SOD酶生物传感器筛选清除超氧阴离子自由基的活性物

目的制备超氧化物歧化酶传感器，建立清除氧自由基药物的体外筛选方法。方法将固定化铜锌超氧化物歧化酶与光纤氧传感器通过特定装置组装为酶传感器。以邻苯三酚的自氧化作为超氧阴离子的发生源，预置的固定化酶响应为内标，以已知有氧自由基清除作用的Vit C为阳性对照，验证测定方法；通过对比加入样品前后邻苯三酚自氧化速度的变化情况考察样品清除超氧阴离子自由基能力。结果酶传感器检测限为7.0 U，使用寿命大于2周。以本传感器对15种样品进行了体外清除氧自由基活性实验，分别验证和发现了部分样品的清除活性。结论所研制的传感器信号稳定性较好，测定方法简便、快速，能直观地获得氧自由基清除的动力学信息，可用于大量药物的体外初筛实验。     

Selenocarbamates are effective superoxide anion scavengers in vitro.

We investigated the superoxide anion-scavenging effects of six selenocarbamates and four thiocarbamates, using a highly sensitive quantitative chemiluminescence method. At 333 nM, six selenocarbamates and four thiocarbamates scavenged in the range of 2.9-68.7% of O(2)*-. Se-methyl N-phenylselenocarbamate and Se-methyl N-(4-methylphenyl)selenocarbamate exhibited the strongest superoxide anion-scavenging activity among the Se-selenocarbamates. In contrast, the corresponding S-thiocarbamates had moderate inhibitory effect. The 50% inhibitory concentrations (IC(50)) of Se-methyl-N-phenylselenocarbamate and Se-methyl-N-(4-methylphenyl)selenocarbamate were determined to be 140 nM and 162 nM, respectively. Thus, these compounds acted in vitro as effective and potentially useful O(2)*- scavengers.     

In vitro interactions between endogenous polyamines and superoxide anion

Endogenous polyamines with anti-inflammatory activity scavenge superoxide and possibly other oxy-radicals produced by xanthine oxidase or from stimulated polymorphonuclear leucocytes. Polyamines incubated with stimulated cells are in part metabolised. Putrescine is converted to metabolites tentatively identified as gamma-aminobutyraldehyde, delta'-pyrolline and gamma-aminobutyric acid. The metabolism of spermidine, spermine and cadaverine was not as extensively studied but metabolites were formed that gave positive reaction to Schiffs reagent on tlc plates. The possible relevance of the results to the anti-inflammatory action of polyamines is discussed.     

Superoxide anion radical scavenging activity of Cassia siamea and Cassia javanica

This study was designed to explore the antioxidant potential of hexane, chloroform, ethyl acetate, methanol, and water extract of bark and leaves of Cassia siamea and Cassia javanica by superoxide anion radical scavenging assay. The different extracts showed significant inhibition of superoxide radicals in a dose-dependant manner. Among all the bark extracts of C. siamea, the methanol extract showed the maximum inhibition of 60.5% at 800 μg/ml concentration and water extract also showed strong antioxidant potential of 51.3% at 1000 μg/ml concentration. The water extract of bark of C. javanica showed strong antioxidant potential of 55% at 1000 μg/ml concentration. The various leaf extracts of C. siamea showed moderate antioxidant potential of 25–50% at 1000 μg/ml, whereas methanol leaf extract of C. javanica showed strong antioxidant potential of 50.4% at 300 μg/ml concentration. This preliminary study indicates the antioxidant activity of the bark and leaves of C. siamea and C. javanica.     

生脉溶液对超氧阴离子自由基作用的影响

目的:研究生脉溶液对超氧阴离子自由基(O-2)的影响.方法:建立能够产生超氧阴离子的二甲基亚砜(DMSO)碱性体系,采用电子顺磁共振(EPR)方法,测定加入不同浓度溶液后体系中超氧阴离子信号的强度,采用Vitc溶液作阳性对照.结果:加入浓度为20%,40%生脉溶液时,体系中超氧阴离子信号增强,且浓度为20%时与相同浓度对照组比较,增强显著(P<0.01);加入生脉溶液浓度增高至60%,80%及100%时,超氧阴离子信号强度逐渐减少并接近零,与相同浓度VC溶液对照组比较,减少显著(P<0.01).结论:生脉溶液对超氧阴离子自由基具有双向调节作用,较低浓度时增加超氧阴离子的产生,较高浓度时具有直接清除作用.     

