in green tea after exposure to air. To find a safe and economical method of preventing the production of H₂O₂ in green tea, effects of Shoyu and Gyoshoyu on H₂O₂ level in bottled green tea were examined. Both Shoyu and Gyoshoyu suppressed the production of H₂O₂. Gyoshoyu decomposed H₂O₂ possibly because of the presence of a thermostable catalase, while Shoyu did not. Some components of Shoyu and Gyoshoyu may be useful to suppress the production of H₂O₂ in green tea.     

Effects of anthocyanins and other phenolics of boysenberry and blackcurrant as inhibitors of oxidative stress and damage to cellular DNA in SH‐SY5Y and HL‐60 cells

There is growing interest both from consumers and researchers in the role that berries play in human health. The objective of this study was to investigate whether anthocyanins and other phenolics present in boysenberries and blackcurrants are effective in protecting cells against the oxidative damage induced by hydrogen peroxide (H₂O₂). The concentrations of polyphenols used were within the human physiological range. The data showed that SH‐SY5Y human neuroblastoma cells were protected against H₂O₂‐induced toxicity by the anthocyanins and phenolic fractions. The concurrent addition of either fractions of these berries with H₂O₂ significantly inhibited the increase in intracellular reactive oxygen species (ROS) production. Pre‐incubation of cells with the same concentrations had no effect on the ROS level—a result that may be due to the metabolic conversion to inactive compounds. Anthocyanins and phenolic fractions of blackcurrant were better at protecting DNA of HL‐60 human promyelocytic cells from damage than similar fractions from boysenberry. The phenolic extract of blackcurrant demonstrated the highest protective effect against H₂O₂‐induced neurotoxicity, oxidative stress and DNA damage and may be a good candidate for inclusion into a processed functional food. Copyright © 2006 Society of Chemical Industry     

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.     

Evaluation of table olive by‐product as a source of natural antioxidants

The main by‐product from the table olive canning industry is the stone with some residual olive flesh. The purpose of this study was to evaluate the composition – phenolic compounds (hydroxytyrosol, tyrosol and oleuropein) and tocopherol – and the antioxidant activity in different fractions (flesh, stone and seed) from the table olive by‐product and the whole by‐product. The highest amounts of phenolic compounds (1710.0 ± 33.8 mg kg^?1) as well as the highest antioxidant activity (8226.9 ± 9.9 hydroxytyrosol equivalents mg kg^?1) were obtained in the seed. The highest amounts of hydroxytyrosol (854.8 ± 66.0 mg kg^?1) and tyrosol (423.6 ± 56.9 mg kg^?1) were found in the whole by‐product from the pepper stuffed olives, while the stone without seed had the maximum oleuropein content (750.2 ± 85.3 mg kg^?1). α‐Tocopherol values were between 79.8 ± 20.8 mg kg^?1 in the seed of the olive stone and 6.2 ± 1.2 mg kg^?1 in the whole by‐product from the anchovy‐stuffed olives. In light of the results obtained, it would seem possible to use table olive by‐product as a source of natural antioxidants in foods, cosmetics or pharmaceutical products, thus contributing to diminishing the environmental impact of table olive by‐product and to its revalorisation.     

Quercitrin protects against oxidative stress-induced injury in lung fibroblast cells via up-regulation of Bcl-xL

The cytoprotective effect of quercitrin (QR) against oxidative stress induced cell damage by hydrogen peroxide (H₂O₂) in Chinese hamster lung fibroblast (V79-4) cells was investigated. QR evidenced a scavenging effect of 1,1-diphenyl-2-picrylhydrazyl (DPPH), superoxide, hydroxyl radicals and on intracellular ROS, and thus prevented lipid peroxidation. As a result, QR reduced H₂O₂-induced cell death and apoptosis in V79-4 cells. Moreover, H₂O₂ induced the cleavage of caspase-3, -9, and poly-ADP-ribose polymerase (PARP) and a reduction in Bcl-xL levels, whereas pretreatment with QR significantly inhibited caspase-3, -9, and PARP cleavage and the reduction in Bcl-xL levels, and ultimately ameliorated H₂O₂-induced apoptosis. Taken together, these results indicate that the treatment of V79-4 cells with QR can block H₂O₂-induced apoptosis via the regulation of Bcl-xL. QR may be exploited as a biopreservative in food applications or as a health supplement to alleviate oxidative stress.     

The component of green tea,L-theanine protects human hepatic L02 cells from hydrogen peroxide-induced apoptosis

L-theanine is a natural amino acid in green tea and it has been well known for its activities of relieving depression and neuroprotection. However, cytoprotective effect and its mechanism of L-theanine on hepatocytes have not been reported. The objective of this work was to investigate the hepatoprotective effect of L-theanine as well as its mechanism by using the human hepatic L02 cells injured by hydrogen peroxide (H₂O₂). Results showed that L-theanine dose dependently decreased H₂O₂-induced cell viability loss and lactate dehydrogenase (LDH) release. L-theanine pretreatment improved nuclear morphology of the cells injured by H₂O₂. By using flow cytometric analysis, we found that L-theanine significantly inhibited H₂O₂-induced cell apoptosis. Further, L-theanine attenuated H₂O₂-induced reduction in pro-caspase3 and cleavage of poly (adenosine diphosphate-ribose) polymerase (PARP). H₂O₂-activated p38 mitogen-activated protein kinase (MAPK) was also inhibited by L-theanine. These data suggest that L-theanine could protect L02 cells against H₂O₂-induced apoptosis via suppression of p38 MAPK. L-theanine might potentially be useful in the prevention and treatment of liver diseases.