Rapid and specific detection of hydroxyl radical using an ultraweak chemiluminescence analyzer and a low-level chemiluminescence emitter: application to hydroxyl radical-scavenging ability of aqueous extracts of Food constituents

With the availability of an ultraweak chemiluminescence analyzer, it is possible to monitor the production of a specific oxygen-derived reactive species, such as hydroxyl radical ((*)OH), whenever a suitable chemiluminescent probe is obtainable. Reported herein is the development of a rapid and specific method for detecting (*)OH production using a specific probe, indoxyl-beta-glucuronide (IBG), a low-level chemiluminescence emitter. Using the Fenton reagent as a source of (*)OH, it was shown that IBG could elicit a very strong intensity of chemiluminescence (CL) (16200 +/- 200 photon counts/s). Conversely, IBG was shown to be insensitive to either superoxide radical or hydrogen peroxide with their CL intensities nearly close to the background values (25 +/- 5 and 180 +/- 20 photon counts/s, respectively). Furthermore, it was also shown that this IBG-based CL production could be effectively quenched by the addition of (*)OH scavengers such as sodium salicylate, dimethyl sulfoxide, and penicillamine to the assay system. Taken together, these data indicate that IBG is a specific CL probe suitable for monitoring the production of (*)OH. This system demonstrated inhibitory activities of various aqueous extracts of food constituents on the CL of hydroxyl radicals generated by Fenton's reagents with the order of scavenging efficiencies being Prunus mume > Cordyceps sinensin > Lilium lancifolium > Astragalus membranceus.     

Biochemical susceptibility of myosin in chicken myofibrils subjected to hydroxyl radical oxidizing systems

Biochemical changes of myosin in chicken myofibrils exposed to nonenzymatic, hydroxyl radical generation systems (HRGS) were investigated by means of cross-linking reaction, ATPase activity, salt solubility, and 40% saturated ammonium sulfate (AS) extractability. HRGS treatment of myofibrils caused cross-linking of myosin heavy chains (MHC) via disulfide bonding, an increase in Ca-ATPase activity, and a decrease in K-ATPase activity, suggesting that thiol groups of myosin including those at the active site were modified. The specific changes depended on the concentrations of H(2)O(2) in HRGS as well as the weight ratio of H(2)O(2) to myofibrils. On the other hand, the decrease in salt solubility or AS extractability of myosin in HRGS-treated samples proceeded slowly when compared with the cross-linking reaction of MHC, indicating that considerable amounts of myosin biopolymers remained hydrophilic in the ionic solutions. The results demonstrated that initial cross-linking of MHC occurred inside the myosin molecule, and this was followed by progressive aggregation of myosin molecules through intermolecular cross-linking. Oxidation under the current experimental condition decreased the gel-forming ability of myofibrillar proteins, which coincided with the progress of the intra- and intermolecular cross-linking reactions as well as with ATPase activity changes.     

Role of hydroxyl radicals and singlet oxygen in the formation of primary radicals in unsaturated lipids: a solid state electron paramagnetic resonance study

Primary radicals were generated by UV photolysis of samples of trilinolein, at 77 K and under a controlled atmosphere. The resulting EPR spectra clearly show that the amount of radicals is dependent on the purity of the lipid, the exposure to visible light in the presence of a photosensitizer and oxygen, and, finally, the presence of an antioxidant. These solid state EPR experiments indicate that if all of the elements for the production of singlet oxygen (Rose Bengal, molecular oxygen, and visible light) are not present, primary radicals are practically not generated. They also point out the various steps of the oxidation mechanism: formation of singlet oxygen, which reacts with the lipid to form a hydroperoxide; and photolytic formation of the hydroxyl radical, which reacts with the frozen lipid to generate primary lipidic radicals. This constitutes a new method for investigating lipid oxidation and studying the influence of photosensitizers and molecules that are likely to react with singlet oxygen.     