In vitro effect of mercury and vanadium on superoxide anion production and plasminogen activator activity of mouse peritoneal macrophages

The in vitro effects of mercuric chloride and vanadate were examined on two functions of mouse peritoneal macrophages, i.e., the superoxide anion production and the plasminogen activator (PA) activity. Vanadate, at concentrations which do not affect the viability of the cells, does not seriously alter any of these functions. High concentrations of mercury depress the respiratory burst; this effect results from loss of the reducing properties of cellular NADPH. Low concentrations of mercury stimulate the effect of phorbol 12-myristate 13-acetate on PA activity. The mechanism of this stimulation does not involve the protein kinase C system. It is hypothesized that mercury could enhance the synthesis of PA, its translocation to the cell surface, or its binding to the membrane receptors.     

Enhancement of rat and human phagocyte superoxide anion radical production by cadmium in vitro

The mechanism by which cadmium produced oxidizing effects in vivo is unknown. We show that cadmium enhances the production of superoxide anion radical (O-(2) .), a reactive oxygen species, in digitonin-stimulated phagocytes from man and rat. Cadmium concentrations ranging from 3.6 X 10(-2)M to 3.6 X 10(-4)M inhibited O-(2) . production in rat alveolar macrophages or human granulocytes. However, when activated in the presence of 3.6 X 10(-5)M cadmium, the production of O-(2) . was increased by a factor of 2.11 +/- 0.25 above control levels in human granulocytes and 3.6 +/- 0.62 above control levels in rat alveolar macrophages. This effect by levels of cadmium within the range of those occurring during in vivo toxicity might provide an explanation for the oxidizing effects of this metal ion.     

Effects of phenolcarboxylic acids on superoxide anion and lipid peroxidation induced by superoxide anion.

The effects of phenolcarboxylic acids, caffeic acid, p-coumaric acid, and ferulic acid on the generation of superoxide anion and the production of lipid peroxide induced by superoxide anion were studied. Only ferulic acid anion among the phenolcarboxylic acids scavenged superoxide. Caffeic acid and ferulic acid inhibited lipid peroxidation induced by superoxide anion. These effects were comparable to those of superoxide dismutase or DL-alpha-tocopherol.     

The contribution of the pyrogallol moiety to the superoxide radical scavenging activity of flavonoids

Sixteen flavonoids including flavonols, flavones, flavanonol and catechins, and five aromatic compounds were examined for their ability to scavenge superoxide radical (O2-*) generated enzymatically in a xanthin-xanthinoxidase system and non-enzymatically in a phenazine methosulfate-NADH system. Pyrogallol, gallic acid and its ester, were much more efficient in scavenging O2-* than catechol. The superiority of pyrogallol over catechol in the flavonoidal nucleus is apparent from the much higher O2-* scavenging activity of myricetin and epigallocatechin, which contain 3',4',5'-trihydroxyl substitution in the B-ring, compared to quercetin and epicatechin, which contain 3',4'-dihydroxyl substitution, respectively. The strong O2-* scavenging ability of pyrogallol appears to function even in the A-ring, as in baicalein, and also in the form of a pyrogalloyl ester at the C-3 position in the C-ring, as in epicatechin gallate and epigallocatechin gallate. It can be concluded that the pyrogallol moiety is an active component of flavonoids for displaying high O2-* scavenging activity. Flavonoids and aromatics were also examined to correlate their O2-* scavenging activity with their oxidizability, which was measured on the basis of electrochemical redox potential and the reducing ability of the Cu2+ ion. Aromatics such as pyrogallol, gallic acid and its ester, and flavonoids such as baicalein, epicatechin gallate and epigallocatechin gallate, in which the O2-* scavenging activity is enhanced by the presence of a pyrogallol moiety which does not belong to the B-ring, reduced the correlation between the higher O2-* scavenging activity and the lower redox potential. The O2-* scavenging activity was well correlated with the Cu2+ reducing ability of flavonoids and aromatics.     

