Scavenging effect of extracts of green tea and natural antioxidants on active oxygen radicals

With the use of the spin trapping methods, the scavenging effects of the extracts of green tea and other natural foods are studied. In stimulated polymorphonuclear leukocytes (PMN) system, water extract fraction 6 (F6) from green tea and green tea polyphenols (GTP) have the strongest scavenging effect on the active oxygen radicals, much stronger than vitamin C (Vc) and vitamin E (VE). Rosemary antioxidants (RA) and Curcumin (Cur) have weaker scavenging effects than Vc, but stronger than VE. In Fenton Reaction, Cur has the strongest scavenging effect (69%) on hydroxyl radicals. In irradiation, riboflavin system F6(74%) and GTP(72%) have very strong scavenging effects that are weaker than Vc, but much stronger than VE (23%). With the use of spin probe oxymetry, the oxygen consumption in respiratory burst of stimulated PMN were measured when the antioxidants existed in these systems. The results demonstrated that these antioxidants did not affect the respiratory burst of human polymorphonuclear leukocytes stimulated with PMA.     

芦丁等天然产物清除活性氧自由基O_(?)~-和·OH的ESR研究

本文用促癌剂PMA(phorbol myristate acetate)刺激人多形核白细胞(PMN)呼吸暴发产生的活性氧自由基,Fenton反应产生的羟自由基·OH,光照核黄素和黄嘌呤/黄嘌呤氧化酶体系中产生的超氧阴离子自由基O(?)为模型,用自旋捕集方法研究天然产物芦丁,槲皮素,异槲皮苷和汉防已甲素对活性氧自由基(?)和·OH的清除作用.除汉防已甲素外,其它药物都能很明显地清除PMN呼吸暴发过程中产生的活性氧自由基.芦丁和异槲皮苷对(?)的清除率分别高达78.1%和79.9%,远远大于维生素E(12.7%)的作用.除汉防已甲素外,其它三种药物对·OH的清除作用也大于维生素E.四种天然产物对O(?)和·OH的清除作用都小于维生素C.     

Lymphocytes can produce respiratory burst and oxygen radicals as polymorphonuclear leukocytes.

The respiratory burst and production of oxygen radicals by lymphocytes stimulated with phorbol myristate acetate (PMA) was studied and compared with that of polymorphonuclear leukocytes (PMN) by electron paramagnetic resonance (EPR) and spin trapping technique. Superoxide anion and hydroxyl radicals spin adducts of DMPO were detected in the stimulated PMN system, but only hydroxyl radical spin adducts of DMPO were detected in the stimulated lymphocyte system. It was proved by superoxide dismutase (SOD) and catalase that the hydroxyl radicals produced in the stimulated lymphocyte system came from superoxide anions, just like the hydroxyl radicals produced in the stimulated PMN.     

Lymphocytes can produce respiratory burst and oxygen radicals as polymorphonuclear leukocytes

The respiratory burst and production of oxygen radicals by lymphocytes stimulated with phorbol myristate acetate (PMA) was studied and compared with that of polymorphonuclear leukocytes (PMN) by electron paramagnetic resonance (EPR) and spin trapping technique. Superoxide anion and hydroxyl radicals spin adducts of DMPO were detected in the stimulated PMN system, but only hydroxyl radical spin adducts of DMPO were detected in the stimulated lymphocyte system. It was proved by Superoxide dismutase (SOD) and catalase that the hydroxyl radicals produced in the stimulated lymphocyte system came from Superoxide anions, just like the hydroxyl radicals produced in the stimulated PMN.     

