Use of red blood cell membranes to evaluate the antioxidant potential of plant extracts.

Antioxidant phytochemicals in fruits and vegetables of a vegetarian diet may account for the reduced risk of aging and stress oxidative associated diseases. In this study, a simple, rapid and accurate new bioassay for the determination of the antioxidant activity of purified or crude plant extracts and thier interactions is described, based on the fluorimetric determination of thiobarbituric acid reactive substances (TBARS) released by UV-B radiated red blood cell (RBC) ghosts. Pure resveratrol, white and red wine and pomegranate juice (PJ) were used as antioxidant source to test the biological method. TBARS production is a function of radiation time, the number of RBC ghosts in the radiated sample and the loaded antioxidant. The antioxidant activity of resveratrol was detected at a submicromolar concentration range [0.02 µg/mL-0.1 µmol/L]. The activity of red wine was almost 10 times higher than that of white wine, and PJ juice had the highest activity. Submaximal protective effects of PJ and red wine were additive.

Online comprehensive RPLC × RPLC with mass spectrometry detection for the analysis of proteome samples.

LC-MS-based shotgun proteomics relies both on the power of the separation techniques and the sensitivity of detection methods. As a viable alternative to classical approaches in this field, we developed a fully automated, comprehensive 2D LC system, in which RPLC × RPLC was coupled to MS detection, for the first time, and applied for the analysis of tryptic digests obtained from α-casein and dephosphorylated α-casein. The use of a significantly different pH in the two dimensions allowed us to attain high peak capacity, despite the employment of novel identical stationary phases. Furthermore, such a combination addresses compatibility issues, thus allowing straightforward interfacing in online 2D LC configuration, as well as direct linkage to a mass spectrometer. A theoretical peak capacity of ca. 8500 was calculated for the setup, employing four serially coupled C18 columns in the first dimension (600 × 2.1 mm, 2.7 µm d.p.), operated under basic conditions, and 3 cm length of the same stationary phase (30 × 4.6 mm, 2.7 µm d.p. column), under acidic conditions, for fast second dimension analysis.

Analysis of anthocyanins in commercial fruit juices by using nano-liquid chromatography-electrospray-mass spectrometry and high-performance liquid chromatography with UV-vis detector.

Nano-LC and conventional HPLC techniques were applied for the analysis of anthocyanins present in commercial fruit juices using a capillary column of 100 µm id and a 2.1 mm id narrow-bore C(18) column. Analytes were detected by UV-Vis at 518 nm and ESI-ion trap MS with HPLC and nano-LC, respectively. Commercial blueberry juice (14 anthocyanins detected) was used to optimize chromatographic separation of analytes and other analysis parameters. Qualitative identification of anthocyanins was performed by comparing the recorded mass spectral data with those of published papers. The use of the same mobile phase composition in both techniques revealed that the miniaturized method exhibited shorter analysis time and higher sensitivity than narrow-bore chromatography. Good intra-day and day-to-day precision of retention time was obtained in both methods with values of RSD less than 3.4 and 0.8% for nano-LC and HPLC, respectively. Quantitative analysis was performed by external standard curve calibration of cyanidin-3-O-glucoside standard. Calibration curves were linear in the concentration ranges studied, 0.1-50 and 6-50 µg/mL for HPLC-UV/Vis and nano-LC-MS, respectively. LOD and LOQ values were good for both methods. In addition to commercial blueberry juice, qualitative and quantitative analysis of other juices (e.g. raspberry, sweet cherry and pomegranate) was performed. The optimized nano-LC-MS method allowed an easy and selective identification and quantification of anthocyanins in commercial fruit juices; it offered good results, shorter analysis time and reduced mobile phase volume with respect to narrow-bore HPLC.

Effects of oxidative stress on mitochondrial content and integrity of human anastomotic colorectal dehiscence: a preliminary DNA study.

