Quantitation and metabolism of mitoquinone, a mitochondria-targeted antioxidant, in rat by liquid chromatography/tandem mass spectrometry

Mitoquinone (MitoQ10 mesylate) is a mitochondria-targeted antioxidant undergoing development for the treatment of neurodegenerative diseases. The aim of this study was to develop and validate an assay based on liquid chromatography/tandem mass spectrometry (LC/MS/MS) to determine mitoquinone and to detect and identify the metabolites of MitoQ10 in rat plasma after an oral dose. After a simple protein precipitation step, plasma samples were analyzed by reversed-phase liquid chromatography using gradient elution with acetonitrile/water/formic acid. Electrospray ionization in the positive ion mode with multiple reaction monitoring (MRM) was used to analyze mitoquinone employing the deuterated compound (d3-MitoQ10 mesylate) as internal standard. The calibration curve for mitoquinone was linear over the concentration range 0.5-250 ng/mL with a correlation coefficient>0.995. The method was sensitive (limit of quantitation 0.5 ng/mL) and had acceptable accuracy (relative error<8.7%) and precision (intra- and inter-day coefficient of variation<12.4%). Recoveries of mitoquinone at concentrations of 1.5, 20 and 200 ng/mL were in the range 87-114%. The method was successfully applied to a pharmacokinetic study in rat after a single oral dose in which four metabolites of MitoQ10 were tentatively identified as hydroxylated MitoQ10, desmethyl MitoQ10 and the glucuronide and sulfate conjugates of the quinol form of MitoQ10.     

In vitro synergistic efficacy of conjugated linoleic acid,oleic acid,safflower oil and taxol cytotoxicity on PC3 cells

The aim of this study was to determine in vitro synergistic efficacy of conjugated linoleic acid (CLA), oleic acid (OLA), safflower oil and taxol (Tax) cytotoxicity on human prostate cancer (PC3) cell line. To determine synergistic efficacy of oil combinations, PC3 treated with different doses of compounds alone and combined with 10 μg/mL Tax. The MTT results indicated that OLA–Tax combinations exhibited cytotoxicity against PC3 at doses of 30 nM+10 μg-Tax, 15 nM+5 μg-Tax and 7.5 nM+2.5 μg-Tax. The treatment of OLA or Tax did not show significant inhibition on PC3, while OLA–Tax combinations showed effective cytotoxicity at treated doses. CLA–Tax combinations demonstrated the same effect on PC3 as combined form with 45.72% versus the alone form as 74.51% viability. Cytotoxic synergy between Tax, OLA and CLA shows enhanced cytotoxicity on PC3 which might be used in the therapy of prostate cancer.     

On-Line Solid Phase Extraction and Spectrophotometric Detection with Flow Technique for the Determination of Nanomolar Level Ammonium in Seawater Samples

The determination of low-level ammonium in seawaters suffers from low sensitivity and high contamination; therefore, it is desirable to develop highly sensitive methods for automatic measurements. A highly sensitive and automated flow technique system for nanomolar level ammonium measurement is described. Reagents for Berthelot reaction were automatically added into seawater samples. After a 10 min reaction at 40°C, the formed indophenol blue compound was on-line extracted onto an Hydrophilic-Lipophilic Balance (HLB) cartridge. The enriched compound was rinsed with water and ethanol solution (30%, v/v) and, in turn, eluted with an eluent containing 30% (v/v) ethanol and 5.0 mM of NaOH, and determined with a spectrophotometer at 640 nm. Parameter, including extraction conditions, reagent concentrations, pH, temperature, and reaction time, were optimized. Under the optimized conditions, the detection limit was 3.5 nM and the linear range was 0–428 nM. The relative standard deviations were 5.7% (n = 8) for 44.6 nM standard solution and less than 6.0% (n = 3–5) for samples within concentrations of about 52.4–288.7 nM; the recovery was in the range 93.6 to 108.5%. The sample throughput was 3 h^?1. The proposed method provides a simple, cheap, and automatic way to determine ammonium in seawater samples without complicated sample treatment.     

