Structural analysis of water-soluble glucans from the root of Angelica sinensis (Oliv.) Diels

Two water-soluble glucans (designated APS-1cI and APS-1cII) were extracted from the roots of Angelica sinensis (Oliv.) Diels and further purified by anion-exchange and gel-filtration chromatography. Their molecular weights were determined to be 1.7 x 10(5) and 3.9 x 10(4)Da, respectively. The structures of the purified glucans were investigated by a combination of chemical and instrumental analysis, such as methylation analysis, periodate oxidation, GC-MS, as well as FTIR and NMR spectroscopy ((1)H, (13)C, H-H COSY, HSQC, HMBC, TOCSY and NOESY). The data obtained indicated that APS-1cI was a linear alpha-glucan composed of only (1-->6)-alpha-D-Glcp, and APS-1cII had a repeating unit consisting of (1-->4)-alpha-D-Glcp and (1-->6)-alpha-D-Glcp in a molar ratio of 4:1. Such glucans isolated from A. sinensis (Oliv.) Diels have not been previously reported.     

Effects on free radical generation by ligands of the peripheral benzodiazepine receptor in cultured neural cells

The effect of peripheral benzodiazepine receptor (PBR) ligands on free radical production was investigated in primary cultures of rat brain astrocytes and neurons as well as in BV-2 microglial cell lines using the fluorescent dye dichlorofluorescein-diacetate. Free radical production was measured at 2, 30, 60 and 120 min of treatment with the PBR ligands 1-(2-chlorophenyl-N-methylpropyl)-3-isoquinolinecarboxamide (PK11195), 7-chloro-5-(4-chlorophenyl)-1,3-dihydro-1-methyl-2H-1,4-benzodiazepin-2-one (Ro5-4864) and protoporphyrin IX (PpIX) (all at 10 nm). In astrocytes, all ligands showed a significant increase in free radical production at 2 min. The increase was short-lived with PK11195, whereas with Ro5-4864 it persisted for at least 2 h. PpIX caused an increase at 2 and 30 min, but not at 2 h. Similar results were observed in microglial cells. In neurons, PK11195 and PpIX showed an increase in free radical production only at 2 min; Ro5-4864 had no effect. The central-type benzodiazepine receptor ligand, clonazepam, was ineffective in eliciting free radical production in all cell types. As the PBR may be a component of the mitochondrial permeability transition (MPT) pore, and free radical production may occur following induction of the MPT, we further investigated whether cyclosporin A (CsA), an inhibitor of the MPT, could prevent free radical formation by PBR ligands. CsA (1 micro m) completely blocked free radical production following treatment with PK11195 and Ro5-4864 in all cell types. CsA was also effective in blocking free radical production in astrocytes following PpIX treatment, but it failed to do so in neurons and microglia. Our results indicate that exposure of neural cells to PBR ligands generates free radicals, and that the MPT may be involved in this process.     

Antioxidant properties and protective effects of a standardized extract of Hypericum perforatum on hydrogen peroxide-induced oxidative damage in PC12 cells

Free radical scavenging and antioxidant activities of a standardized extract of Hypericum perforatum (SHP) were examined for inhibition of lipid peroxidation, for hydroxyl radical scavenging activity and interaction with 1,1-diphenyl-2-picrylhydrazyl stable free radical (DPPH). Concentrations between 1 and 50 microg/ml of SHP effectively inhibited lipid peroxidation of rat brain cortex mitochondria induced by Fe2+/ascorbate or NADPH system. The results showed that SHP scavenged DPPH radical in a dose-dependent manner and also presented inhibitory effects on the activity of xanthine oxidase. In contrast, hydroxyl radical scavenging occurs at high doses. The protective effect of the standardized extract against H2O2-induced oxidative damage on the pheochromocytoma cell line PC 12 was investigated by measuring cell viability via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), lactate dehydrogenase (LDH) assays, caspase-3-enzyme activity and accumulation of reactive oxygen species [2',7'-dichlorofluorescin (DCF) assay]. Following 8-h cell exposure to H2O2 (300 microM), a marked reduction in cell survival was observed, which was significantly prevented by SHP (pre-incubated for 24 h) at 1-100 microg/ml. In a separate experiment, different concentrations of the standardized extract (0.1-100 microg/ml) also attenuated the increase in caspase-3 activity and suppressed the H2O2 -induced reactive oxygen species generation. Taken together, these results suggest that SHP shows relevant antioxidant activity both in vitro and in a cell system, by means of inhibiting free radical generation and lipid peroxidation.     