大黄中蒽醌类成分清除氧自由基作用的研究

目的通过研究大黄中游离蒽醌类成分大黄酸、芦荟大黄素、大黄素、大黄素甲醚和大黄酚清除超氧阴离子自由基（O2.）的作用,从分子生物学的角度探讨大黄延缓衰老作用的可能机理。方法采用电子自旋共振技术（ESR）分别检测大黄中游离蒽醌类成分对O2.的清除作用。结果大黄中游离蒽醌类成分对O2.的清除作用,依次为大黄酸〉大黄酚〉大黄素〉大黄素甲醚〉芦荟大黄素。结论大黄所具有的延缓衰老的作用,可能与其所含的游离蒽醌类成分大黄酸、芦荟大黄素、大黄素、大黄素甲醚和大黄酚具有清除O2.的能力有关。     

Antioxidant activity of Hypericum androsaemum infusion: scavenging activity against superoxide radical,hydroxyl radical and hypochlorous acid

Hypericum androsaemum is a medicinal plant species containing many polyphenolic compounds, namely flavonoids and phenolic acids. Since polyphenolic compounds have high antioxidant potential, the ability of H. androsaemum infusion to act as a scavenger of reactive oxygen species (superoxide radical, hydroxyl radical and hypochlorous acid) was investigated. Superoxide radical was generated by the xanthine/xanthine oxidase and phenazine methosulphate/NADH systems. The infusion-mediated prevention of nitroblue tetrazolium reduction by the superoxide radical was used as the measured endpoint. Hydroxyl radical was generated by the Fe3+-EDTA/ascorbate Fenton system, and assayed by evaluating deoxyribose degradation using the thiobarbituric acid method. Hypochlorous acid scavenging activity was tested by measuring the inhibition of hypochlorous acid-induced 5-thio-2-nitrobenzoic acid oxidation to 5,5'-dithiobis(2-nitrobenzoic acid). The tested infusion mainly exhibited a potent scavenging effect on superoxide radicals (although a noncompetitive inhibitory effect on xanthine oxidase was also observed). The infusion also acted as a moderate scavenger of hydroxyl radicals and hypochlorous acid. A phytochemical study of the infusion was also undertaken, and nine phenolic compounds were identified.     

Discriminative protection against hydroxyl and superoxide anion radicals by quercetin in human leucocytes in vitro.

Antioxidants play a vital role in the cellular protection against oxidative damage. Quercetin is a well-investigated antioxidant and known to be able to protect against cellular oxidative DNA damage. In this study, we tried to relate the protection by quercetin pre-treatment against oxidative DNA damage in human leucocytes in vitro to the interaction of quercetin in solution with hydroxyl and superoxide anion radicals as measured by electron spin resonance (ESR) spectrometry, using DMPO as a spin trap. Further, scavenging capacity of quercetin-treated leucocytes in vitro was evaluated by ESR spectrometry. Quercetin appears capable of protecting human leucocytes against oxidative DNA damage caused by hydrogen peroxide in a dose-dependent manner. The protection of leucocytes against superoxides is ambiguous. Incubation concentrations of quercetin (1, 10, and 50 microM) reduced levels of superoxide-induced oxidative DNA damage, while at 100 microM the amount of damage was increased. These results are supported by ESR-findings on quercetin in solution, also showing a prooxidant effect at 100 microM. ESR spectroscopy showed rate constant values for the reaction kinetics of quercetin in lowering iron-dependent hydroxyl radical formation and NADH-dependent superoxide anion formation of respectively 3.2 x 10(12)M(-1)s(-1) and 1.1 x 10(4)M(-1)s(-1). This shows that quercetin is a more potent inhibitor of hydroxyl radical formation than a scavenger of superoxide anions.     