Evaluation of antioxidants on the thermo-oxidative stability of soybean biodiesel

This work shows the evaluation of three antioxidants (2,6-di-t-butyl-4-methylphenol (BHT)—synthetic antioxidant, hydrogenated cardanol (HC), and alkyl hydrogenated cardanol (AHC)—both derived from cashew nut shell liquid) on the thermo-oxidative stability of the soybean biodiesel. The antioxidants were added at concentrations of 200, 300, and 400 ppm, and the oxidative stability of the biofuel with and without antioxidants were investigated by thermogravimetric analysis (TG-DTG and IPDT) and Metrohm 743 Rancimat per the EN 14112 method. The results showed that all antioxidants contributed for the thermo-oxidative stability of the soybean biodiesel as follows: soybean biodiesel < soybean biodiesel + BHT < soybean biodiesel + HC < soybean biodiesel + AHC. In the Rancimat method, the results showed that the antioxidants influenced the biodiesel stability with an increase of at least 71 %.     

Fractionation of Protein Hydrolysates of Fish and Chicken Using Membrane Ultrafiltration: Investigation of Antioxidant Activity

In this work, chicken and fish peptides were obtained using the proteolytic enzymes α-Chymotrypsin and Flavourzyme. The muscle was hydrolyzed for 4 h, and the resulting peptides were evaluated. Hydrolysates were produced from Argentine croaker (Umbrina canosai) with a degree of hydrolysis (DH) of 25.9 and 27.6 % and from chicken (Gallus domesticus) with DH of 17.8 and 20.6 % for Flavourzyme and α-Chymotrypsin, respectively. Membrane ultrafiltration was used to separate fish and chicken hydrolysates from Flavourzyme and α-Chymotrypsin based on molecular weight cutoff of >1,000, <1,000 and >500, and <500 Da, to produce fractions (F1,000, F1,000–500, and F500) with antioxidant activity. Fish hydrolysates produced with Flavourzyme (FHF) and α-Chymotrypsin showed 60.8 and 50.9 % of peptides with a molecular weight of <3 kDa in its composition, respectively. To chicken hydrolysates produced with Flavourzyme and α-Chymotrypsin (CHC) was observed 83 and 92.4 % of peptides with a molecular weight of <3 kDa. The fraction that showed, in general, higher antioxidant potential was F1,000 from FHF. When added 40 mg/mL of FHF and CHC, 93 and 80 % of lipid oxidation in ground beef homogenates was inhibited, respectively. The composition of amino acids indicated higher amino acids hydrophobic content and amino acids containing sulfuric residues for FHF, which showed antioxidant potential.     

Olive oil and its phenolic constituent tyrosol attenuates dioxin-induced toxicity in peripheral blood mononuclear cells via an antioxidant-dependent mechanism

Olive oil (OO) and its phenolic compounds are reported to possess many potential biological effects, which are ascribed to its powerful antioxidant property. In this study, we have assessed whether OO and its phenolic compound tyrosol (TY) could mitigate 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induced oxidative damages in peripheral blood mononuclear cells (PBMC). The results showed that exposure of PBMC to 10 nM TCDD caused significant cell death and elevated cellular concentrations of reactive oxygen species and lipid peroxidation. Comet assay indicated that OO and TY protected DNA damage against dioxin toxicity. In addition, alterations in levels of antioxidant enzymes were substantially prevented by OO and TY. TCDD-induced CYP1A1 activity and loss of mitochondrial membrane potential were significantly reduced by the administration of OO and TY. The results suggested that dietary modifications incorporating diets rich in OO and associated phenolics could prove beneficial in protecting individuals against toxicity induced by dioxins.     