Glutathione-dependent protection by rat liver microsomal protein against lipid peroxidation

GSH is an important cellular defense against oxidant injury. Its effect in the rat liver microsomal lipid peroxidation system has been examined. Incubation of fresh rat liver microsomes with ascorbic acid and ADP-chelated iron leads to the peroxidation of microsomal lipids (production of thiobarbituric acid-reactive substances and destruction of polyunsaturated fatty acids) following a 2 to 5 min lag. Addition of 0.1 mM GSH to the system lengthened the lag period by 5 to 15 min without affecting the rate or the extent of lipid peroxidation. GSH could not be replaced in prolonging the lag by cysteine, mercaptoethanol, dithiothreitol, propylthiouracil, or GSSG. The GSH effect on the lag was abolished by heating or trypsin digestion of the microsomes, indicating that microsomal protein is required for its expression. Progressively longer lags were observed as the GSH concentration was increased from 0.1 to 5 mM, but there was no evidence of GSH oxidation as a consequence of the protection against lipid peroxidation. GSH protected against heat inactivation of the microsomal protein responsible for the GSH effect. Experiments with an oxygen electrode revealed that the GSH protection did not alter the ratio of O₂ consumed to thiobarbituric acid-reactive substances produced. This implicated free radical scavenging as the mechanism of protection. These results indicate the existence of a GSH-dependent rat liver microsomal protein which scavenges free radical. This protein may be an important defense against free radical injury to the microsomal membrane.     

Fucoxanthin as the major antioxidant in Hijikia fusiformis, a common edible seaweed

The radical scavenging activity of Japanese edible seaweeds was screened by the DPPH (1-diphenyl-2-picrylhydrazyl) assay to evaluate the DPPH radical scavenging activity in organic extracts. The fresh brown alga Hijikia fusiformis showed the strongest DPPH radical scavenging activity, followed by Undaria pinnatifida and Sargassum fulvellum. The major active compound from Hijikia fusiformis in its acetone extract was identified as fucoxanthin by 13C-NMR spectroscopy.     

Anti-plasmodial and antioxidant activities of constituents of the seed shells of Symphonia globulifera Linn f

A xanthone derivative, named gaboxanthone (1), has been isolated from the seed shells of Symphonia globulifera, together with known compounds, symphonin (2), globuliferin (3), guttiferone A (4), sistosterol, oleanolic acid and methyl citrate. The structure of the compound was assigned as 5,10-dihydroxy-8,9-dimethoxy-2,2-dimethyl-12-(3-methylbut-2-enyl) pyrano [3,2-b]xanthen-6(2H)-one, by means of spectroscopic analysis. The anti-plasmodial and antioxidant activities of the phenolic compounds were evaluated, respectively, in culture against W2 strain of Plasmodium falciparum and using the free radical scavenging activity of the DPPH radical, respectively. Compounds 1-4 were found to be active against the Plasmodium parasites (IC(50) of 3.53, 1.29, 3.86 and 3.17 microM, respectively). Guttiferone A (4) showed a potent free radical scavenging activity compared to the well-known antioxidant caffeic acid.     

