油茶总皂甙抑菌活性及清除活性氧能力初步研究

用溶剂法提取油茶饼中油茶总皂甙,经柱层析制备标准品标定有效成分含量为82.5%。以油茶皂甙为材料对枯草杆菌、大肠杆菌、金黄色葡萄球菌进行抑菌试验,结果表明均有抑制作用;其中对枯草杆菌和金黄色葡萄球菌抑制作用较强,20%浓度对三种细菌均具有致死作用,抑菌圈直径大小与油茶总皂甙浓度呈正相关;且初步测定油茶总皂甙清除活性氧能力强于维生素C。     

In vitro scavenging capacity of annatto seed extracts against reactive oxygen and nitrogen species

Bixa orellana L. (annatto), from Bixaceae family, is a native plant of tropical America, which accumulates several carotenoids (including bixin and norbixin), terpenoids, tocotrienols and flavonoids with potential antioxidant activity. In the present study, the in vitro scavenging capacity of annatto seed extracts against reactive oxygen species (ROS) and reactive nitrogen species (RNS) was evaluated and compared to the bixin standard. Annatto extracts were obtained using solvents with different polarities and their phenolic compounds and bixin levels were determined by high performance liquid chromatography coupled to diode array detector. All annatto extracts were able to scavenge all the reactive species tested at the low μg/mL range, with the exception of superoxide radical. The ethanol:ethyl acetate and ethyl acetate extracts of annatto seeds, which presented the highest levels of hypolaetin and bixin, respectively, were the extracts with the highest antioxidant capacity, although bixin standard presented the lowest IC₅₀ values.     

Effect of nitric oxide on reactive oxygen species and antioxidant enzymes in kiwifruit during storage

BACKGROUND: The accumulation of reactive oxygen species (ROS) in kiwifruit can cause oxidative damage during storage. Little research has been carried out on the effects of nitric oxide (NO) on oxidative damage to kiwifruit. Therefore the aim of the present study was to evaluate the ability of 0.5, 1 and 2 µmol L^?1 NO aqueous solutions to alleviate oxidative damage to kiwifruit during storage. RESULTS: The most marked effect was obtained with 1 µmol L^?1 NO solution, which significantly reduced the accumulation of malondialdehyde, superoxide and hydrogen peroxide, delayed the decrease in vitamins C and E, maintained the content of soluble solids, inhibited the activity of lipoxygenase and peroxidase and increased the activity of superoxide dismutase and catalase in kiwifruit during storage. The 0.5 µmol L^?1 NO solution was too weak to significantly affect the content of ROS and the activity of enzymes. However, treatment with 2 µmol L^?1 NO solution promoted the accumulation of ROS, decreased the activity of antioxidant enzymes and accelerated peroxidation in kiwifruit during storage. CONCLUSION By increasing the activity of antioxidant enzymes and maintaining the content of vitamins C and E, treatment with 1 µmol L^?1 NO aqueous solution could protect kiwifruit against oxidative damage caused by ROS during storage. Copyright © 2008 Society of Chemical Industry     

三种贮藏低温对厚皮甜瓜果实活性氧产生和清除的比较

本文以厚皮甜瓜‘金红宝’为试材,研究了三种贮藏低温（3、7和9℃）对果实O-2·产生速率和H2O2含量,超氧化物歧化酶（SOD）、过氧化氢酶（CAT）、谷胱甘肽还原酶（GR）和抗坏血酸过氧化物酶（APX）活性的影响。结果表明,7℃和9℃下果实O-2·产生速率在前期（第4d）显著增加,而3℃下在后期（第12d）逐渐积累;3℃下果实的H2O2含量前期明显升高,而7℃和9℃下在中期（第8d）和后期逐渐升高。贮藏前期7℃下果实SOD、GR和APX的活性明显高于3℃和9℃处理,而3℃下果实的CAT活性最低。上述结果显示,3℃下大量的H2O2积累以及较低的抗氧化酶活性是导致甜瓜果实冷害发生的重要原因,7℃下果实维持了良好的活性氧产生和清除平衡,为厚皮甜瓜贮藏的适宜温度。      