Enhanced Resveratrol Production in Vitis vinifera Cell Suspension Cultures by Heavy Metals Without Loss of Cell Viability

The effects of heavy metal ions (Co²⁺, Ag⁺, Cd²⁺) on cell viability and secondary metabolite production, particularly anthocyanins and phenolic acids in Vitis vinifera cell suspension cultures, were investigated. Of these, Co at all three used concentrations (5.0, 25, and 50 μM), Ag, and Cd at low concentration (5.0 μM) were most effective to stimulate the phenolic acid production, increasing the 3-O-glucosyl-resveratrol up to 1.6-fold of the control level (250.5 versus 152.4 μmol/g), 4 h after the treatments. Meanwhile, the elicitors at effective concentrations did not suppress cell growth, while the cell viability maintained. In contrast, Ag and Cd at high concentrations (25 and 50 μM) remarkably reduced the cell viability, decreasing the cell viability up to about 15 % of the control level, 24 h after the treatments. The heavy metal ions did not affect the anthocyanin production. These observations show how, in a single system, different groups of secondary products can show distinct differences in their responses to potential elicitors. The 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity, peroxidase activity, medium pH value, and conductivity were only slightly elevated by the heavy metal ions. The results suggest that some of the secondary metabolites production was stimulated by the used elicitors, but there was not a stress response of the cells.     

Silver–palm pollen nanocomposite exhibits antiproliferative,antioxidant, and proapoptotic properties on MCF-7 breast cancer cells

The aim of the present study is to evaluate the antioxidant and proapoptotic effects of silver–palm pollen (Ag/PP) nanocomposite. The percentage of live cells after treatment with various concentrations of Ag/PP (0, 5, 10, 20, and 40 µM/mL) was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. The antioxidant potential of Ag/PP was measured via the scavenging effects of 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and molecular analysis. Apoptosis was assessed by morphological analysis, fluorescent dye, and flow cytometry, and its fundamental mechanism studied based on evaluation of Bax and Bcl2 gene expression. Ag/PP nanocomposite suppressed the viability of MCF-7 cells (dose and time dependently) and showed antioxidant properties. Morphological changes associated with cell death were observed in treated cells. Accumulation of dead cells in sub-G1 phase confirmed the occurrence of apoptosis in exposed cells. In addition, NPs induced cell death by altering Bcl-2 expression in MCF-7 cells. These results indicate that Ag/PP nanocomposite is an effective combination for removal of cancer cells by induction of apoptosis and could be useful for human health due to its antioxidant effects.     

Antioxidant and antiproliferative activity of Stachys glutinosa L. ethanol extract

Ethanol extracts of Stachys glutinosa L. (Lamiaceae) were investigated for antioxidative properties, as well as antiproliferative action on various cell lines. The antioxidant activities were investigated by ABTS (2,2′-azinobis-3-ethylbenzothiazoline-6-sulphonic acid) assay, DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging, β-carotene/linoleic acid assay, scavenging of hydrogen peroxide (horseradish peroxidase test), superoxide anion scavenging, and hypochlorous acid scavenging (taurine test). The antioxidant activity was reported as IC₅₀ and reveals antioxidant effects. Antiproliferative effects were measured in vitro on three cell lines: HepG2 (human hepatocarcinoma), MCF7 (breast human adenocarcinoma) and C2C12 (mouse myoblast) cell lines by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The ethanol extract induced variations in cell viability on all cell lines tested. At 200 μg/mL, the effects on cell viability were ? 23%, ? 27% and ? 37%, respectively, for C2C12, MCF7 and HepG2.     

Rice husk ash nanosilica to inhibit human breast cancer cell line (3T3)

NaOH treatment on phase, purity and particle size of rice husk nanosilica were investigated employing alkaline-extraction–acid-precipitation method. Mild reaction at lower NaOH (1.5 N) revealed small and fine particles (600 nm) due to moderate reaction rate. Morphology of human breast cancer cell line (3T3) revealed that suggestive cell distress effects were more pronounced at 500 µg/ml silica dosages which are in agreement with cell viability decrease (~50 %). Among different silica dosages, 10 µg/ml was found to have highest cell viability (～70 ± 2 %) capable of increasing dissolved oxygen level while viability was almost decreased to 27 ± 5 % due to toxic effect caused at higher SiO₂ dosage and could be a potential candidate best suited for biomedical applications through development of sustainable materials.     

