Superoxide radicals scavenging and xanthine oxidase inhibitory activity of magnesium lithospermate B from Salvia miltiorrhiza

In this study we investigated the superoxide radicals scavenging effect and xanthine oxidase inhibitory activity by magnesium lithospermate B, which was originally isolated from the roots of Salvia miltiorrhiza (also named Danshen or Dansham), an important herb in Oriental medicine. Superoxide radicals were generated both in β-NADH/PMS system and xanthine/ xanthine oxidase system. Magnesium lithospermate B significantly inhibited the reduction of NBT induced by superoxide radicals with an IC₅₀ of 29.8 μg/mL and 4.06 μg/mL respectively in the two systems. Further study suggested that magnesium lithospermate B can directly inhibit xanthine oxidase and exhibits competitive inhibition. Magnesium lithospermate B was also found to have the hypouricemic activity in vivo against potassium oxonate-induced hyperuricaemia in mice. After oral administration of magnesium lithospermate B at doses of 10, 20 and 30 mg/kg, there was a significant decrease in the serum urate level when compared to the hyperuricemia control. In addition, magnesium lithospermate B significantly protected HL-60 cells from superoxide radicals-induced apoptosis in the xanthine/ xanthine oxidase reactions. This study provided evidence that magnesium lithospermate B exhibits direct superoxide radicals scavenging and xanthine oxidase inhibitory activity.     

Effect of Rhus coriaria L. (Anacardiaceae) on superoxide radical scavenging and xanthine oxidase activity

Rhus coriaria L. (Anacardiaceae) is a well-known spice in the Middle-East and grown in the Central and East Anatolia region of Turkey. A methanolic extract (water-soluble part constituents) of R. coriaria, was found to be an uncompetitive inhibitor of xanthine oxidase and scavenger of superoxide radical in vitro with IC50 values of 172.5 microg/mL and 232 microg/mL respectively. Superoxide radicals were generated either by an enzymatic or a non-enzymatic system, and scavenging ability was evaluated by the inhibition of nitroblue tetrazolium reduction. This study provides evidence that a crude extract of R. coriaria exhibits interesting antioxidant properties, expressed either by the capacity to scavenge superoxide radical or to uncompetitively inhibit xanthine oxidase.     

EFFECT OF RHUS CORIARIA L. (ANACARDIACEAE) ON SUPEROXIDE RADICAL SCAVENGING AND XANTHINE OXIDASE ACTIVITY

Rhus coriaria L. (Anacardiaceae) is a well-known spice in the Middle-East and grown in the Central and East Anatolia region of Turkey. A methanolic extract (water-soluble part constituents) of R. coriaria, was found to be an uncompetitive inhibitor of xanthine oxidase and scavenger of superoxide radical in vitro with IC 50 values of 172.5 μg/mL and 232 μg/mL respectively. Superoxide radicals were generated either by an enzymatic or a non-enzymatic system, and scavenging ability was evaluated by the inhibition of nitroblue tetrazolium reduction. This study provides evidence that a crude extract of R. coriaria exhibits interesting antioxidant properties, expressed either by the capacity to scavenge superoxide radical or to uncompetitively inhibit xanthine oxidase.     

Lithospermic acid as a novel xanthine oxidase inhibitor has anti-inflammatory and hypouricemic effects in rats

Lithospermic acid (LSA) was originally isolated from the roots of Salvia mitiorrhiza, a common herb of oriental medicine. Previous studies demonstrated that LSA has antioxidant effects. In this study, we investigated the in vitro xanthine oxidase (XO) inhibitory activity, and in vivo hypouricemic and anti-inflammatory effects of rats. XO activity was detected by measuring the formation of uric acid or superoxide radicals in the xanthine/xanthine oxidase system. The results showed that LSA inhibited the formation of uric acid and superoxide radicals significantly with an IC50 5.2 and 1.08 microg/ml, respectively, and exhibited competitive inhibition. It was also found that LSA scavenged superoxide radicals directly in the system beta-NADH/PMS and inhibited the production of superoxide in human neutrophils stimulated by PMA and fMLP. LSA was also found to have hypouricemic activity on oxonate-pretreated rats in vivo and have anti-inflammatory effects in a model of gouty arthritis. These results suggested that LSA is a competitive inhibitor of XO, able to directly scavenge superoxide and inhibit superoxide production in vitro, and presents hypouricemic and anti-inflammatory actions in vivo.     