三种贮藏低温对厚皮甜瓜果实活性氧产生和清除的比较

本文以厚皮甜瓜‘金红宝’为试材,研究了三种贮藏低温(3、7和9℃)对果实O-2·产生速率和H2O2含量,超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽还原酶(GR)和抗坏血酸过氧化物酶(APX)活性的影响。结果表明,7℃和9℃下果实O-2·产生速率在前期(第4d)显著增加,而3℃下在后期(第12d)逐渐积累;3℃下果实的H2O2含量前期明显升高,而7℃和9℃下在中期(第8d)和后期逐渐升高。贮藏前期7℃下果实SOD、GR和APX的活性明显高于3℃和9℃处理,而3℃下果实的CAT活性最低。上述结果显示,3℃下大量的H2O2积累以及较低的抗氧化酶活性是导致甜瓜果实冷害发生的重要原因,7℃下果实维持了良好的活性氧产生和清除平衡,为厚皮甜瓜贮藏的适宜温度。     

Reactive oxygen species scavenging activities of naturally occurring colorants

Twenty-five kinds of plant extract used as natural dyeing agents were tested for their reactive oxygen species (ROS) scavenging activities using several assay systems. Among plant extracts examined in this study the hot water extracts from logwood, wattle, pomegranate, dragon’s blood, and quebracho showed relatively strong antioxidant activity. In particular, the pigments extracted from pomegranate and wattle effectively suppressed intracellular generation of ROS induced by hydrogen peroxide in mouse hepatoma hepa1c1c7 cells as well as showed a strong oxygen radical scavenging activity in in vitro assay systems, suggesting their potential usefulness as health functional food ingredients and natural food colorants.     

Fullerol-sensitized production of reactive oxygen species in aqueous solution

The relative production rate of reactive oxygen in aqueous solution sensitized by fullerol (a polyhydroxylated, water-soluble form of the fullerene C60) was measured and compared to known reactive oxygen sensitizers using an oxygen consumption method. The solutions were irradiated by polychromatic visible and ultraviolet light. Reactive oxygen species were generated under both visible and ultraviolet light sources. The greatest rates of oxygen consumption were observed at acidic pH. We show for the first time evidence of both singlet oxygen and superoxide production by fullerol under both UV and polychromatic light sources.     

Chemistry and reactions of reactive oxygen species in foods

Reactive oxygen species (ROS) are formed enzymatically, chemically, photochemically, and by irradiation of food. They are also formed by the decomposition and the inter-reactions of ROS. Hydroxy radical is the most reactive ROS, followed by singlet oxygen. Reactions of ROS with food components produce undesirable volatile compounds and carcinogens, destroy essential nutrients, and change the functionalities of proteins, lipids, and carbohydrates. Lipid oxidation by ROS produces low molecular volatile aldehydes, alcohols, and hydrocarbons. ROS causes crosslink or cleavage of proteins and produces low molecular carbonyls from carbohydrates. Vitamins are easily oxidized by ROS, especially singlet oxygen. The singlet oxygen reaction rate was the highest in ss-carotene, followed by tocopherol, riboflavin, vitamin D, and ascorbic acid.     

Polyphenolic compounds characterization and reactive nitrogen species scavenging capacity of Oenothera paradoxa defatted seed extracts

Biological investigations have revealed high scavenging capacity of Oenothera paradoxa defatted seed extract on reactive nitrogen species such as NO and ONOO⁻. The characteristics of the polyphenols present in the extracts were checked using high performance liquid chromatography coupled with electrospray negative ionization ion trap mass spectrometry. Extracts contained five groups of compound: phenolic acids (gallic acid, ethyl gallate, ellagic acid and ferulic acid pentoside), flavanols (catechin, catechin gallate) and oligomeric procyanidins, flavonols (quercetin-3-O-glucoside, quercetin-3-O-glucuronide, quercetin-3-O-pentoside and quercetin), and gallotannins (tetragalloyl glucose, pentagalloyl glucose and hexagalloyl glucose). Penta-O-galloyl-β-d-glucose were present in the extracts in concentrations from 9.44 to 16.75 mg/g, which demonstrated a significant NO and ONOO⁻ scavenging activity with IC₅₀ 0.20 and 0.06 μM, respectively, may be considered as an O. paradoxa extract quality marker.     

The role of reactive oxygen species in the induction of Ty1 retrotransposition in Saccharomyces cerevisiae

Here we provide evidence for a dependence between the increased production of reactive oxygen species and the activation of Ty1 retrotransposition. We have found that the strong activator of Ty1 mobility, methylmethane sulphonate, can not induce Ty1 retrotransposition in cells with compromised mitochondrial oxidative phosphorylation (rho^?; sco1Δ), which is the major source for production of reactive oxygen species (ROS) in Saccharomyces cerevisiae. The quantitative estimation of superoxide anions in living cells showed that rho⁺ cells exposed to methylmethane sulphonate increase Ty1 retrotransposition and superoxide levels. The increase of superoxide anions by the superoxide generator menadione is accompanied by induction of Ty1 mobility without any treatment with a DNA‐damaging agent. Higher frequencies of retrotransposition were found in rho⁺ and rho^? cells treated with exogenously added hydrogen peroxide or in cells with disrupted YAP1 gene characterized by increased intracellular levels of hydrogen peroxide. These data indicate that increased levels of ROS may have an independent and key role in the induction of Ty1 retrotransposition. Copyright © 2010 John Wiley & Sons, Ltd.     