The differences in thermal profiles between normal and leukemic cells exposed to anticancer drug evaluated by differential scanning calorimetry

Chronic lymphocytic leukemia (CLL) is a heterogenous disease with an imbalance between apoptosis and cell proliferation. Therefore, the main goal in CLL therapy is to induce apoptosis and effectively support this process in transformed B lymphocytes. In the current study, we have compared differential scanning calorimetry (DSC) profiles of nuclei isolated from CLL cells and normal mononuclear cells exposed to cladribine or fludarabine combined with mafosfamide (CM; FM), and additionally to CM combined with monoclonal antibody—rituximab (RCM) for 48 h, as well as in culture medium only (controls). Under current study, the mononuclear cells from peripheral blood (PBMCs) of healthy individuals have been included. The obtained results have shown the presence of thermal transition at 95 ± 5 °C in most of nuclear preparations (92.2 %) isolated from blood of CLL patients. This thermal characteristic parameter was changed after drug exposure, however, to a different extent. These thermal changes were accompanied by the decrease of cell viability, an elevation of apoptosis rate and the changes in expression/proteolysis of poly(ADP-ribose)polymerase-1—main marker of apoptosis. Importantly, in DSC profiles of nuclear preparations of PBMCs from blood of healthy donors exposed to investigated drug combinations and control CLL cells, the lack of such changes was observed. Our results confirmed that DSC technique complemented with other biological approaches could be helpful in tailoring therapy for CLL patients.     

Ethylene Epoxidation over Alumina-Supported Silver Catalysts in Low-Temperature AC Corona Discharge

In this paper, the epoxidation of ethylene over different catalysts—namely Ag/(low-surface-area, LSA)α-Al₂O₃, Ag/(high-surface-area, HSA)γ-Al₂O₃, and Au–Ag/(HSA)γ-Al₂O₃—in a low-temperature corona discharge system was investigated. In a comparison among the studied catalysts, the Ag/(LSA)α-Al₂O₃ catalyst was found to offer the highest selectivity for ethylene oxide, as well as the lowest selectivity for carbon dioxide and carbon monoxide. The selectivity for ethylene oxide increased with increasing applied voltage, while the selectivity for ethylene oxide remained unchanged when the frequency was varied in the range of 300–500 Hz. Nevertheless, the selectivity for ethylene oxide decreased with increasing frequency beyond 500 Hz. The optimum Ag loading on (LSA)α-Al₂O₃ was found to be 12.5 wt.%, at which a maximum ethylene oxide selectivity of 12.9% was obtained at the optimum applied voltage and input frequency of 15 kV and 500 Hz, respectively. Under these optimum conditions, the power consumption was found to be 12.6 × 10^?16 W s/molecule of ethylene oxide produced. In addition, a low oxygen-to-ethylene molar ratio and a low feed flow rate were also experimentally found to be beneficial for the ethylene epoxidation.     

Effects of ELF Magnetic Field in Combination with Iron(III) Chloride (FeCl3) on Cellular Growth and Surface Morphology of Escherichia coli (E. coli)

This study investigated the effects of extremely low frequency (ELF) magnetic field with/without iron(III) chloride (FeCl₃) on bacterial growth and morphology. The ELF exposures were carried out using a pair of Helmholtz coil-based ELF exposure system which was designed to generate 50 Hz sinusoidal magnetic field. The field was approximately uniform throughout the axis of the coil pair. The samples which were treated or non-treated with different concentrations FeCl₃ were exposed to 50 Hz, 2 millitesla (mT) magnetic field for 24 h. ELF effect on viability was assessed in terms of viable colony counts (in colony-forming unit per milliliter) with the standard plate count technique. Scanning electron microscopy was used to investigate the magnetic field effect on surface morphology of Escherichia coli. No significant results were seen in terms of cell viability between ELF and sham-exposed bacterial strains. Similarly, FeCl₃ treatment did not change cell viability of E. coli samples. However, we observed some morphological changes on E. coli cell surfaces. Pore formations and membrane destruction were seen on the surface of 24 h ELF field-exposed cells. We concluded that ELF magnetic field exposure at 2 mT does not affect cell viability; however, it may affect bacterial surface morphology.     