Inhibitory activity of xanthine oxidase and superoxide-scavenging activity in some taxa of the lichen family Graphidaceae

Results on the screening of species of the lichen family Graphidaceae for superoxide-scavenging activity (SSA) and xanthine-oxidase inhibitory (IXO) activity have been presented. The potential of the extracts for scavenging of superoxide and inhibition of xanthine-oxidase under various physiological conditions has been evaluated. The methanolic extracts of the species of family Graphidaceae showed inhibitory properties of xanthine oxidase (IC50 = 2.0 to 5.26 microg/ml) with an additional superoxide scavenging capacity (IC50 = 3.63 to 13.88 microg/ml). The potential of the methanolic extracts for scavenging of superoxide and inhibition of xanthine oxidase remained stable at 4 degrees C. Thus the extracts can be maintained for longer periods for their therapeutic uses.     

Decrease in heart mitochondrial creatine kinase activity due to oxygen free radicals.

This study was undertaken to examine the effects of oxygen free radicals on mitochondrial creatine kinase activity in rat heart. Xanthine plus xanthine oxidase (superoxide anion radical generating system) reduced mitochondrial creatine kinase activity both in a dose- and a time-dependent manner. Superoxide dismutase showed a protective effect on depression in creatine kinase activity due to xanthine plus xanthine oxidase. Hydrogen peroxide inhibited creatine kinase activity in a dose-dependent manner, this inhibition was protected by the addition of catalase. In order to understand the detailed mechanisms by which oxygen free radicals inhibit mitochondrial creatine kinase activity, the effects of oxygen free radicals on mitochondrial sulfhydryl groups were examined. Mitochondrial sulfhydryl groups contents were decreased by xanthine plus xanthine oxidase or hydrogen peroxide; this depression in sulfhydryl groups contents was prevented by the addition of superoxide dismutase or catalase. N-Ethylmaleimide (sulfhydryl group reagent) expressed inhibitory effects on the creatine kinase activity both in a dose- and a time-dependent manner; dithiothreitol or cysteine (sulfhydryl group reductant) showed protective effects on the creatine kinase activity depression induced by N-ethylmaleimide. Dithiothreitol or cysteine also blocked the depression of mitochondrial creatine kinase activity caused by xanthine plus xanthine oxidase or hydrogen peroxide. These results lead us to conclude that oxygen free radicals may inhibit mitochondrial creatine kinase activity by modifying sulfhydryl groups in the enzyme protein.     

Antioxidant activity in vitro of two aromatic compounds from Caesalpinia sappan L

Two antioxidant compounds were isolated from C. sappan L by multiple steps of column chromatography and thin layer chromatography in succession with superoxide scavenging assay as activity monitor. Structures of the two compounds were determined by spectroscopic methods as 1',4'-dihydro-spiro[benzofuran-3(2H),3'-[3H-2]benzopyran]-1',6',6',7'-tetrol (compound 1) and 3-[[4,5-dihydroxy-2(hydroxymethyl) phenyl]-methyl]-2,3-dihydro-3,6-benzofurandiol (compound 2). Characterization of antioxidant properties of these two compounds was done by determining the inhibitory effect on xanthine oxidase activity as well as scavenging effect on superoxide anion and hydroxyl radicals. Our results indicated that compounds 1 and 2 inhibited xanthine oxidase activity and scavenged superoxide anion and hydroxyl radicals. Compounds 1 and 2 possessed similar radical scavenging activities as ascorbic acid, and they were more effective than other well-known antioxidants such as alpha-tocopherol, beta-carotene, and BHT. As inhibitors of free radical formation, compounds 1 and 2 were more effective than all the other antioxidants tested. In conclusion, compounds 1 and 2 can be regarded as primary antioxidants with radical-scavenging and chain-breaking activities as well as secondary antioxidants with inhibitory effect on radical generation.     

Effect of zinc on superoxide-dependent hydroxyl radical production in vitro

Trace elements play an important role in oxygen metabolism and therefore in the formation of free radicals. Whereas iron and copper are usually the main enhancers of free radical formation, other trace elements, such as zinc and selenium, protect against the harmful effects of these radicals. To investigate the different protective mechanisms of zinc on radical formation, we examined the effects of added zinc and copper on superoxide dismutase activity. We also studied the effects of copper and iron on xanthine oxidase activity and on the Haber-Weiss cycle (iron, superoxide, and hydrogen peroxide), which generates hydroxyl radicals in vitro. The hypoxanthine/xanthine oxidase radical generating system contained a variety of different physiological ligands for binding the iron. This study confirmed the inhibitory effect of copper on xanthine oxidase activity. Moreover, it demonstrated that zinc inhibited hydroxyl radical formation when this formation was catalyzed by a citrate-iron complex in the hypoxanthine/xanthine oxidase reaction. Finally, human blood plasma inhibited citrate-iron-dependent hydroxyl radical formation under the same conditions. Although trace elements seemed responsible for this antioxidant activity of plasma, it is likely that zinc played no role as a plasma antioxidant. Indeed, calcium appeared to be responsible for most of this effect under our experimental conditions.     