Free radical scavenging and antioxidant activities of EPS2, an exopolysaccharide produced by a marine filamentous fungus Keissleriella sp. YS 4108

The reactive oxygen species (ROS) and free radical-initiated reactions are ascertained to play multiple roles in degenerative or pathological events such as aging, cancer, heart dysfunction and Alzheimer's disease. EPS2 with a mean molecular weight of 1.3 x 10(5) was characterized as an antioxidant exopolysaccharide from the broth of a marine filamentous fungus Keissleriella sp. YS 4108. Compositionally, it is composed of galactose, glucose, rhamnose, mannose and glucuronic acid in an approximate proportion of 50:8:1:1:0.4. The radical eliminating and antioxidant actions of the glycan was assessed in different in vitro systems showing that EPS2 exhibited profound scavenging activities in superoxide radical. As a reinforcement of the action, similar radical scavenging effects of EPS2 were also discerned with both site-specific and non site-specific hydroxyl radical using the deoxyribose assay method. Moreover, EPS2 effectively blocked as well the non site-specific strand-breaking of DNA induced by the Fenton reaction at concentrations of 0.1 and 1 mg/mL. Further investigation of the effect of EPS2 on human low density lipoprotein (LDL) system demonstrated that it significantly inhibited copper-mediated oxidation of LDL in a dose-dependent manner. These results suggest that EPS2, possessing pronounced free radical scavenging and antioxidant activities, could be of considerable preventive and therapeutic significance to some life-threatening health problems such as cancer, atherogenesis and Alzheimer's disease which pathologically initiated by the presence of free radicals leading to the inevitable peroxidation of important biomolecules.     

Vialinin A, a novel 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenger from an edible mushroom in China

While screening for bioactive compounds from edible mushrooms, a new potent antioxidant, vialinin A (1), together with a known compound, ganbajunin B (2), and a mixture of ganbajunins D (3) and E (4), were isolated from the dry fruiting bodies of Thelephora vialis. The structure of 1, 5',6'-bis(phenylacetoxy)-1,1':4',1'-terphenyl-2',3',4,4'-tetraol, was elucidated by spectroscopic and chemical methods. This compound had strong 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical-scavenging activity with an EC(50) value of 14.0 microM, nearly equal to that of butylated hydroxytoluene (BHT; EC(50) = 10.0 microM). A radical scavenging experiment using 1 and DPPH radicals indicated that 1 donated two hydrogen atoms to two molecules of the DPPH radical under hydrophobic conditions.     

高速逆流色谱仪分离纯化芦荟多糖的研究

采用紫外-可见分光光度计法进行了高速逆流色谱技术分离芦荟多糖的溶剂系统研究,得出了高速逆流色谱分离芦荟多糖的溶剂系统为w(PEG600)∶w(KH2PO4)∶w(K2HPO4)∶w(H2O)=5∶15∶15∶65,加入NaCl的质量分数为2%。在水浴温度30℃,转速600 r/min,下相流速为2 mL/min的条件下,采用高速逆流色谱技术成功分离出芦荟多糖粗品,得到APS-1和APS-2两个组分,经Sephadex G-100凝胶柱层析技术和高效凝胶渗透色谱技术初步分析:APS-1和APS-2均为单一组分。     

DPPH free radical scavenger components from the fruits of Alpinia rafflesiana Wall. ex. Bak. (Zingiberaceae)

The methanol extract of the dried ripe fruits of Alpinia rafflesiana was investigated for its DPPH free radical scavenger constituents. 2',3',4',6'-Tetrahydroxychalcone (7), which has never been isolated from natural sources was found to be most active as a DPPH free radical scavenger with the IC50 value of 55 microM. Other known compounds isolated from this species include 5,6-dehydrokawain (1), flavokawin B (2). 1,7-diphenyl-5-hydroxy-6-hepten-3-one (3), (-)-pinocembrin (4), cardamonin (5) and (-)-pinostrobin (6). The DPPH free radical scavenger compounds were detected using TLC autographic analysis. The percentage inhibition of DPPH free radical scavenging activity was measured on isolates (5-7) using colorimetric analysis.     

An electron spin resonance study on alkylperoxyl radical in thin-sliced renal tissues from ferric nitrilotriacetate-treated rats: the effect of alpha-tocopherol feeding.