Antidiabetic,cytotoxic and antioxidant activities of oil extracted from Acrocomia aculeata pulp

This study evaluated the hypoglycemic effect of the oil extracted from the Acrocomia aculeata pulp (OPAC) in normoglycemic rats and streptozotocin (STZ), fructose-induced diabetic rat models and its in vitro antioxidant and cytotoxic potential. OPAC (3, 30 or 300 mg/kg, v.o.) significantly decreased (p < 0.05) the high glucose levels induced by a high fructose-diet in rats. Persistent treatment with OPAC for 24 days also reduced the high plasmatic glucose induced by STZ. In normoglycemic animals, OPAC significantly decreased glucose levels. While A. aculeata oil exhibited good in vitro antioxidant activity, no sign of cytotoxicity was observed in LLC-PK1 cells (5–500 μg/mL). OPAC has antidiabetic and antioxidant activities without causing in vitro cytotoxicity.     

Evaluation of in vitro antioxidant activities of soyasaponins from soy hypocotyls in human HepG2 cell line

Soyasaponins were shown to have a wide range of biological activities in previous studies; however, the activities of their monomeric compounds are unclear. The aim of this study was to evaluate the in vitro antioxidant activities of soyasaponins in HepG2 cells. Four soyasaponins were isolated from soy hypocotyls and identified as soyasaponin Aa, Ab, Ba, and Bb. The protective effects of these soyasaponins against production of hydrogen peroxide-induced reactive oxygen species in cells were investigated. The cellular antioxidant activity of soyasaponins was found to be in a dose-dependent manner at concentrations ranging between 25 and 400 μg/mL in 24 h. Finally, based on cell morphology observations, group A soyasaponins showed better cellular antioxidant activity and anti-oxidative enzyme activity than group B ones, with an optimal concentration of 100 μg/mL.     

LC with Coulometric Detection for Analysis of 5-Methyltetrahydrofolate in Human Plasma

An isocratic high-performance liquid chromatographic method with coulometric electrochemical detection has been used for analysis of 5-methyltetrahydrofolate (5-MTHF) in human plasma. A 250 mm × 4.6 mm i.d., 5-μm particle, C₁₈ column was used with 12:88 (v/v) acetonitrile ?35 mM sodium phosphate buffer pH 3.8 as mobile phase at a flow rate of 1.0 mL min^?1. The method was validated for 5-MTHF plasma concentrations in the range 2.5–100.0 nM. The method was characterized by a good linearity (regression coefficient r ≥ 0.9989) and limits of detection and quantification of 0.72 and 2.16 nM, respectively. Mean recovery at low and high concentrations ranged from 89.1 to 96.3%, respectively, with a relative standard deviation <4.6%. Between-run imprecision (4.2%) was higher than within-run imprecision (3.4%). The proposed separation and detection procedures were successfully applied to analysis of 5-MTHF in human plasma.     

Electrochemical study of Aloe-emodin on an ionic liquid-type carbon paste electrode

An ionic liquid-type carbon paste electrode (CPIE) was fabricated from 1-butyl-3-methylimidazolium hexafluorophosphate mixed with paraffin oil as the binder. The electrochemistry of Aloe-emodin (AE) at the electrode was investigated at pH 1.0 by cyclic voltammetry and differential pulse voltammetry. The ionic liquid as a binder distinctly enhances the electron transfer rate between AE and the electrode. A redox mechanism is discussed. The CPIE showed good sensitivity, selectivity and stability, and was applied to determine the concentration of AE. Under the optimal conditions, the oxidative peak current increased linearly with the concentration of AE in the range from 10 nM to 12.4 µM, with a correlation coefficient of 0.9973. The detection limit is 3.0 nM (at S/N?=?3).     