EPR studies on the superoxide-scavenging capacity of the nutraceutical resveratrol

Resveratrol (3,4',5-trihydroxystilbene), a polyphenolic compound found in mulberries, grapes, and red wine, has received considerable attention because of its apparent protective effects against various degenerative diseases due to its potential antioxidant activities. However, direct evidence for the superoxide-scavenging capacity of resveratrol is lacking in literature. In this study, electron paramagnetic resonance spectroscopy in combination with 5-(diethoxyphosphoryl)-5-methylpyrroline-N-oxide (DEPMPO)-spin trapping technique was utilized to determine the ability of resveratrol in scavenging superoxide anions generated from both potassium superoxide and the xanthine oxidase/xanthine system. We have demonstrated here for the first time that the presence of resveratrol resulted in decreased formation of DEPMPO-superoxide adduct (DEPMPO-OOH) in both the potassium superoxide and xanthine oxidase/xanthine systems, indicating that resveratrol could directly scavenge superoxide anions. The inhibition of DEPMPO-OOH in the xanthine oxidase/xanthine system, however, was found to be much potent as compared to that observed in potassium superoxide system. It was further shown that resveratrol could also directly inhibit xanthine oxidase activity as assessed by oxygen consumption and formation of uric acid. Taken together, the dual role of resveratrol in directly scavenging superoxide and inhibiting its generation via xanthine oxidase reported in this study may explain, at least in part, the protective role of this compound against oxidative injury in various disease processes.     

黄伞胞外多糖抗自由基作用的研究

目的:以乙醇沉淀的黄伞发酵浓缩液得到的胞外多糖为研究对象,检验其对自由基的清除作用。方法:采用电子顺磁共振(EPR)波谱仪检测黄伞胞外多糖清除羟自由基OH.和超氧阴离子自由基O2-.的作用。结果:在样品浓度均为100mg/mL的条件下,利用Fenton反应体系,黄伞胞外多糖对羟自由基OH.的清除率仅为38.5%;而利用次黄嘌呤-黄嘌呤氧化酶体系,黄伞胞外多糖对超氧阴离子自由基O2-.的清除率可达到80.6%。结论:黄伞胞外多糖在体外对自由基有一定的清除作用且其对超氧阴离子自由基O2-.的清除作用明显优于对羟自由基OH.的清除作用。     

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.     

Antioxidative activities of galloyl glucopyranosides from the stem-bark of Juglans mandshurica

Two phenolics, 1,2,6-trigalloylglucose (1) and 1,2,3,6-tetragalloylglucose (2), isolated from the stem-bark of Juglans mandshurica were evaluated for their antioxidative activities. The results showed that compounds 1 and 2 exhibited strong scavenging activities against 1,1'-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis-(3-ethylbenzenthiazoline-6-sulphonic) acid (ABTS(*+)), and superoxide radicals (O(2)(*-)), and also had a significant inhibitory effect on lipid peroxidation and low-density lipoprotein (LDL) oxidation. The strong superoxide radical scavenging of 1 and 2 resulted from the potential competitive inhibition with xanthine at the active site of xanthine oxidase (OX). In addition, compounds 1 and 2 displayed significant lipoxygenase inhibitory activity, the mode of inhibition also being identified as competitive. In comparison, the antioxidative activities of compounds 1 and 2, together with gallic acid, indicated that the number of galloyl moieties could play an important role in the antioxidative activity.     