Photoperiod-induced differential expression of angiogenesis genes in testes of adult Peromyscus leucopus

Non-pathological angiogenesis in adults is rare and is largely thought to be restricted to wound healing and female reproductive cycles. Adult male rodents, however, display seasonal angiogenesis to support seasonal changes in reproductive tissue morphology. Non-tropical rodents use photoperiod (day length) to determine the time of year. During short days, the reproductive system undergoes involution and mating behaviours stop, adaptations which presumably allow energy resources to be shifted to processes necessary for winter survival. We compared the patterns of gene expression involved in angiogenesis in testes of white-footed mice (Peromyscus leucopus) following 7, 14, 21 or 34 weeks of long or short day lengths. Short days decreased body mass, reproductive tract mass and seminiferous tubule diameter. Potential genes involved in seasonal angiogenesis were screened by hybridizing testicular RNA from each group to angiogenesis-specific microarrays. Genes that were > or =6-fold different between long- and short-day testes (i.e. hypoxia-inducible factor 1alpha(Hif1alpha), plasminogen activator inhibitor 1 (Serpine1), transforming growth factor beta receptor 3 (Tgfbetar3) and tumour necrosis factor (Tnf )) were sequenced and expression differences were compared throughout gonadal regression and recrudescence using quantitative RT-PCR. Our results suggest that short days trigger expression of Hif1alpha, Serpine1, and Tgfbetar3 to inhibit angiogenesis or promote apoptosis during testicular regression, and also trigger expression of Tnf to promote angiogenesis during testicular recrudescence.     

番茄红素纳米分散体体外清除活性氧自由基研究

采用乳化-蒸发工艺（超声乳化）制备了番茄红素纳米分散体,用分光光度法研究了番茄红素纳米分散体体外清除活性氧自由基（过氧化氢、羟自由基）的作用,并与溶于四氢呋喃的番茄红素进行比较。结果表明,番茄红素纳米分散体对这两种自由基均有不同程度的清除作用,且均呈现一定的量效关系;在相同浓度下,其清除能力均强于番茄红素四氢呋喃溶液,其中对过氧化氢清除能力远远高于番茄红素四氢呋喃溶液。      

青椒果实冷害与活性氧清除酶和脂氧合酶活性变化的关系

青椒在9℃下冷藏8周,未出现冷害,膜透性变化不大。而在2℃下贮藏1周后即出现冷害症状,超过4周发生严重冷害,膜透性迅速上升。与9℃贮藏青椒相比,2℃贮藏的青椒SOD活性降低,而CAT和APX活性降低更多;POD活性也明显低于9℃贮藏青椒,但7周时骤升至峰值。9℃下青椒的LOX活性变化很小,而2℃下青椒的LOX活性2周时迅速上升,此后保持较高的水平。低温促进了膜脂过氧化产物MDA的积累,使2℃下青椒的MDA含量高于9℃的。可见青椒的冷害与低温下活性氧清除酶,特别是CAT和APX活性的降低以及LOX活性增强,导致活性氧化代谢失调和膜脂过氧化有关。     

The role of reactive oxygen species in the electrochemical inactivation of microorganisms

Electrochemical disinfection has emerged as one of the most promising alternatives to the conventional disinfection of water in many applications. Although the mechanism of electrochemical disinfection has been largely attributed to the action of electro-generated active chlorine, the role of other oxidants, such as the reactive oxygen species (ROS) *OH, O3, H2O2, and *O2- remains unclear. In this study, we examined the role of ROS in the electrochemical disinfection using a boron-doped diamond (BDD) electrode in a chloride-free phosphate buffer medium, in order to avoid any confusion caused by the generation of chlorine. To determine which species of ROS plays the major role in the inactivation, the effects of several operating factors, such as the presence of *OH scavenger, pH, temperature, and the initial population of microorganisms, were systematically investigated. This study clearly showed that the *OH is the major lethal species responsible for the E. coli inactivation in the chloride-free electrochemical disinfection process, and that the E. coli inactivation was highly promoted at a lower temperature, which was ascribed to the enhanced generation of O3.     