Selective growth inhibition of human leukemia and human lymphoblastoid cells by resveratrol via cell cycle arrest and apoptosis induction

There is great interest in the potential chemopreventive activity of resveratrol against human cancers. However, there are conflicting results on its growth inhibitory effect on normal cells. This project examined the differential effect of resveratrol at physiologically relevant concentrations on nonmalignant (WIL2-NS) and malignant (HL-60) cell lines and compared the underlying mechanisms via cell cycle modulation, apoptosis induction, and genotoxicity potential. Twenty-four hours of exposure to resveratrol was toxic to WIL2-NS and HL-60 cells in a dose-dependent manner. WIL2-NS cells regrew 5 times more than HL-60 cells by 120 h after the removal of 100 microM resveratrol (p < 0.05). Furthermore, significant alterations in cell cycle kinetics were induced by resveratrol in HL-60 cells, but were to a lesser extent for WIL2-NS cells. The proportion of apoptosis was also 3 times higher in HL-60 cells as compared to WIL2-NS cells for 100 microM resveratrol (p < 0.05). In conclusion, resveratrol preferentially inhibited the growth of HL-60 cells via cell cycle modulation and apoptosis induction and subsequently directed the cells to irreversible cell death, whereas the effect on WIL2-NS cells was largely reversible.     

Semicarbazide formation in azodicarbonamide-treated flour: a model study

Semicarbazide was previously found in foods that were in contact with rubber gaskets foamed at high temperatures with a blowing agent azodicarbonamide. Because azodicarbonamide is an approved flour additive in certain countries, we set out to ascertain if semicarbazide is formed during the baking process from flours containing that additive. The levels of semicarbazide in baking flour treated with azodicarbonamide and bread baked from such flours were determined by isotope dilution (13C15N2-semicarbazide) liquid chromatography electrospray tandem mass spectrometry (LC-MS/MS). The samples were homogenized with HCl, extracted with n-pentane, derivatized with 2-nitrobenzaldehyde, and the derivative was extracted with ethyl acetate. After solvent exchange to 10% acetonitrile in water containing 0.1% acetic acid, the samples were analyzed using a 2.1 mm x 150 mm C18 column eluted with 2 mM ammonium formate in water/methanol (40:60). Semicarbazide was formed during the dry heating of commercial azodicarbonamide-containing flours at temperatures of 150-200 degrees C reaching levels of 0.2 mg/kg. Similar levels of semicarbazide were found in the crusts of breads made from azodicarbonamide-treated flour.     

Detection of free radical transfer in lipoxygenase I-B-catalyzed linoleic acid-soybean protein interaction by electron spin resonance spectroscopy (ESR)

Effects of lipoxygenase I-B (LOX)-catalyzed oxidation of linoleic acid on soybean proteins was evaluated by electron spin resonance (ESR) and fluorescence spectroscopy in different model systems in the presence or absence of antioxidants. A strong central singlet signal was detected by ESR spectroscopy and identified as the carbon radical (g value range 2.0041-2.0054). A downfield shoulder attributed to the sulfur radical (g value 2.019-2.028) was also observed. The changes in soybean proteins were accompanied by an increase in fluorescence, indicating the formation of cross-links. Natural antioxidants such as ascorbic acid and alpha-tocopherol as well as synthetic antioxidants butyl hydroxytoluene (BHT) inhibited the development of both the free radical signal and the fluorescence when added to soybean proteins prior to incubation with linoleic acid and lipoxygenase I-B; the central singlet signal attributed to the carbon radical was reduced by 35-65%. This paper clearly indicates direct free radical transfer from oxidizing linoleic acid catalyzed by LOX to soybean proteins.     

Concurrent phenomena contributing to the formation of the aroma of wine during aging in oak wood: an analytical study

Red wine was stored in different oak barrels or in stainless steel, and samples were taken for two years to determine 79 aroma compounds. Aging in oak affects 41 compounds. The type of wood affects 11 compounds. At least seven different processes seem to take place concurrently in aroma evolution, and five such processes, affecting 37 compounds, are linked to the oak cask. These are extraction from the wood, oxidation of wine alcohols and amino acids, microbiological formation of ethyl phenols, sorption processes, and condensation of acetaldehyde with polyphenols. The wood can release linear gamma- and delta-lactones, beta-damascenone, and ionones. Some compounds are released very fast from wood, which suggests they lie in the external part of the wood. Some extraction profiles are too complex to be explained by physical processes. Finally, the levels of 2,5-dimethyl-4-hydroxy-3(2H)-furanone and 2-ethyl-5-methyl-4-hydroxy-3(2H)-furanone increase even in the reference wine, which suggests the presence of a precursor.     