Inhibition of cardiac phosphatidylethanolamine N-methylation by oxygen free radicals

This study was undertaken to examine the effects of oxygen free radicals on phosphatidylethanolamine (PE) N-methylation in rat heart sarcolemmal (SL) and sarcoplasmic reticular (SR) membranes. Three catalytic sites involved in the sequential methyl transfer reaction were studied by assaying the incorporation of radiolabeled methyl groups from S-adenosyl-L-methionine (0.055, 10, and 150 microM) into SL or SR PE molecules under optimal conditions. In the presence of xanthine + xanthine oxidase (superoxide anion radicals generating system), PE N-methylation was inhibited at site I and III in the heavy SL fraction isolated by the hypotonic shock-LiBr treatment method. In the light SL fraction isolated by sucrose-density gradient, a significant inhibition of PE N-methylation was seen at all three sites. These inhibitory effects of xanthine + xanthine oxidase on PE N-methylation were prevented by the addition of superoxide dismutase. Hydrogen peroxide showed a significant inhibition of PE N-methylation at site I in the heavy SL fraction, and at site I and II in the light SL fraction. Catalase blocked the inhibitory effects of hydrogen peroxide. The effects of both xanthine + xanthine oxidase and hydrogen peroxide on the SR membranes were similar to those seen for the heavy SL fraction. These results suggest that, in addition to lipid peroxidation, the oxygen free radicals may affect the function of cardiac membranes by decreasing the phospholipid N-methylation activity.     

Free radical scavenging efficiency of Nano-Se in vitro

In this study, we showed that smaller size particles of Nano-Se have better scavenging effects on the following free radicals: carbon-centered free radicals (R*) generated from 2,2'-azo-bis-(2-amidinopropane) hydrochloride (AAPH), the relatively stable free radical 1,1-diphenyl-2-picryhydrazyl (DPPH), the superoxide anion (O2*-) generated from the xanthine/xanthine oxidase (X/XO) system, singlet oxygen (1O2) generated by irradiated hemoporphyrin. Furthermore, the three sizes of Nano-Se studied also show protective effects against the oxidation of DNA. The three samples all have potential size-dependent characteristics on scavenging the free radicals. Although in this study we regarded Nano-Se as a whole without considering interactions between BSA and the red selenium nano-particles, there is the possibility that the apparent free radical scavenging effects may be partially contributed by such interactions.     

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.     

Redox cycling of potential antitumor aziridinyl quinones

The formation of reactive oxygen intermediates (ROI) during redox cycling of newly synthesized potential antitumor 2,5-bis (1-aziridinyl)-1,4-benzoquinone (BABQ) derivatives has been studied by assaying the production of ROI (superoxide, hydroxyl radical, and hydrogen peroxide) by xanthine oxidase in the presence of BABQ derivatives. At low concentrations (< 10 microM) some BABQ derivatives turned out to inhibit the production of superoxide and hydroxyl radicals by xanthine oxidase, while the effect on the xanthine-oxidase-induced production of hydrogen peroxide was much less pronounced. Induction of DNA strand breaks by reactive oxygen species generated by xanthine oxidase was also inhibited by BABQ derivatives. The DNA damage was comparable to the amount of hydroxyl radicals produced. The inhibiting effect on hydroxyl radical production can be explained as a consequence of the lowered level of superoxide, which disrupts the Haber-Weiss reaction sequence. The inhibitory effect of BABQ derivatives on superoxide formation correlated with their one-electron reduction potentials: BABQ derivatives with a high reduction potential scavenge superoxide anion radicals produced by xanthine oxidase, leading to reduced BABQ species and production of hydrogen peroxide from reoxidation of reduced BABQ. This study, using a unique series of BABQ derivatives with an extended range of reduction potentials, demonstrates that the formation of superoxide and hydroxyl radicals by bioreductively activated antitumor quinones can in principle be uncoupled from alkylating activity.     

Nitric oxide-scavenging activity of polyhydroxylated fullerenol, C60(OH)24.

Investigation of the possible nitric oxide-scavenging activity of hydroxylated derivative of fullerene, fullerenol C60(OH)24, demonstrated that it expressed direct scavenging activity toward nitric oxide radical (NO) liberated within solution of sodium nitroprusside (SNP), a well known NO donor. In parallel, pre-treatment (30') with intratesticular injection of fullerenol (60 microg/each testis) prevented NO-induced decrease of catalase, glutathione transferase and glutathione peroxidase activities in the denucleated fraction of interstitial testicular cells of adult rats 2 h after intratesticular injection of SNP (20 microg/each testis). In addition, fullerenol decreased formation of thiobarbituric acid-reactive substances (TBA-RS) with similar efficiency as butylated hydroxy toluen (BHT), a well known antioxidant. Also, fullerenol expressed certain scavenging activity toward superoxide anion (O2-) in xanthine/xanthine oxidase system. In summary, results obtained in this study confirmed free radical-scavenging activity of fullerenol, and according to our knowledge, it is the first evidence of direct NO-quenching activity of hydroxylated C60 derivative in different milieu.     