Environmentally persistent free radicals (EPFRs). 1. Generation of reactive oxygen species in aqueous solutions

Reactive oxygen species (ROS) generated by environmentally persistent free radicals (EPFRs) of 2-monochlorophenol, associated with CuO/silica particles, were detected using the chemical spin trap, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), in conjunction with electron paramagnetic resonance (EPR) spectroscopy. Yields of hydroxyl radical ((?)OH), superoxide anion radical (O(2)(?-)), and hydrogen peroxide (H(2)O(2)) generated by EPFR-particle systems were reported. Failure to trap superoxide radicals in aqueous solvent, formed from reaction of EPFRs with molecular oxygen, results from fast transformation of the superoxide to hydrogen peroxide. However, formation of superoxide as an intermediate product in hydroxyl radical formation in aprotic solutions of dimethyl sulfoxide (DMSO) and acetonitrile (AcN) was observed. Experiments with superoxide dismutase (SOD) and catalase (CAT) confirmed formation of superoxide and hydrogen peroxide, respectively, in the presence of EPFRs. The large number of hydroxyl radicals formed per EPFR and monotonic increase of the DMPO-OH spin adduct concentration with incubation time suggest a catalytic cycle of ROS formation.     

Generation of reactive oxygen species mediated by humic-like substances in atmospheric aerosols

Particulate matter (PM)-mediated reactive oxygen species (ROS) generation has been implicated in health effects posed by PM. Humic-like substances (HULIS) are an unresolved mixture of water-extracted organic compounds from atmospheric aerosol particles or isolated from fog/cloudwater samples. In this study, we use a cell-free dithiothreitol (DTT) assay to measure ROS production mediated by HULIS. The HULIS samples are isolated from aerosols collected at a rural location and a suburban location in the Pearl River Delta, China. In our experiments, ROS activities by residue metal ions in the HULIS fraction are suppressed by including a strong chelating agent in the DTT assay. Under conditions of DTT consumption not exceeding 90%, the HULIS-catalyzed oxidation of DTT follows the zero-order kinetics with respect to DTT concentration, and the rate of DTT oxidation is proportional to the dose of HULIS. The ROS activity of the aerosol HULIS, on a per unit mass basis is 2% of the ROS activity by a reference quinone compound, 1,4-naphthoquinone and exceeds that of two aquatic fulvic acids. The HULIS fraction in the ambient samples tested exhibits comparable ROS activities to the organic solvent extractable fraction, which would contain compounds such as quinones, a known organic compound class capable of catalyzing generation of ROS in cells. HULIS was found to be the major redox active constituent of the water-extractable organic fraction in PM. It is plausible that HULIS contains reversible redox sites, thereby serving as electron carriers to catalyze the formation of ROS. Our work suggests that HULIS could be an active PM component in generating ROS and further work is warranted to characterize its redox properties.     

Identification of the reactive oxygen species responsible for carbon tetrachloride degradation in modified Fenton's systems

The reactive oxygen species responsible for the transformation of carbon tetrachloride (tetrachloromethane, CT) by modified Fenton's reagent using hydrogen peroxide (H2O2) concentrations >0.1 M was investigated. Addition of the hydroxyl radical scavenger 2-propanol to modified Fenton's reactions did not significantly lower CT transformation rates. Scavenging by 2-propanol not only confirmed that hydroxyl radicals are not responsible for CT destruction, but also suggested that a major product of an iron (III)-driven initiation reaction, superoxide radical anion (O2-), is the species responsible for CT transformation. To investigate this hypothesis, CT degradation was studied in aqueous KO2 reactions. Minimal CT degradation was found in CT-KO2 reactions; however, when H2O2 was added to the KO2 reactions at concentrations similar to those in the modified Fenton's reactions (0.1, 0.5, and 1 M), CT degradation increased significantly. Similar results were obtained when 1 M concentrations of other solvents were added to aqueous KO2 reactions, and the observed first-order rate constant for CT degradation correlated strongly (R2 = 0.986) with the empirical solvent polarity (E(T)N) of the added solvents. The results indicate that even dilute concentrations of solvents, including H202, can increase the reactivity of O2- in water, probably by changing its solvation sphere. The higher reactivity of O2- generated in modified Fenton's reagent, which has a less polar nature due to the presence of H2O2, may result in a wider range of contaminant degradation than previously thought possible.     