Thymoquinone is a potent superoxide anion scavenger

The antioxidant and pro-oxidant effects of thymoquinone (TQ), a natural main constituent of the volatile oil of Nigella saliva seeds, and a synthetic structurally-related tert-butylhydroquinone (TBHQ), were examined in vitro. Both TQ and TBHQ efficiently inhibited iron-dependent microsomal lipid peroxidation in a concentration-dependent manner with median inhibitory concentration (IC50) values of 16.8 and 14.9 microM, respectively. TBHQ was stronger than TQ as a scavenger of 2,2'-diphenyl-p-picrylhydrazyl radical (DPPH) (IC50 = 5 microM, 200 times more active than TQ) and as a scavenger of hydroxyl radical (OH*) with an IC50 of 4.6 microM (approximately 10 times more active than TQ). TQ was more active than TBHQ as a superoxide anion scavenger with IC50 of 3.35 microM compared to 18.1 microM for TBHQ. Only TBHQ significantly promoted DNA damage in the bleomycin-Fe(III) system. The results suggest that both TQ and TBHQ have strong antioxidant potentials through scavenging ability of different free radicals. Moreover, the data indicate that TQ is acting mainly as a potent superoxide anion scavenger.     

Interactions between carnosine and captopril on free radical scavenging activity and angiotensin-converting enzyme activity in vitro

Interactions between carnosine (beta-alanyl-L-histidine), being plentiful in skeletal muscles and neuronal tissues, and captopril, a widely used angiotensin-converting enzyme (ACE) inhibitor, were examined concerning free radical scavenging activity and ACE activity in vitro. Not only captopril, but also carnosine, at concentrations less than those ordinarily found in muscles and neuronal tissues, significantly scavenged 2,2'-azinobis (3-ethylbenzothiazoline-6-sulphonate) (ABTS) radical cations, and inhibited ACE activity. Cupric ions reversed the ABTS scavenging activity of carnosine and captopril, whereas cupric ions strengthened the inhibitory action of carnosine on ACE activity. In contrast, cupric ions antagonized the inhibition of ACE activity induced by ethylenediaminetetraacetic acid, indicating that the inhibitory effect of carnosine on ACE activity is not related to the chelating action of carnosine. On the other hand, carnosine and captopril synergistically enhanced the free radical scavenging activity, but not the inhibitory effect on the ACE activity. These data suggest that carnosine in its concurrent use with captopril could act as a beneficial free radical scavenger, with less danger of overdose, in the inhibition of ACE activity.     

In vitro modulation of human neutrophil superoxide anion generation by various calcium channel antagonists used in ischemia-reperfusion resuscitation

Generation of toxic oxygen species by activated polymorphonuclear leukocytes (PMNs) may represent an important mechanism of ischemia-reperfusion injury. Concentration-response data concerning inhibition of superoxide anion (O2-) generation by NADPH oxidoreductase (NADPH OR) from isolated human PMN were generated for five calcium channel antagonists commonly utilized in ischemia-reperfusion investigational therapeutics. Regression analysis derived IC50 values for verapamil, nimodipine, nicardipine and lidoflazine were 45, 20, 12 and 7 microM respectively. Inhibition of the extent of reaction at 5 min paralleled inhibition of initial velocity. No inhibition by flunarizine was noted at concentrations less than or equal to 25 microM (where it did not alter reaction mixture composition). Only nicardipine demonstrated a significant concentration-response effect relative to prolonging lag time preceding O2- synthesis. Inhibition appeared at least partially reversible for all five agents. Neither PMN activation/desensitization, free-radical scavenging, nor PMN cytotoxicity appeared to be involved in the inhibition of PMN O2- synthesis by these agents. Ca2+ antagonist inhibition of PMN NADPH OR appears to involve more than simple inhibition of Ca2+ flux across the PMN plasma membrane. Direct inhibition of the intracellular events involved in the activation and/or activity of NADPH OR may be operative.     