BACKGROUND: Anastomotic dehiscence is one of the most severe complications of colorectal surgery. Gaining insight into the molecular mechanisms responsible for the development of anastomotic dehiscence following colorectal surgery is important for the reduction of postoperative complications. OBJECTIVE: Based on the close relationship between surgical stress and oxidative stress, the present study aimed to determine whether a correlation exists between increased levels of reactive oxygen species and colorectal anastomotic dehiscence. METHODS: Patients who underwent surgical resection for colorectal cancer were divided into three groups: patients with anastomotic dehiscence (group 1); patients without dehiscence who underwent neoadjuvant radiochemotherapy (group 2); and patients without anastomotic dehiscence who did not undergo neoadjuvant radiochemotherapy (group 3). Quantitative polymerase chain reaction and real-time polymerase chain reaction assays were performed to measure nuclear DNA and mitochondrial DNA (mtDNA) content, and possible oxidative damage to nonmalignant colon and rectal tissues adjacent to the anastomoses. RESULTS: mtDNA content was reduced in the colon tissue of patients in groups 1 and 2. Rectal mtDNA was found to be more damaged than colonic mtDNAs in all groups. The 4977 bp common deletion was observed in the mtDNA of tissues from both the colon and rectum of all patients. DISCUSSION: Patients in groups 1 and 2 were more similar to one another than to group 3, probably due to higher levels of reactive oxygen species in the mitochondria; the greater damage found in the rectum suggests that dehiscence originates primarily from the rectal area. CONCLUSIONS: The present study of mtDNA analyses of normal human colon and rectal tissues from patients with colorectal cancer is among the first of its kind.

Sulphurous mineral water oral therapy: effects on erythrocyte metabolism.

The ingestion of water containing hydrogen sulphide (H(2)S) is common in spring sulphurous mineral water (SMW) therapy. We hypothesized that observed detrimental effects are related to the alteration of erythrocytes metabolism caused by H(2)S. To verify our hypothesis, we treated 20 healthy volunteers with SMW and evidenced an increase of methemoglobin concentration, an inhibition of both erythrocyte glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and glucose-6-phosphate dehydrogenase (G6PDH) activities. To investigate the mechanism of H(2)S effect on GAPDH activity, an in vitro study was performed by incubating both erythrocytes from 12 healthy volunteers and purified GAPDH with buffered [(35)S]-H(2)S labelled sulphurous water. The interaction between H(2)S and NAD(+)(H), was also investigated. The results indicate that a direct reaction between GAPDH and H(2)S does not occur and the observed decrease of GAPDH activity is to ascribe to the reaction between H(2)S and NAD(+)(H). This may lead to GAPDH inhibition by two ways, namely (i) cellular NAD(+)(H) reduced availability and (ii) catalytic site blockage. In conclusion, our results show that among the detrimental effects of SMW administration are erythrocyte GAPDH and G6PDH activity inhibition and increased methemoglobin concentration. A mechanism to explain the occurrence of these biochemical events is also proposed.

Effect of quercetin on oxidative nuclear and mitochondrial DNA damage.

Quercetin is a well-investigated antioxidant known to protect cells against oxidative nuclear DNA damage. There is no knowledge regarding its effect on oxidative mitochondrial DNA damage. In this study we investigated the effect of quercetin on oxidatively-injured DNA. Cell-free and cell studies were performed. Cell-free analyses carried out on plasmidic DNA showed that quercetin protects from all oxidative challenges used. Cellular studies were carried out on NCTC 2544 cells which were insulted with hydrogen peroxide and UVC radiations. Nuclear and mitochondrial DNAs were analysed by measuring DNA damage with a quantitative polymerase chain reaction. Quercetin supplementation showed significant genoprotective activity on mitochondrial DNA when hydroperoxide was used. The evidence of the protection afforded by quercetin suggests that this flavonoid may play an important role on mitochondrial genome stability.

Intracellular flavonoids as electron donors for extracellular ferricyanide reduction in human erythrocytes.

Reduction of extracellular ferricyanide [Fe(CN)(6)](-3) to ferrocyanide by intact cells reflects the activity of a trans-plasma membrane oxidoreductase that, in human red blood cells, utilizes ascorbic acid as an electron donor. We herein report that the flavonoids quercetin and myricetin, while inhibiting dehydroascorbic acid uptake-and thus the erythrocyte ascorbic acid content-effectively stimulate the extracellular reduction of ferricyanide. Other flavonoids such as rutin, acacetin, apigenin, and genistein do not show the same effect. The notion that quercetin or myricetin may serve as an intracellular donor for a trans-plasma membrane oxidoreductase is supported by the following lines of evidence: (i) they afford direct reduction of ferricyanide; (ii) extracellular reduction of ferricyanide was not mediated by direct effects of the flavonoids released by the cells and was abolished by the sulphydryl reagent parachloromercuribenzenesulfonic acid (pCMBS); (iii) the intracellular concentrations of quercetin or myricetin well correlate with increases in ferricyanide reduction; (iv) the intracellular concentration of the flavonoids dramatically declines after ferricyanide exposure. Taken together, the results presented in this study demonstrate that myricetin and quercetin, which accumulate in large amounts in red blood cells, act as intracellular substrates of a pCMBS-sensitive trans-plasma membrane oxidoreductase. This may represent a novel mechanism whereby these flavonoids exert beneficial effects under oxidative stress conditions.