Svf1 inhibits reactive oxygen species generation and promotes survival under conditions of oxidative stress in Saccharomyces cerevisiae

Aberrant regulation of apoptosis, or programmed cell death, contributes to the aetiology of several diseases, including cancers, immunodeficiencies and neurodegenerative illnesses. We hypothesized that key features of mammalian cell death regulation may be conserved in single celled organisms such as the budding yeast Saccharomyces cerevisiae. We previously identified the yeast gene SVF1 in a screen for mutations that could be functionally complemented by exogenous expression of the human anti-apoptotic gene Bcl-x(L). Anti-apoptotic Bcl-2 family members have been shown to promote redox stability through upregulation of antioxidant pathways in mammalian cells. Here we demonstrate that the Svf1 protein is required for yeast survival under conditions of oxidative stress, including cold stress. Cells lacking SVF1 are hypersensitive to conditions associated with increased reactive oxygen species (ROS) generation and to direct chemical precursors of ROS, and demonstrate increased levels of ROS under these conditions. Hypersensitivity to oxidative stress can be reversed by treatment with the antioxidant N-acetylcysteine or expression of exogenous SVF1, although exogenous expression of Bcl-x(L) did not protect cells from cold stress. Exogenous SVF1 expression in mammalian cells confers resistance to H(2)O(2) exposure. Our data are consistent with previous observations suggesting a key role of oxidative stress response in mammalian apoptotic regulation and validate the use of S. cerevisiae as a model for studying programmed cell death.     

Low abundance of mitophagy markers is associated with reactive oxygen species overproduction in cows with fatty liver and causes reactive oxygen species overproduction and lipid accumulation in calf hepatocytes

Mitochondria are the main site of fatty acid oxidation and reactive oxygen species (ROS) formation. Damaged or dysfunctional mitochondria induce oxidative stress and increase the risk of lipid accumulation. During the process of mitophagy, PTEN induced kinase 1 (PINK1) accumulates on damaged mitochondria and recruits cytoplasmic Parkin to mitochondria. As an autophagy receptor protein, sequestosome-1 (p62) binds Parkin-ubiquitinated outer mitochondrial membrane proteins and microtubule-associated protein 1 light chain 3 (LC3) to facilitate degradation of damaged mitochondria. In nonruminants, clearance of dysfunctional mitochondria through the PINK1/Parkin-mediated mitophagy pathway contributes to reducing ROS production and maintaining metabolic homeostasis. Whether PINK1/Parkin-mediated mitophagy plays a similar role in dairy cow liver is not well known. Thus, the objective of this study was to investigate mitophagy status in dairy cows with fatty liver and its role in free fatty acid (FFA)-induced oxidative stress and lipid accumulation. Liver and blood samples were collected from healthy dairy cows (n = 10) and cows with fatty liver (n = 10) that had a similar number of lactations (median = 3, range = 2 to 4) and days in milk (median = 6 d, range = 3 to 9 d). Calf hepatocytes were isolated from 5 healthy newborn female Holstein calves (1 d of age, 30–40 kg). Hepatocytes were transfected with small interfering RNA targeted against PRKN for 48 h or transfected with PRKN overexpression plasmid for 36 h, followed by treatment with FFA (0.3 or 1.2 mM) for 12 h. Mitochondria were isolated from fresh liver tissue or calf hepatocytes. Serum concentrations of β-hydroxybutyrate were higher in dairy cows with fatty liver. Hepatic malondialdehyde (MDA) and hydrogen peroxide (H₂O₂) were greater in cows with fatty liver. The lower protein abundance of PINK1, Parkin, p62, and LC3-II in hepatic mitochondrial fraction of dairy cows with fatty liver indicated the mitophagy was impaired. In hepatocytes, knockdown of PRKN decreased protein abundance of p62 and LC3-II in the mitochondrial fraction, and increased contents of triacylglycerol (TG), MDA, and H₂O₂. In addition, protein abundances of PINK1, Parkin, p62, and LC3-II were lower in the mitochondrial fraction from hepatocytes treated with 1.2 mM FFA than the hepatocytes treated with 0.3 mM FFA, whereas the content of TG, MDA, and H₂O₂ increased. In 1.2 mM FFA-treated hepatocytes, PRKN overexpression increased protein abundance of p62 and LC3-II in the mitochondrial fraction and decreased contents of TG, MDA, and H₂O₂. Together, our data demonstrate that low abundance of mitophagy markers is associated with ROS overproduction in dairy cows with fatty liver and impaired mitophagy induced by a high concentration of FFA promotes ROS production and lipid accumulation in female calf hepatocytes.