CZE Determination of Quinolinic Acid in Rat Brain Tissue and Plasma

In this paper, a capillary zone electrophoretic method for the determination of the excitotoxic quinolinic acid in rat brain tissue (cerebellum, cortex, hippocampus, striatum) and plasma samples is described. Optimum separation of the excitotoxic quinolinic acid was achieved with a 14.4 mM boric acid/5.6 mM sodium tetraborate electrolyte solution at pH 8.84. The applied voltage was 30 kV and the capillary temperature was kept constant at 25 °C. The regression equations revealed a good linear correlation between the peak area and the concentration. The method was linear over the concentration range of 0.50 to 600 nM. All correlation coefficients were higher or equal to 0.9998. To optimize the analysis conditions, the effects of electrolyte solution pH, the concentration, and the use of methanol as an organic modifier were systematically studied. The amount of quinolinic acid in the rat brain tissue and plasma under control conditions were found to be: cerebellum 30.2 ± 1.7 nM (mean ± standard deviation); cortex 5.6 ± 0.7 nM; hippocampus 64.2 ± 9.4 nM; striatum 4.3 ± 0.6 nM, and plasma 40.1 ± 2.3 nM. The limits of detection and quantification were 0.47 nM (signal/noise = 3) and 1.58 nM, respectively. The method was successfully applied to quantify quinolinic acid in the rat brain striata under two neurotoxicity models with good repeatability (RSD < 10%) and recovery (98–102%). The proposed analytical method could be useful to clarify the role of quinolinic acid in neurodegenerative entities such as Alzheimer’s and Huntington’s diseases.     

Rapid speciation of trace iron in rainwater by reverse flow injection analysis coupled to a long path length liquid waveguide capillary cell and spectrophotometric detection

A method for speciation and determination of low levels of dissolved iron in rainwater was established by coupling reverse flow injection analysis with a 2-m liquid waveguide capillary cell and spectrophotometric detection. Ferrozine solution was injected into a sample stream to form an Fe(II)-ferrozine complex with Fe(II), and the absorbance of this complex was detected at both 562 nm and 625 nm with a reference wavelength at 700 nm. Fe(III) was analyzed in the same manner after being reduced to Fe(II) by ascorbic acid. The optimum conditions and the interference of Cu(I), Ni(II) and Co(II) were investigated. The limits of detection were 0.1 nM for Fe(II) and 0.2 nM for Fe(III), while the linear ranges were 0.4 – 200 nM for Fe(II) and 0.8 – 287 nM for Fe(III) at 562 nm, and can be extended to higher concentrations with the detection at a less sensitive wavelength of 625 nm. The sample throughput was 6 h^?1, and the total sample volume consumed was 10 mL. This method has been successfully applied to analyze dissolved iron in rainwater of Xiamen from August to November, 2008. The lowest level of iron in rainwater was observed during typhoon events. By adopting reverse flow injection analysis coupled with liquid waveguide long path length capillary cell, the reagent consumption was low and the sensitivity was enhanced. The other advantages of this method are high sample throughput, wide linear dynamic range and high selectivity for Fe(II).     

Growth, Osmolyte Concentration and Antioxidant Enzymes in the Leaves of Sesuvium portulacastrum L. Under Salinity Stress

In this study, growth and osmolyte concentration in the leaves of halophyte, Sesuvium portulacastrum, were studied with respect to salinity. Therefore, the changes in shoot growth, leaf tissue water content, osmolyte concentration (proline content, glycine betaine) and antioxidant enzymes [polyphenol oxidase (PPO), superoxide dismutase (SOD) and catalase (CAT)] were investigated. The 30-day old S. portulacastrum plants were subjected to 100, 200, 300, 400, 500 and 600 mM NaCl for 28 days. The plant growth was steadily increased up to 500 mM NaCl stress at 28 days. TWC was higher in 300 mM NaCl treated leaves than that of 600 mM NaCl. Salinity stress induced the accumulation of osmolyte concentration when compared to control during the study period. The antioxidant enzymes PPO, CAT and SOD were increased under salinity.