Effects of a static magnetic field on cell growth and gene expression in Escherichia coli.

Escherichia coli cultures exposed to a 300mT static magnetic field (SMF) were studied in order to analyse possible induced changes in cellular growth and gene expression. Biomass was evaluated by visible-light spectrometry and gene expression analyses were carried out by use of RNA arbitrarily primed PCR. The bacterial strain XL-1Blue, cultivated in traditional and modified Luria-Bertani medium, was exposed to SMF generated by permanent neodymium magnetic disks. The results show alterations induced by SMF in terms of increased cell proliferation and changes in gene expression compared with control groups. Three cDNAs were found to be expressed only in the exposed cells, whereas one cDNA was more expressed in the controls. One clone, expressed only in the exposed cells, corresponds to a putative transposase. This is of particular interest in that it suggests that exposure to a magnetic field may stimulate transposition activity.

Antibacterial effect of a magnetic field on Serratia marcescens and related virulence to Hordeum vulgare and Rubus fruticosus callus cells.

The exposure to a static magnetic field of 80+/-20 Gauss (8+/-2 mT) resulted in the inhibition of Serratia marcescens growth. Callus cell suspensions from Hordeum vulgare and Rubus fruticosus were also examined and only the former was found to be affected by the magnetic field, which induced a decreased viability. S. marcescens was shown to be virulent only toward H. vulgare and this virulence was reduced by the presence of the magnetic field. The modification of glutathione peroxidase activity under the different experimental conditions allowed us to speculate on the possibility of an oxidative-stress response of H. vulgare both to S. marcescens infection and magnetic field exposure. Since the control of microbial growth by physical agents is of interest for agriculture, medicine and food sciences, the investigation presented herein could serve as a starting point for future studies on the efficacy of static magnetic field as low-cost/easy-handling preservative agent.

In vitro protective effect of Rhodiola rosea extract against hypochlorous acid-induced oxidative damage in human erythrocytes.

Rhodiola rosea L. (Crassulaceae) is a plant living at high altitudes in Europe and Asia. Its roots have long been used in the traditional medical system of these geographical areas to increase the organism resistance to physical stress; today, it has become an important component of many dietary supplements. In this study we investigate the antioxidant capacity of the R. rosea aqueous extract evaluating its ability to counteract some of the main damages induced by hypochlorous acid (HOCl), a powerful oxidant generated by activated phagocytes, to human erythrocytes. Ascorbic acid was used as a reference substance because of its physiological HOCl-scavenging ability. Our study demonstrates that R. rosea is able to significantly protect, in a dose-dependent manner, human RBC from glutathione (GSH) depletion, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) inactivation and hemolysis induced by the oxidant. Furthermore, we demonstrate that R. rosea aqueous extract acts from the inside of the erythrocyte suggesting a probable involving of cell components. The protection on GSH afforded by the R. rosea extract with respect to ascorbic acid, occurred also if added 2 or 5 min. later than the oxidant, suggesting a more rapid or powerful effect.

Aqueous extract from Vitis vinifera tendrils is able to enrich keratinocyte antioxidant defences.

An aqueous extract of V. vinifera L. tendrils was evaluated for its ability to enrich the antioxidant capacity of cultured cells. The long-time antioxidant capability of the extract was measured by in vitro chemical methods, and its influence on reduced glutathione levels and plasma membrane oxido reductase activity was determined in cultured human keratinocytes (NCTC 2544). Keratinocytes are cells normally exposed to oxidative stress, and for this reason adequately equipped with antioxidant defences. However, it has long been suggested that exogenous antioxidants may play an important role in minimizing the adverse effects of oxidative stress on skin.We demonstrated that V. vinifera tendril aqueous extract was able to increase, in a time- and dose-dependent manner, the reduced glutathione concentration and activity of trans plasma membrane oxido reductase as an indirect evaluation of the intracellular redox status of the cells demonstrating a relevant antioxidant activity of this phytocomplex.

Chemical characterization of Sacha Inchi (Plukenetia volubilis L.) oil.