Scavenging effect of schizandrins on active oxygen radicals

The reactive oxygen radicals produced from human polymorphonuclear leukocytes (PMN) stimulated with PMA (phorbol myristate acetate), hydroxyl radicals generated by a Fenton reaction, and superoxide anion radicals produced by irradiating solutions of riboflavin in the presence of EDTA have been taken as the models for production of oxygen radicals. With the use of the electron spin resonance spin trapping method, the scavenging effects of schizandrol A (solA) (5 x 10(-4) M) and schizandrin B (sinB) (5 x 10(-4) M) have been studied and compared with the effects of vitamin E (5 x 10(-4) M) and vitamin C (5 x 10(-4) M). It has been found that in cell system the scavenging effects of sinB and solA, as judged by ESR spin trappings, on hydrpxyl radicals (.OH) are greater than vitamin E and vitamin C and the scavenging effects on superoxide anion (O2) are greater than vitamin E but lower than vitamin C. With respect to the Fenton reaction, sinB has the strogest scavenging effect on .OH (77%) and solA has strong scavenging effect on .OH (63%), both of them larger than that of vitamin E (35%) and vitamin C (56%). In the riboflavin/EDTA system, the scavenging effect of sinB (46%) is smaller than that of vitamin C (96%) but larger than that of vitamin E (23%); the scavenging effect of solA is not obvious (14%). With the use of spin probe oximetry, the oxygen consumption during the respiratory burst of stimulated PMN has been measured when exposed to schizandrins. The experiment results demonstrated that they do not affect the activity of production of active oxygen radicals in the respiratory burst of PMN stimulated with PMA.     

Scavenging effects on active oxygen radicals by schizandrins with different structures and configurations

We have studied the scavenging effects of different structures and configurations of schizandrins isolated from Fructus Schizandrae, a traditional Chinese herb, on active oxygen radicals with the method of spin-trapping technique. The active oxygen radicals were produced from human polymorphonuclear leukocytes (PMN) stimulated with phorbol myristate acetate (PMA). In addition, the scavenging effects of schizandrins on hydroxyl radicals (.OH) in Fenton's reaction and the scavenging effects on superoxide anions (O2-.) in both riboflavin/EDTA and xanthine/xanthine oxidase systems have also been studied. They are compared with the scavenging effects of both Vitamin C (Vc) and Vitamin E (VE). The experimental results have shown that the scavenging effect of schizandrin B (Sin B) on the active oxygen radicals is stronger than that of S(-) Sin B and R(+) Sin B. For schizandrins of the same molecular structures with different stereoconfigurations the scavenging effects of S type of the benzene ring on active oxygen radicals are stronger than those of R type and for schizandrins of the same stereoconfigurations with different structures the scavenging effects of schizandrin C (Sin C) on the active oxygen radicals are stronger than those of Sin B.     

ESR studies on active oxygen radicals produced in the respiratory burst of human polymorphonuclear leukocytes

The mechanism and process of production of active oxygen radicals in the respiratory burst of polymorphonuclear leukocytes (PMN) stimulated with PMA (phorbol myristate acetate) was studied in this paper. The experimental results indicate that when the PMA was dilute enough or at the beginning of stimulation even when the PMA concentration was high, the spectrum of hydroxyl radical spin adducts, DMPO-OH, was dominant in the ESR spectra. However, at the maximum level of the respiratory burst, the spectrum of superoxide anion spin adducts, DMPO-OOH, was dominant.     

Effects of oxygen radicals on the conformation of sulfhydryl groups on human polymorphonuclear leukocyte membranes.

The healthy intact polymorphonuclear leukocytes (PMNs) were labeled with 4-maleimide-TEMPO spin labeling compound (MAL) to study the effects of oxygen radicals produced by phorbol myristate acetate (PMA)-stimulated PMNs on the conformation of sulfhydryl (SH) groups of PMN membrane proteins. The lipid peroxidation induced by PMA-stimulated PMNs was detected by evaluating the formation of malonaldehyde (MDA) with the thiobarbituric acid (TBA) test. From the experiments of luminol-dependent chemiluminescence (CL) and fluorometry, it was found that Chinese herbs schizandrin B (Sin B) and quercetin (Q) possessed scavenging properties for oxygen radicals produced during the PMN respiratory burst. These two herbs can also inhibit the conformation changes in SH binding sites on the PMN membrane proteins caused by oxygen radicals produced by the PMNs themselves. They also decreased the amount of MDA, which was a final product formed during lipid peroxidation.     