Effect of black soybean extract on the suppression of the proliferation of human AGS gastric cancer cells via the induction of apoptosis

Black soybean is known to have a health-promoting effect because of its high content of polyphenolic compounds and antioxidant activities. The objective of the present study was to investigate the chemopreventive effects of black soybean extract against human AGS gastric cancer cells and its possible mechanism in inducing apoptosis. Black soybean extract was obtained by extracting black soybean with acidified aqueous acetone, and its phytochemical constituents, as determined by HPLC-DAD methods, were demonstrated to contain various phenolics. The black soybean extract inhibited AGS cell growth in a dose-dependent manner with an IC(50) of 3.69 mg/mL as measured by the MTT assay. This growth inhibition effect was further confirmed by the CFDA-SE assay. Flow cytometry analysis showed that black soybean extract dose-dependently induced apoptosis of AGS cells. Moreover, the involvement of black soybean extract in inducing apoptosis was confirmed by the expression of Bax, Bcl-2, caspase-3, and PARP. The results of the present study indicated that black soybean extract could be used as an apoptosis inducer in AGS cells and a natural chemopreventive agent in the treatment of human gastric cancer.     

Metabolic profiling: a new tool in the study of wood formation

In the realm of plant genomics, metabolic profiling has become a valuable tool with which to assess the effect of genetic and/or environmental factors on plant development. This paper reports the first application of metabolic profiling on differentiating xylem tissue of loblolly pine. A protocol is presented for the analysis of loblolly pine xylem tissue. The effects of sample preparation, extraction, and derivatization on the corresponding metabolite profiles and yields have been investigated and are reported. Gas chromatography-mass spectroscopy has been used to quantify >60 polar and lipophilic metabolites from wood-forming tissue. It was possible to assign chemical structures to approximately half of these compounds. Comparison of six loblolly pine genotypes, three high cellulose (50-52%) and three medium (45-48%) cellulose, showed distinct metabolic profiles. Principal component analysis enabled the assignment of metabolic phenotypes using these large data sets. Metabolic phenotype clustering occurred in which the three high-cellulose genotypes were segregated from the medium-cellulose genotypes. These results demonstrate the use of metabolic profiling for the study of wood-forming tissue and as a tool in functional genomics.     

Determination of tea polyphenols and caffeine in tea flowers (Camellia sinensis) and their hydroxyl radical scavenging and nitric oxide suppressing effects

The native occurrence of tea polyphenols, namely, (-)-epicatechin, (+)-catechin, (-)-epigallocatechin 3-gallate, (-)-epicatechin, and (-)-epicatechin 3-gallate, and caffeine in tea flowers was assessed by an isocratic HPLC procedure. The levels of total catechins and caffeine were determined in tea flowers collected from 10 different species of Camellia sinensis. The results showed the levels of total catechin ranged from 10 to 38 mg/g, whereas the level of caffeine ranged from 3 to 8 mg/g. Levels of catechins and caffeine in tea leaves and various teas were also determined and ranged from 2 to 126 mg/g and from 23 to 49 mg/g, respectively. Both tea flower and tea leaf extracts exert their strong hydroxyl radical scavenging effects in the Fenton reaction system and nitric oxide suppressing effects in LPS-induced RAW 264.7 cells. Most tea flowers contain less caffeine, but comparable amounts of total catechins, compared to tea leaves and teas. The present study demonstrates that both tea flowers and tea leaves contain appreciable amounts of catechins and caffeine. It is likely that tea flowers might be useful for making alternative tea beverages.     

Electron spin resonance (ESR) studies on the formation of roasting-induced antioxidative structures in coffee brews at different degrees of roast