A chemical characterization of the major components, namely, triacylglycerols (TAGs), polyphenols, and tocopherols in a Sacha inchi oil derived from cold pressing of the seed, is hereby reported. To tackle such a task, high-performance liquid chromatography in combination with photodiode array (PDA), fluorescence (RF), and mass spectrometry (MS) detection was employed. The latter was interfaced with atmospheric pressure chemical ionization and with electrospray ionization for the analysis of TAGs and polyphenols, respectively, whereas RF detection was tested for the determination of tocopherol content. Furthermore, fatty acid methyl esters (FAMEs) were evaluated by gas chromatography-flame ionization detector. A 93% amount of total fatty acids was represented by unsaturated FAMEs with the greatest percentage represented by linoleic (L) and linolenic (Ln) accounting for approximately 50 and 36%, respectively. The main TAGs (>10%) were represented by LLnL, LnLnLn, and LnLLn; the latter was present in the oil sample at the highest percentage (22.2%). Among tocopherols, γ-tocopherol was detected to be the most abundant component (over 50%). The polyphenolic composition was also investigated, and a total of 15 compounds were positively identified, through the complementary analytical information coming from PDA and MS data. To the best of our knowledge, this is the first report providing a thorough chemical characterization of a Plukenetia volubilis L. oil.

Effect of surgical stress on nuclear and mitochondrial DNA from healthy sections of colon and rectum of patients with colorectal cancer.

Surgical resection at any location in the body leads to stress response with cellular and subcellular change, leading to tissue damage. The intestine is extremely sensitive to surgical stress with consequent postoperative complications. It has been suggested that the increase of reactive oxygen species as subcellular changes plays an important role in this process. This article focuses on the effect of surgical stress on nuclear and mitochondrial DNA from healthy sections of colon and rectum of patients with colorectal cancer. Mitochondrial DNA copy number, mitochondrial common deletion and nuclear and mitochondrial 8-oxo-2'-deoxyguanosine content were measured. Both the colon and rectal tissue were significantly damaged either at the nuclear or mitochondrial level. In particular, mitochondrial DNA was more damaged in rectum than in colon. The present investigation found an association between surgical stress and nuclear and mitochondrial DNA damage, suggesting that surgery may generate an increase in free radicals, which trigger a cascade of molecular changes, including alterations in DNA.

Rhodiola rosea ability to enrich cellular antioxidant defences of cultured human keratinocytes.

Keratinocytes are cells strongly exposed to oxidative stress, but normally good equipped for antioxidant responses. However, it has long been suggested that exogenous antioxidants could play a useful role in minimizing the adverse skin responses associated with such oxidant species. In this work it was paid attention to the extract of Rhodiola rosea L. roots by using the phytocomplex as a whole because of the important activity of its composition and mutual distribution of its components. We have measured the protection afforded by the extract to reduced glutathione levels, glyceraldehyde-3-phosphate dehydrogenase activity, and thiobarbituric acid reactive substances levels in cultured human keratinocytes (NCTC 2544) exposed to different oxidative insults: Fe(II)/ascorbate, Fe(II)/H(2)O(2), and tert-butyl-hydroperoxide. We also have investigated the influence of the R. rosea extract on the production of intracellular reactive oxygen species and on the activity of antioxidant enzymes (catalase, superoxide dismutase, glutathione peroxidase, and glutathione reductase). Furthermore, we have demonstrated that R. rosea extract was able to increase in a time- and dose-dependent manner the activity of the trans plasma membrane oxido reductase activity as an indirect evaluation of the intracellular redox status and this effect was already evident with small concentration of the extract and in a long time. As a result, NCTC 2544 are able to better counteract to several oxidative insults if incubated with R. rosea extract demonstrating a very good antioxidant activity of this phytocomplex.

Effects of high static magnetic field exposure on different DNAs.

The effects of magnetic fields produced by permanent magnets on different DNA sources were investigated in vivo and in vitro. Escherichia coli DNA, plasmid, and amplification products of different lengths were used as the magnetic field target. The in vivo assays did not reveal any DNA alterations following exposure, demonstrating the presence of cell dependent mechanisms, such as the repair system and the buffering action of the heat shock proteins DNA K/J (Hsp 70/40). The in vitro assays displayed interactions between the magnetic field and DNA, revealing principally that magnetic field exposure induces DNA alterations in terms of point mutations. We speculate that the magnetic field can perturb DNA stability interacting with DNA directly or potentiating the activity of oxidant radicals. This genotoxic effect of the magnetic field, however, is minimized in living organisms due to the presence of protective cellular responses.