Antioxidant effect of hydroxytyrosol, a polyphenol from olive oil: scavenging of hydrogen peroxide but not superoxide anion produced by human neutrophils

Hydroxytyrosol (HT) (also known as dihydroxyphenylethanol (DPE)) is a polyphenol extracted from virgin olive oil. HT is known to exert an antioxidant effect but the mechanism of action and the identity of the reactive oxygen molecule(s) targeted are not known. In this study, we show that HT inhibits luminol-amplified chemiluminescence of human neutrophils stimulated with N-formyl-methionyl-leucyl-phenylalanine (fMLP), phorbol myristate acetate (PMA) and opsonized zymosan. This effect was dose-dependent and occurred immediately after the addition of HT. However, HT had no effect on lucigenin-amplified chemiluminescence, suggesting that it does not inhibit NADPH oxidase activation or scavenge superoxide anions. Furthermore, HT inhibited H(2)O(2)-dependent-dichlorofuoroscein (DCFH) fluorescence of activated neutrophils, as measured by flow cytometry. Finally, HT inhibited luminol-amplified chemiluminescence in a cell-free system consisting of H(2)O(2) and HRPO. These results suggest that HT, a polyphenol derived from olive oil, could exert its antioxidant effect by scavenging hydrogen peroxide but not superoxide anion released during the respiratory burst.     

Direct scavenging of nitric oxide and superoxide by green tea

In the present study, we investigated the free radical scavenging effects of green tea extract and green tea tannin mixture and its components using a nitric oxide (NO) and superoxide (O₂⁻) generating system in vitro. Green tea extract showed direct scavenging activity against NO and O₂⁻ and green tea tannin mixture, at the same concentration, showed high scavenging activity. Comparison of the activities of seven pure compounds isolated from green tea tannin mixture showed that (−)-epigallocatechin 3-O-gallate (EGCg), (−)-gallocatechin 3-O-gallate (GCg) and (−)-epicatechin 3-O-gallate (ECg) had higher scavenging activities than (−)-epigallocatechin (EGC), (+)-gallocatechin (GC), (−)-epicatechin (EC) and (+)-catechin (C), showing the importance of the structure of flavan-3-ol linked to gallic acid for this activity. Among the gallate-free tannins, EGC and GC were more effective O₂⁻ scavengers than EC and C, indicating the O-trihydroxy structure in the B ring is an important determinant of such activity. However, this structure did not affect the NO scavenging activity. These findings confirm that green tea tannin has excellent antioxidant properties, which may be involved in the beneficial effect of this compound.     

Nicotine potentiates superoxide anion generation by human neutrophils

Cytotoxic neutrophil-derived oxygen radicals have been implicated in the pathogenesis of a variety of cardiovascular, pulmonary, and neoplastic disorders for which cigarette smoking is a prominent risk factor. Although nicotine alone failed to provoke neutrophil oxidative metabolism, the alkaloid caused dose-dependent (0.1 to 10 microM) potentiation of superoxide anion release induced by either phorbol myristate acetate or N-formyl-methionyl-leucyl-phenylalanine. The potentiating effect of nicotine was not attenuated by either atropine or hexamethonium nor was it mimicked by acetylcholine, suggesting involvement of noncholinergic receptors or a membrane-fluidizing effect of the alkaloid. Nicotine-induced exacerbation of neutrophil superoxide anion production may be involved with the enhanced risk of cardiovascular, pulmonary, or neoplastic disease in individuals who smoke.     

Captopril has no significant scavenging antioxidant activity in human plasma in vitro or in vivo

1 Captopril has been reported to possess hydroxyl radical (OH) and hypochlorous acid (HOCl) scavenging effects, which could contribute to its therapeutic activity in the clinical setting.
2 The objective of the present study was to determine whether therapeutically achievable captopril concentrations could augment antioxidant properties of human plasma and protect it against OH- and HOCl-driven oxidant injury in vitro . Possible drug influences on systemic oxidative stress status in vivo were also investigated in subjects taking 50  mg captopril orally by measuring plasma and red blood cell peroxidation, as well as plasma protein thiols.
3 The results show that captopril is incapable of enhancing antioxidant properties of human plasma, of protecting it against specific oxidative attack and of decreasing systemic oxidant load in vivo .
4 The present data, therefore, do not support the contention of a beneficial action of captopril through systemic antiradical-antioxidant effects in human beings.