The antioxidative properties of coffee brew fractions were studied using electron spin resonance spectroscopy using 2,2,6,6-tetramethyl-1-piperidin-1-oxyl (TEMPO) and Fremy's salt (nitrosodisulfonate) as stabilized radicals. TEMPO was scavenged by antioxidants formed during roasting and not by chlorogenic acid, whereas Fremy's salt was scavenged by all antioxidants tested including chlorogenic acid. The stabilized radical TEMPO allowed the exclusive measurement of roasting-induced antioxidants. The roasting-induced antioxidant activity of coffee brews increased with increasing degree of roast, and most of these antioxidants were formed during the initial roasting stage. The majority of these roasting-induced antioxidants were present in the high molecular weight fractions, indicating that the formation of these antioxidants preferably occurs at specific high molecular weight structures, likely being arabinogalactan and/or protein moieties which might be part of the melanoidin complex. It was found that chlorogenic acids most probably do not lose their antioxidant activity and phenolic characteristics upon incorporation in coffee melanoidins. The parameter fast reacting antioxidants (FRA) was introduced as an alternative for the antioxidative potential. FRA levels showed that coffee fractions rich in roasting-induced antioxidants exposed their antioxidant activity relatively slowly, which must be a consequence of its complex structure. Finally, the melanoidin content and the roasting-induced antioxidant activity showed a positive and linear correlation for the coffee brew fractions, showing that roasting-induced antioxidants are present within melanoidins. This is the first time that the formation of roasting-induced antioxidants could be directly correlated with the extent of Maillard reaction and melanoidin formation in a complex product such as coffee.     

Reproducible induction of cavity spot in carrots and physiological and microbial changes occurring during cavity formation

Inoculation of carrots with 40 types of bacteria, both aerobic and anaerobic, including clostridia isolated from cavity spots, failed to induce cavity spot in carrots. A combined stress of minimum 6 h flooding and temperatures higher than 28°C clearly induced cavity formation. Sugars, amino acids, lipids and minerals leaked from the carrots after flooding and heating the roots. A longer growth period following stress markedly increased cavity spots. Soil types (sandy loam and loess) and several carrot cultivars tested had no marked effect on spot formation. Cavities were formed in stressed carrots grown in sterilized soil containing only one type of bacterium, a Gram-negative short rod. Scanning electron micrographs revealed that after carrots were subjected to combined stress, microscopic cavities nearly free of bacteria were formed under the epidermis. Proliferation of bacteria was concommitant with the appearance of visible cavities. Cell-free extracts of infected carrots showed higher protease and pectinase-specific activities, as well as significantly higher peroxidase and polyphenoloxidase activities and total phenol content as compared to healthy carrots.     

Effect of hesperetin against oxidative stress via ER- and TrkA-mediated actions in PC12 cells

Hesperetin is known to activate estrogen receptors (ERs). Estrogen-mediated neuroprotection could be via both ER and tyrosine kinase receptor (Trk) signaling. This study tested whether hesperetin protected PC12 cells from hydrogen peroxide induced oxidative damage via ER- and/or TrkA-mediated actions. Hesperetin (0.1, 1, and 50 μM) inhibited cell viability decreases and reactive oxygen species, intracellular calcium level, and caspase-3 activity increases in H(2)O(2)-induced PC12 cells. Such actions were significantly (p < 0.05) suppressed by ICI 182,780 (an ER antagonist) or K252a (a TrkA antagonist) at low concentrations (0.1 or 1 μM) only. Hesperetin also stimulated the activation of Akt, ERK, and CREB as well as induced brain-derived neurotrophic factor, PPARγ coactivator 1α (PGC-1α), and seladin-1 (selective Alzheimer's disease indicator-1) via both ER and TrkA in the cells. This study demonstrates that the neuroprotective effects of hesperetin, at low concentrations, are attributed to its stimulation on receptor signaling. Moreover, ER and TrkA are known to be expressed in most Alzheimer's disease (AD) vulnerable brain regions. This study thus suggests that hesperetin might have potential for intervention in neurodegenerative disorders, particularly for AD.     

Mechanisms involved in the antiplatelet activity of rutin, a glycoside of the flavonol quercetin, in human platelets

The aim of this study was to systematically examine the inhibitory mechanisms of rutin, a well-known flavonoid in platelet aggregation. In this study, rutin concentration-dependently (250 and 290 microM) inhibited platelet aggregation in human platelets stimulated by agonists (i.e., collagen). Rutin (250 and 290 microM) did not significantly interfere with the binding of FITC-triflavin to the glycoprotein IIb/IIIa complex in human platelets. Rutin (250 and 290 microM) markedly inhibited intracellular Ca(2+) mobilization and thromboxane A(2) formation in human platelets stimulated by collagen. Rapid phosphorylation of a platelet protein of M(r) 47000 (P47), a marker of protein kinase C activation, was triggered by collagen (1 microg/mL). This phosphorylation was markedly inhibited by rutin (250 and 290 microM). On the other hand, rutin (250 and 290 microM) did not significantly increase the formations of cyclic AMP and nitric oxide/cyclic GMP in platelets. In conclusion, these results indicate that the antiplatelet activity of rutin may involve the following pathways: rutin inhibited the activation of phospholipase C, followed by inhibition of protein kinase C activity and thromboxane A(2) formation, thereby leading to inhibition of the phosphorylation of P47 and intracellular Ca(2+) mobilization, finally resulting in inhibition of platelet aggregation.     