ESR studies on oxygen consumption during the respiratory burst of human polymorphonuclear leukocytes

Oxygen consumption during the respiratory burst of human polymorphonuclear leukocytes (PMN) stimulated with phorbol myristate acetate (PMA) was studied with spin probe oxymetry and using the transition metal ion CrOX (potassium trioxalatochromate) as a widening agent. The experimental results demonstrated that during the respiratory burst of PMN stimulated with PMA, oxygen consumption was found mainly in the intercellular medium but no change of oxygen concentration was found in the intracellular medium.     

咖啡和绿茶的抗氧化活性研究

茶和咖啡是全世界消费最多的饮料。该工作利用电子自旋共振(ESR)技术和ABTS〔2,2'-Azinobis(3-ethylbenzothiazoline-6-sulfonic acid)〕等方法,研究和比较了3种绿茶(龙井、碧螺春和毛峰)和3种咖啡的抗氧化活性,以及对ABTS 、DPPH、超氧阴离子、羟基自由基、单线态氧的清除作用和对脂质过氧化的抑制作用。利用高压液相色谱(HPLC)分析了绿茶中的抗氧化成分之一EGCG((-)-epigallocatechin gallate)和咖啡中的抗氧化成分之一绿原酸(chlorogenic acid5,CQA)的含量。结果发现,如果按每克剂量比较,对于清除ABTS 的能力而言,茶和咖啡水提取液对ABTS 清除能力各有千秋,咖啡水提取液的比龙井、毛峰水提取液的高,但与碧螺春无显著差异,咖啡B比咖啡A和C强;按每杯茶(3g/200mL)和咖啡(2.4g/200mL)对ABTS 的清除能力进行比较,咖啡的比龙井和毛峰的高但比碧螺春低,咖啡B比咖啡A和C强。对超氧阴离子、羟基和DPPH自由基及单线态氧的清除作用和对脂质过氧化的抑制作用的研究表明,如果按每克剂量比较,茶和咖啡水提取液没有明显区别;按每杯量进行比较时,茶水提取液比咖啡水提取液对自由基的清除能力稍强。HPLC结果表明,EGCG含量在毛峰中最高,5CQA含量在咖啡B中最高。该研究结果表明茶和咖啡中均含有丰富的抗氧化成分,并都具有较强的抗氧化活性和清除自由基能力。     

Ability of antioxidants to prevent oxidative mutations in Salmonella typhimurium TA102

An assay for the ability of antioxidants to prevent mutations induced by various oxidants in Salmonella typhimurium TA102 cells was developed. Protection against hydrogen-peroxide-induced mutagenicity was observed for quercetin, caffeic acid, ascorbic acid and dimethyl sulfoxide (used as a solvent for water-insoluble antioxidants). No protective effect was observed for green tea extract (weakly pro-oxidative), catechin, rutin, sinigrin, ferulic acid and alpha-tocopherol. Mutagenicity caused by tert-butyl hydroperoxide (tBOOH) was prevented most effectively by quercetin and ascorbic acid, whereas weaker effects were observed for green tea extract and for rutin, and no effect being observed for the other antioxidants tested. The results for hydrogen peroxide indicate iron chelation to be the most important protective mechanism. Radical scavenging appeared to be effective only with dimethyl sulfoxide and ascorbic acid, which are effective scavengers of hydroxyl radicals and were used here in high concentrations. It is proposed that the hydrogen-peroxide-induced mutations in the Salmonella cells are caused by hydroxyl radicals generated by iron ions closely associated with DNA. Protection against mutagenicity caused by tert-butyl hydroperoxide appears to occur mainly through the scavenging of alkoxyl and possibly of alkyl radicals.     