Formation of excess free radical causes cellular oxidative stress, which has been shown to be associated with a variety of pathologic conditions. While electron spin resonance (ESR) spectroscopy has been the only method to demonstrate the presence of free radicals, its application to tissue samples has been challenging. We report here the successful ESR detection in thin-sliced fresh tissues or frozen sections in a rat model. Ferric nitrilotriacetate (Fe-NTA) induces oxidative renal tubular damage that ultimately leads to high incidence of renal carcinoma in rodents. Twenty minutes after administration of 5 mg iron/kg Fe-NTA to rats, a thin-slice of the kidney was mounted on a tissue-type cell and analyzed by ESR spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). An ESR signal from alkylperoxyl radical adduct was obtained, and the signal was inversely proportional to renal alpha-tocopherol content which was modulated through diet. Furthermore, we undertook ex vivo study using frozen sections. Fe-NTA (1 mM) was added to a rat kidney frozen section for 10 min. After washing the specimen was mounted on a tissue-type cell and analyzed with ESR spin trapping using DMPO. Alkylperoxyl radical signal was dependent on thickness, incubation time and renal tissue levels of alpha-tocopherol, and was reduced by preincubation with catalase or dimethyl sulfoxide but not with alpha-tocopherol outside tissue. This versatile method facilitates identification of free radicals in pathologic conditions, and may be useful for selection of antioxidants.     

In the neuronal cell line SH-SY5Y, oxidative stress-induced free radical overproduction causes cell death without any participation of intracellular Ca(2+) increase

Adding the membrane-permeant oxidant tert-butylhydroperoxide (t-BOOH) to the incubation medium, in SH-SY5Y human neuroblastoma cells, induced a marked and progressive concentration-dependent (300, 500 and 1000 microM) increase of free radical production, as evaluated by the fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) and of the intracellular Ca(2+) ion concentrations [Ca(2+)](i). The removal of extracellular Ca(2+) ions did not prevent t-BOOH-induced [Ca(2+)](i) elevation, whereas the intracellular Ca(2+) ion chelator 1,2-bis(o-aminophenoxy) ethane-N,N, N',N'-tetraacetic acid (BAPTA) (10 microM) was shown to be effective. Both t-BOOH-induced free radical formation and the [Ca(2+)](i) increase were completely prevented by the peroxyl scavenger alpha-tocopherol (50 microM). t-BOOH induced a time-dependent SH-SY5Y cell injury, monitored by a 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay (approximately 25% at 1 h, 50% at 3 h, 80% at 5 h) and by fluorescein diacetate (FDA)-propidium iodide (PI) fluorescent staining. The entity of t-BOOH-induced cell damage was the same both in the absence and in the presence of the intracellular Ca(2+) ion chelator BAPTA. By contrast, the peroxyl scavenger alpha-tocopherol (50 microM) completely prevented cell injury due to oxidative stress. Finally, superoxide dismutase (SOD) (500 ng/ml) caused a 30% reduction of t-BOOH-induced 2', 7'-dichlorofluorescein (DCF) fluorescence, whereas it did not modify the extent of cell injury produced by the oxidant. Collectively, the results of the present study demonstrated that in SH-SY5Y human neuroblastoma cells, the rise of [Ca(2+)](i) which occurs during oxidative stress is not involved in cell injury. Therefore, oxidative stress-induced cell death may be exclusively attributed to free radical overproduction.     