In vitro study of the relationship between the structure of ginsenoside and its antioxidative or prooxidative activity in free radical induced hemolysis of human erythrocytes

Ginsenoside, the major active component in Panax ginseng, which has been used in traditional Chinese medicine, contains a series of derivatives of the triterpene dammarane being attached by some sugar moieties. To clarify the relationship between the structure of ginsenoside and its properties, 11 individual ginsenosides, along with the central structures of ginsenoside, protopanaxadiol and protopanaxatriol, are used in 2,2'-azobis(2-amidinopropane hydrochloride) (AAPH) induced hemolysis of human erythrocytes, a good experimental model to research free radical induced membrane damage and to evaluate the antioxidative or prooxidative activities of various antioxidants conveniently. It is found that the central structures of ginsenosides, either protopanaxadiol or protopanaxatriol, play a prooxidative role in AAPH-induced hemolysis of erythrocytes. As to the individual ginsenoside, if there are no sugar moieties attached to the 20-position of the triterpene dammarane, the ginsenoside acts as a prooxidant, that is, Rg3, Rh2, and Rg2. A glucose attached to the 6-position instead of the 20-position sugar moieties can make the ginsenoside an antioxidant, that is, Rh1. The antioxidants among ginsenosides follow two different mechanisms that can be expressed mathematically by the Boltzmann equation, that is, Rc and Rb1, and a polynomial equation, that is, Re, Rd, R1, Rg1, Rb3, and Rh1. The orders of antioxidative ability are Rc > Rb1 and Re > Rd > R1 > Rg1 > Rb3 > Rh1, respectively.     

Artifact formation during smoke trapping: an improved method for determination of N-nitrosamines in cigarette smoke

Studies in our laboratory revealed artifactual formation of N-nitrosamines during trapping of mainstream and sidestream tobacco smoke by the method of Hoffmann and coworkers (2, 4). Both volatile and tobacco-specific N-nitrosamines were produced. This artifact formation took place on the Cambridge filter, which is part of the collection train used in the previously published procedure. When the filter was treated with ascorbic acid before smoke collection, artifact formation was inhibited. The improved method resulting from these studies was applied to a comparative analysis of N-nitrosamines in smoke from cigarettes that heat, but do not burn, tobacco (the test cigarette) and several reference cigarettes. Concentrations of volatile and tobacco-specific N-nitrosamines in both mainstream and sidestream smoke from the test cigarette were substantially lower than in the reference cigarettes.     

Evolution of wheat gliadins conformation during film formation: a fourier transform infrared study

The secondary structures of wheat gliadins (a major storage protein fraction from gluten) in film-forming solutions and their evolution during film formation were investigated by Fourier transform infrared spectroscopy. In the film-forming solution, wheat gliadins presented a mixture of different secondary structures, with an important contribution of beta-turns induced by proline residues. The presence of plasticizer did not have any influence on protein secondary structure in the film-forming solution. The evolution of protein conformation was followed during drying; the major feature of this evolution was a clear growing of the infrared band at 1622 cm(-1), characteristic of intermolecular hydrogen-bonded beta-sheets. This revealed the formation of protein aggregates during film drying. The influence of the drying temperature on film properties and gliadin secondary structures was also investigated. Higher drying temperatures induced an increase of both the tensile strength of the films and the amount of beta-sheets aggregates. Although the appearance of heat-induced disulfide bridge cross-links has already been described, there is clear evidence that hydrogen-bonded beta-sheets aggregates are also induced by thermal treatment. It was not possible, however, to determine whether there is a direct relationship between the occurrence of these aggregates and the increase of the tensile strength of the films.     