Sesquiterpene lactone from Wunderlichia crulsiana inhibits the respiratory burst of leukocytes triggered by distinct biochemical pathways

The sesquiterpene lactone tubiferin was chemically purified from the brazilian native plant Wunderlichia crulsiana and identified by NMR and GC/MS data. Its ability to inhibit the respiratory burst of peritoneal inflammatory polymorphonuclear leukocytes (PMN) stimulated upon addition of phorbol miristate acetate (PMA), opsonized zymosan (OZ), and N-formyl-methionyl-leucyl-phenylalanine (fMLP) was evaluated. The tubiferin inhibition was more pronounced when PMN were stimulated through the protein kinase C pathway (PMA) compared to the alternative complement pathway (OZ). The inhibition when PMN were triggered by a chemoattractant stimulus (fMLP) was similar to that achieved with OZ-stimulated phagocytes. Tubiferin showed dose-dependent effects on the PMN respiratory burst triggered by the three different substances, and also decreased substantially the carrageenan-induced mice paw edema.     

Functional activity of enucleated human polymorphonuclear leukocytes

Enucleated human polymorphonuclear leukocytes (PMN) were prepared by centrifuging isolated, intact PMN over a discontinuous Ficoll gradient that contained 20 microM cytochalasin B. The enucleated cells (PMN cytoplasts) contained about one-third of the plasma membrane and about one-half of the cytoplasm present in intact PMN. The PMN cytoplasts contained no nucleus and hardly any granules. The volume of the PMN cytoplasts was about one-fourth of that of the original PMN. Greater than 90% of the PMN cytoplasts had an "outside-out" topography of the plasma membrane. Cytoplasts prepared from resting PMN did not generate superoxide radicals (O2-) or hydrogen peroxide. PMN cytoplasts incubated with opsonized zymosan particles or phorbol-myristate acetate induced a respiratory burst that was qualitatively (O2 consumption, O2- and H2O2 generation) and quantitatively (per unit area of plasma membrane) comparable with that of intact, stimulated PMN. Moreover, at low ratios of bacteria/cells, PMN cytoplasts ingested opsonized Staphylococcus aureus bacteria as well as did intact PMN. At higher ratios, the cytoplasts phagocytosed less well. The killing of these bacteria by PMN cytoplasts was slower than by intact cells. The chemotactic activity of PMN cytoplasts was very low. These results indicate that the PMN apparatus for phagocytosis, generation of bactericidal oxygen compounds, and killing of bacteria, as well as the mechanism for recognizing opsonins and activating PMN functions, are present in the plasma membrane and cytosol of these cells.     

Murexide bleaching: a new direct assay method for characterizing reactive oxygen species

The murexide (5,5'-nitrilodibarbituric acid, monoammonium salt) is an efficient scavenger for superoxide and hydroxyl radicals. When exposed to oxygen radicals, murexide is converted to a colorless alloxan derivative and its absorbance at 520 nm decreases in proportion to the radicals produced. It is used to detect these reactive oxygen species in biochemical systems such as acetaldehyde oxidation by xanthine oxidase and the respiratory burst of polymorphonuclear leukocytes induced by phorbol 12-myristate, 13-acetate. The method was sensitive enough to allow direct monitoring of the production of superoxides from 10(6) phorbol 12-myristate, 13-acetate polymorphonuclear leukocyte-stimulated cells. Moreover, murexide bleaching is inhibited in the presence of radical scavengers, allowing a comparison of their scavenging activities.     

Oxygen-derived radicals from Trypanosoma cruzi-stimulated human neutrophils

This study provides biochemical and electron spin resonance spectroscopic evidence that contract of human polymorphonuclear leukocytes with antibody-coated Trypanosoma cruzi triggers the respiratory burst. Oxygen consumption, superoxide anion and hydrogen peroxide release were stimulated under conditions of polymorphonuclear leukocyte-mediated killing. This stimulation did not occur under non-killing conditions when antibody was omitted. A common mechanism of cytotoxicity of human polymorphonuclear leukocytes against different T. cruzi forms is suggested by the triggering of the respiratory burst by antibody-coated epimastigotes and trypomastigotes.     