Phenylpropanoid glucosides from leaves of Coussarea hydrangeifolia (Rubiaceae)

Phenylpropanoid glycosides, 1'-O-benzyl-alpha-L-rhamnopyranosyl-(1'-->6')-beta-D-glucopyranoside (1) and alpha-L-xylopyranosyl-(4'-->2')-(3-O-beta-D-glucopyranosyl)-1'-O-E-caffeoyl-beta-D-glucopyranoside (2), together with the known derivatives, 1,6-di-O-caffeoyl-beta-D-glucopyranoside (3), 1-O-(E)-caffeoyl-beta-D-glucopyranoside (4) and 1-O-(E)-feruloyl-beta-D-glucopyranoside (5), were isolated from leaves of Coussarea hydrangeifolia. Their structures were determined by IR, HRESIMS, and 1D and 2D NMR experiments, and their antioxidant activities, evaluated by assaying the free radical scavenging capacity using the DPPH (1,1-diphenyl-2-picrylhydrazyl) radical as substrate. The antioxidant activities of 3 and 4 (IC50 values of 15.0 and 19.2 microM, respectively) were comparable to that of the standard positive control caffeic acid, whilst 2 and 5 were only weakly active and 1 was inactive.     

An aqueous extract of Rubus chingii fruits protects primary rat hepatocytes against tert-butyl hydroperoxide induced oxidative stress

Different in vitro free radical generating systems were used to assess the antioxidative activity of aqueous extracts of the five herbal components of Wu-zi-yan-zong-wan, a traditional Chinese medicinal formula with a long history of use for tonic effects. Fructus Rubi [Rubus chingii (Rosaceae) fruits] was found to be the most potent. It was further investigated using the primary rat hepatocyte system. tert-Butyl hydroperoxide (t-BHP) was used to induce oxidative stress. Being a short chain analog of lipid hydroperoxide, t-BHP is metabolized into free radical intermediates by the cytochrome P450 system in hepatocytes, which in turn, initiate lipid peroxidation, glutathione depletion and cell damage. Pre-treatment of hepatocytes with Fructus Rubi extract (50 microg/ml to 200 microg/ml) for 24 h significantly reversed t-BHP-induced cell viability loss, lactate dehydrogenase leakage and the associated glutathione depletion and lipid peroxidation. The amount of reactive oxygen species formed was also decreased as visualized by the fluorescence probe 2',7'-dichlorofluorescin diacetate. These results suggested that Fructus Rubi was useful in protecting against t-BHP-induced oxidative damage and may also be capable of attenuating cytotoxicity of other oxidants.     

Comparison of microbial diversity of edible flowers and basil grown with organic versus conventional methods

The consumption and use of edible flowers as food is growing; however, no study has been conducted to evaluate their role in the cause of food-borne illness or in food safety. Recent food-borne outbreaks traced to fresh herbs have raised concern about their processing and handling. Basil, one of the most commonly used fresh herbs, has been identified as a source of food-borne illness. Baseline assessments of microflora were performed, and the microbial diversity between growing methods (organic vs. conventional) was compared. DNA sequencing was used to identify the microbial flora present on fresh edible flower and basil samples. The most predominant species identified were Enterobacter hormaechei (10%), Acinetobacter calcoaceticus (10%), Enterobacter ludwigii (10%), Enterobacter asburiae (6%), and Enterobacter cowanii (6%). Pseudomonas aeruginosa (6%), Salmonella enterica (6%), and Bacillus amyloliquefaciens (2%) were also isolated. Phylogenetic analysis showed that most species of isolated bacteria belonged to the phyla Gammaproteobacteria (81.2%) and Firmicutes (18.8%). Statistical analysis, diversity index for species richness, and lineage-per-time plots showed that for basil, organically grown samples had a higher microbial diversity than conventionally grown samples. Edible flowers and basil are often grown using organic methods and are commonly consumed raw without any washing or cooking, to preserve aesthetic value, but these practices may pose a potential risk for food-borne illness. The baseline assessment, together with phylogenetic and statistical analyses, indicated possible microbial contamination in edible flowers and basil. The use of statistical estimation of molecular diversity based on the 16S rRNA sequences and lineage-per-time plots with phylogenetic analysis well served as a means for comparing microbial diversity in food samples between the growing methods (organic vs. conventional).     