Identification of adducts between an odoriferous volatile thiol and oxidized grape phenolic compounds: kinetic study of adduct formation under chemical and enzymatic oxidation conditions

HPLC-MS and (1)H, (13)C, and 2D NMR analyses were used to identify new addition products between 3-sulfanylhexan-1-ol (3SH) and o-quinones derived from (+)-catechin, (-)-epicatechin, and caftaric acid. The kinetics of formation of these adducts were monitored in a wine model solution and in a must-like medium by HPLC-UV-MS with the aim of understanding the chemical mechanism involved in reactions between volatile thiols and o-quinones. One o-quinone-caftaric acid/3SH adduct, three o-quinone-(+)-catechin/3SH adducts, and three o-quinone-(-)-epicatechin/3SH adducts were characterized. Caftaric acid was oxidized faster than (-)-epicatechin and (+)-catechin when these phenolic compounds were incubated in a one-component mixture with polyphenoloxidase (PPO) in the presence of 3SH. Consequently, o-quinone-caftaric acid formed adducts with 3SH more rapidly than o-quinone-(+)-catechin and o-quinone-(-)-epicatechin in the absence of other nucleophilic species. Furthermore, o-quinone-(-)-epicatechin reacted faster than o-quinone-(+)-catechin with 3SH. Sulfur dioxide decreased the yield of adduct formation to a significant extent. Under chemical oxidation conditions, the rates and yields of adduct formation were lower than those observed in the presence of PPO, and o-quinone-caftaric acid was slightly less reactive with 3SH, compared to oxidized flavan-3-ols. The identification of o-quinone-caftaric acid/3SH and o-quinone-(+)-catechin/3SH adducts in a must matrix suggests that the proposed reaction mechanism is responsible for 3SH loss in dry wines during their vinification and aging process.     

Hemolytic anemia and induction of phase II detoxification enzymes by diprop-1-enyl sulfide in rats: dose-response study

Epidemiological evidence indicates that a high dietary intake of plants of the Allium family, such as garlic and onions, is associated with a decreased risk of cancer in humans. It has been suggested that this chemopreventative effect involves the ability of the aliphatic sulfides derived from these vegetables to increase tissue activities of phase II detoxification enzymes. Several highly effective inducers from garlic have been identified, but most of the previously studied compounds from onion have proved to be only weakly active. In the present study, the inductive activity of another onion-derived sulfide, diprop-1-enyl sulfide, has been investigated. This substance was a potent inducer of phase II enzymes in rats, showing significant effects in the lungs and in the lower part of the gastrointestinal tract, suggesting that diprop-1-enyl sulfide could be a useful chemopreventative agent at these sites. At high dose levels, diprop-1-enyl sulfide caused hemolytic anemia, which may be due to in vivo conversion of the sulfide to active metabolites.     

New insights into an ancient antibrowning agent: formation of sulfophenolics in sodium hydrogen sulfite-treated potato extracts

The effect of sodium hydrogen sulfite (S), used as antibrowning agent, on the phenolic profile of potato extracts was investigated. This extract was compared to one obtained in the presence of ascorbic acid (A). In the presence of A, two major compounds were obtained, 5-O-caffeoylquinic acid (5-CQA) and 4-O-caffeoyl quinic acid. With S, their 2'-sulfo-adducts were found instead, the structures of which were confirmed by nuclear magnetic resonance spectroscopy and mass spectrometry. Also, for minor caffeoyl derivatives and quercetin glycosides, the corresponding sulfo-adducts were observed. Feruloyl and sinapoyl derivatives were not chemically affected by the presence of S. Polyphenol oxidase (PPO) was thought to be responsible for the formation of the sulfo-adducts. This was confirmed by preparing 2'-sulfo-5-O-caffeoyl quinic acid in a model system using 5-CQA, sodium hydrogen sulfite, and PPO. This sulfo-adduct exhibited a small bathochromic shift (λmax 329 nm) as compared to 5-CQA (λmax 325 nm) and a strong hypochromic shift with an extinction coefficient of 9357±395 M(-1) cm(-1) as compared to 18494±196 M(-1) cm(-1), respectively. The results suggest that whenever S is used as an antibrowning agent, the O-quinone formed with PPO reacts with S to produce sulfo-O-diphenol, which does not participate in browning reactions.