Antimutagenic activity of green tea polyphenols

For centuries green tea has been a widely consumed beverage throughout the world. It is known to contain a number of pharmacologically active compounds. In this study water extracts of green tea (WEGT) and their major constituents, green tea polyphenols (GTP), were examined for antimutagenic activity. WEGT and GTP were found to significantly inhibit the reverse mutation induced by benzo[alpha]pyrene (BP), aflatoxin B1 (AFB1), 2-aminofluorene, and methanol extracts of coal tar pitch in Salmonella typhimurium TA100 and/or TA98 in the presence of a rat-liver microsomal activation system. GTP also inhibited gene forward mutation in V79 cells treated with AFB1 and BP, and also decreased the frequency of sister-chromatid exchanges and chromosomal aberrations in V79 cells treated with AFB1. The addition of GTP during and after nitrosation of methylurea resulted in a dose-dependent inhibition of mutagenicity. Studies to define the mechanism of the antimutagenic activity of GTP suggest that it may affect carcinogen metabolism, DNA adduct formation, the interaction of ultimate carcinogen or the scavenging of free radicals.     

Temperature influence on stimulated PMN respiratory burst.

Body temperature can modulate the pathogenesis of infectious, metabolic and autoimmune diseases. This effect has been attributed to several hypothesized mechanisms. Body temperature could play an important role in influencing some cellular functions of human white blood cells. In this work we examined the temperature effect on the respiratory burst in human neutrophils. Human polymorphonuclear leucocytes (PMN) were obtained from heparinized venous blood by dextran sedimentation and erythrocyte lysis with NH4Cl (0.87%). Granulocytes were stimulated with opsonized zymosan (OZ), formyl-methionyl-leucyl-phenylalanine (FMLP), phorbol myristate acetate (PMA), and monosodium urate (MSU) crystals at different temperatures (26, 37, 39, 40, 42 degrees C). The technique of luminol dependent chemiluminescence (CL) was used as indicator of oxygen free radicals (OFR) release by stimulated cells. OFR production from PMN stimulated with OZ, PMA, FMLP was higher at 37 degrees C than at 26, 39, 40, 42 degrees C (p < 0.001 OZ stimulated PMN at 40-42 degrees C; p < 0.05 PMA stimulated PMN at 42 degrees C. Significantly different from 37 degrees C value). OFR release from PMN stimulated with MSU crystals was significantly increased at 39 degrees C compared to 37 degrees C value (p < 0.001). This effect could not only be attributed to temperature influence on neutrophil activity. The specific polymorphonuclear leukocyte response to the microcrystals and the temperature influence on chemical and physical characteristics of the crystals may play an important role. We are now studying the temperature effect on activity of PMN exposed to others crystals.     

Mac-1 (CD11b/CD18) as accessory molecule for Fc alpha R (CD89) binding of IgA

IgA, the principal ligand for FcalphaRI, exists in serum as monomeric IgA and at mucosal sites as secretory IgA (SIgA). SIgA consists of dimeric IgA linked by joining chain and secretory components. Human polymorphonuclear leukocytes (PMN) and mouse PMN transgenic for human FcalphaRI exhibited spreading and elicited respiratory burst activity upon interaction with either serum or SIgA. However, PMN devoid of the beta(2) integrin Mac-1 (Mac-1(-/-)) were unable to bind SIgA, despite expression of FcalphaRI. Consistent with this, serum IgA stimulated Mac-1(-/-) PMN oxygen radical production, in contrast to SIgA. Binding studies showed the secretory component, by itself, to interact with Mac-1-expressing PMN, but not with Mac-1(-/-) PMN. These data demonstrate an essential role for Mac-1 in establishing SIgA-FcalphaRI interactions.     

Direct scavenging of free radicals by captopril, an angiotensin converting enzyme inhibitor

Captopril, an angiotensin converting enzyme (ACE) inhibitor, was hypothesized to be a potential scavenger of free radicals because of the presence of a thiol group. The scavenging action of captopril was examined against superoxide anion (O2-), hydroxyl radical (OH.), hypohalite radical (HOCL) either generated biochemically, or derived from activated polymorphonuclear leukocytes (PMN). Our results indicate that captopril is an extremely potent free radical scavenger, scavenging power being as effective as superoxide dismutase (SOD) against O2-, or dimethylthiourea against OH., but better than allopurinol against OCL. plus HOCL. Free radical scavenging action of captopril against PMN-derived free radical is equivalent to the combined effects of SOD, catalase and allopurinol.