Bacteriological quality of crops irrigated with wastewater in the Xochimilco plots, Mexico City, Mexico

Xochimilco county plots (Mexico City), one of the most fertile agricultural areas in the Valley of Mexico, produce a large portion of the fresh vegetables consumed in the city. These plots are generally irrigated with domestic wastewater, and for this reason, it was deemed important to examine and evaluate the bacteriological quality of the water, soil, and vegetables from these plots that are harvested and marketed. The soils were also examined for the classical parameters such as nitrates, ammonia, etc., and organic matter and texture. The crops selected for this study were radishes, spinach, lettuce, parsley, and celery because they are usually consumed raw. The highest bacterial counts were encountered in leafy vegetables, i.e., spinach (8,700 for total coliform and 2,400 for fecal coliform) and lettuce (37,000 for total coliform and 3,600 for fecal coliform). Statistically significant differences in bacterial counts between rinsed and unrinsed edible portions of the crops were observed even in rinsed vegetables, and high densities of fecal coliform were detected, indicating that their consumption represents a potential health hazard. The total coliform values found in irrigation water ranged from 4 X 10(4) to 29 X 10(4), and for fecal coliform the values ranged from 5 X 10(2) to 30 X 10(2).     

Antivirus and immune enhancement activities of sulfated polysaccharide from Angelica sinensis

This study is to synthesize sulfated Angelica polysaccharides (APSs) and investigate the activity of one of the sulfated derivatives APS-1 on murine leukemia virus in vivo. Six sulfated derivatives with degree of sulfation ranging from 0.68 to 1.91 were obtained. And the virus replication was inhibited by APS-1 at the dose of 10 and 30 mg/kg (26% and 30% inhibition respectively). Furthermore, both the percentage of CD4(+) cells and CD4(+)/CD8(+) ratio in peripheral blood cells were significantly enhanced by APS-1 at 3-30 mg/kg. In addition, the reduced thymus/body weight index by murine leukemia virus infection was increased by ASP-1 in a dose dependent manner. These results suggest that APS-1 could not only inhibit virus replication, but also improve the immune function. APS-1 may be a potential new and better antiviral drug.     

Angelica Sinensis Polysaccharides Stimulated UDP-Sugar Synthase Genes through Promoting Gene Expression of IGF-1 and IGF1R in Chondrocytes: Promoting Anti-Osteoarthritic Activity

Background

Osteoarthritis (OA) is a chronic joints disease characterized by progressive degeneration of articular cartilage due to the loss of cartilage matrix. Previously, we found, for the first time, that an acidic glycan from Angelica Sinensis Polysaccharides (APSs), namely the APS-3c, could protect rat cartilage from OA due to promoting glycosaminoglycan (GAG) synthesis in chondrocytes. In the present work, we tried to further the understanding of ASP-3c’s anti-OA activity.

Methodology/Principal Findings

Human primary chondrocytes were treated with APS-3c or/and recombinant human interleukin 1β (IL-1β). It turned out that APS-3c promoted synthesis of UDP-xylose and GAG, as well as the gene expression of UDP-sugar synthases (USSs), insulin like growth factor 1 (IGF1) and IGF1 receptor (IGF1R), and attenuated the degenerative phenotypes, suppressed biosynthesis of UDP-sugars and GAG, and inhibited the gene expression of USSs, IGF1 and IGF1R induced by IL-1β. Then, we induced a rat OA model with papain, and found that APS-3c also stimulated GAG synthesis and gene expression of USSs, IGF1 and IGF1R in vivo. Additionally, recombinant human IGF1 and IGF1R inhibitor NP-AEW541 were applied to figure out the correlation between stimulated gene expression of USSs, IGF1 and IGF1R induced by APS-3c. It tuned out that the promoted GAG synthesis and USSs gene expression induced by APS-3c was mediated by the stimulated IGF1 and IGF1R gene expression, but not through directly activation of IGF1R signaling pathway.

Conclusions/Significances

We demonstrated for the first time that APS-3c presented anti-OA activity through stimulating IGF-1 and IGF1R gene expression, but not directly activating the IGF1R signaling pathway, which consequently promoted UDP-sugars and GAG synthesis due to up-regulating gene expression of USSs. Our findings presented a better understanding of APS-3c’s anti-OA activity and suggested that APS-3c could potentially be a novel therapeutic agent for OA.     

13C NMR, GC and HPLC characterization of lipid components of the salted and dried mullet (Mugil cephalus) roe "bottarga"

13C NMR spectroscopy, in conjunction with HPLC and GC techniques, has been used to study the molecular composition of lipids extracted from commercial products of bottarga. To this goal, both the saponifiable and unsaponifiable fractions of lipid extracts were also examined by 13C NMR. Among the major lipid classes wax esters (WE) showed a concentration of more than 50mol%, triacylglycerols (TAG) and phospholipids (PL) represented a minor fraction. Concentrations up to 29mol% of free fatty acids (FFA) were found. The most represented fatty alcohol was 16:0 that accounted for more than 50%, among fatty acids the most represented were 16:1 n-7, 22:6 n-3, 18:1 n-9, 16:0, and 20:5 n-3, in particular the n-3 polyunsaturated fatty acids (PUFA) averaged 40mg/g of the edible portion. 13C NMR spectroscopy put in evidence that cholesterol was present in its free and esterified forms and its total content was measured as ca. 10mg/g of the edible portion.     

The quantitative characterization of free radical sources and traps by electromigration applications

Nowadays, very diverse human activities generate urgent demands for fast, sensitive reliable innovative tools capable of detecting major industrial, military, and other dangerous products. An important part of these compounds are free radicals. Capillary electrophoresis (CE), especially in its miniaturized format (lab-on-a-chip), and other electromigration methods offer special possibilities to resolve this problem. These measurements have a great opportuness because of very wide chemical and biological role of free radicals. Several compounds, e.g. monomers and some biologically important groups (as are nitrones) oppose oxidative challenges by virtue of their trap very rapidly oxygen- or carbon-centered radicals and generating other radical species which are stable and biochemically less harmful than the original ones. In many cases, conventionally, the relative trap capacity is measured against tert.-butylhydroperoxide (TBH). In this lecture are presented numerous important free radical species (active oxygen–, nitrogen- and carbon-centered ones, as HO, NO etc) and their adequate in vitro and in vivo applied bioanalytical methods, including liquid chromatography with electrochemical detection and mass spectrometry, gas chromatography with mass spectrometry, capillary electrophoresis, electron spin resonance and chemiluminescence analysis. A simple and highly sensitive method is the capillary zone electrophoresis with amperometric detection (CZE-AD); It was introduced to determine indirectly OH by analysing its reaction products with salicylic and dihydroxybenzoic acids. Hydroxylated radical products of these acids are often used as a relative measurement in free radical research. Accurate determination of pK(a) values is important for proper characterization of newly synthesized molecules. CZE method was used for determination of their values. Are initiated new research fields as Fenton-, electro-Fenton and photoelectro-Fenton chemistry and foreseen their perspectives.

Nitric oxide is an important cell signaling molecule in physiology and pathophysiology. An indirect method for monitoring nitric oxide (NO) by determining nitrate and nitrite by microchip capillary electrophoresis (CE) with electrochemical (EC) detection has been developed. The amount of nitrite formed in this reaction (analyzed by capillary electrophoresis) was compared with the amount of oxygen consumed (measured by polarography). Were observed a linear relationship between the amount of consumed oxygen and the amount of nitrite formed in the measured range. These results demonstrate that polarographic measurements of the amount of oxygen consumed in the reaction with NO could be used to estimate the concentration of dissolved NO in authentic media. Polarography is an adequate method also to quantitative kinetic study of the free radical activity and of the trapping capacity of different compounds. This method is based on measure of the catalytic polarografic current of Fe(III) in the presence of free radical sources (TBH, hydrogen-peroxydes), and their traps. Personal contribution of the authors in this field is discussed.