Free radical scavenging profile and myeloperoxidase inhibition of extracts from antidiabetic plants: Bauhinia forficata and Cissus sicyoides

There is abundant evidence that reactive oxygen species are implicated in several physiological and pathological processes. To protect biological targets from oxidative damage, antioxidants must react with radicals and other reactive species faster than biological substrates do. The aim of the present study was to determine the in vitro antioxidant activity of aqueous extracts from leaves of Bauhinia forficata Link (Fabaceae-Caesalpinioideae) and Cissus sicyoides L. (Vitaceae) (two medicinal plants used popularly in the control of diabetes mellitus), using several different assay systems, namely, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) decolorization, superoxide anion radical (O2(.-)) scavenging and myeloperoxidase (MPO) activity. In the ABTS assay for total antioxidant activity, B. forficata showed IC50 = 8.00+/-0.07 microg/mL, while C. sicyoides showed IC50 = 13.0+/-0.2 microg/mL. However, the extract of C. sicyoides had a stronger effect on O2(.-) (IC50 = 60.0+/-2.3 microg/mL) than the extract of B. forficata (IC50 = 90.0+/-4.4 microg/mL). B. forficata also had a stronger inhibitory effect on MPO activity, as measured by guaiacol oxidation, than C. sicyoides. These results indicate that aqueous extracts of leaves of B. forficata and C. sicyoides are a potential source of natural antioxidants and may be helpful in the prevention of diabetic complications associated with oxidative stress.     

Antioxidant activities of cultured Armillariella mellea

This study aimed to evaluate the antioxidant activities of a cultured medicinal fungus--Armillariella mellea (Vahl. ex Fr.) Karst. (AM). Three antioxidant assay systems, namely cytochrome c, xanthine oxidase inhibition and FeCl2-ascorbic acid stimulated lipid peroxidation in rat tissue homogenate tests, were used. Total flavonoid and phenol contents of AM extracts were also analyzed. Results showed that both aqueous (AM-H2O) and ethanolic (AM-EtOH) extracts of solid state cultured AM showed antioxidant activities in a concentration-dependent manner. At concentrations 1-100 microg/ml, the free radical scavenging activity was 73.7-92.1% for AM-H2O, and 60.0-90.8% for AM-EtOH. These extracts also showed an inhibitory effect on xanthine oxidase activity, but with a lesser potency (IC50 - 9.17 microg/ml for AM-H2O and 7.48 microg/ml for AM-EtOH). In general, AM-H2O showed a stronger anti-lipid peroxidation activity on different rat's tissues than AM-EtOH. However, both AM extracts displayed a weak inhibitory effect on lipid peroxidation in plasma. Interestingly, the anti-lipid peroxidation activity of AM-H2O (IC50 - 6.66 microg/ml) in brain homogenate was as good as alpha-tocopherol (IC50 - 5.42 microg/ml). AM-H2O (80.0 mg/g) possessed a significant higher concentration of total flavonoids than AM-EtOH (30.0 mg/g), whereas no difference was noted in the total phenol content between these two extracts. These results conclude that AM extracts possess potent free radical scavenging and anti-lipid peroxidation activities, especially the AM-H20 in the brain homogenate.     

Antioxidative Activity of 5,6,7,8-Tetrahydrobiopterin and its Inhibitory Effect on Paraquat-Induced Cell Toxicity in Cultured Rat Hepatocytes

The in vitro antioxidative activity of 5,6,7,8-tetrahydrobiopterin (BPH4) was measured and the ability of BPH4 to prevent paraquat-induced cell damage was examined in cultured hepatocytes. The scavenging activity of BPH4 against superoxide anion radicals was assayed in two systems, i.e., xanthine/xanthine oxidase (X/XOD) and rat macrophage/phorbol myristate acetate (MξPMA) radical-generating systems. BPH4 showed an extremely strong superoxide anion radical-scavenging activity in both assay systems. Biopterin (BP) itself did not show any activity in the X/XOD system, but was effective in the MξPMA system. The antioxidative activities of BPH4 against both superoxide anion and hydroxyl radicals were confirmed by spin trapping-ESR spectrometry. BPH4 also protected rat brain homogenate against auto-oxidation. We further examined the effect of BPH4 on paraquat-induced cell toxicity in cultured rat hepatocytes. The paraquat-induced elevation of the release of lactate dehydrogenase (LDH), a marker enzyme for cytotoxicity from cultured hepatocytes was suppressed by BPH4 in a dose-dependent manner. The elevation of lipid peroxides simultaneously induced by paraquat was also inhibited by BPH4 in the same manner. These results suggest that BPH4 might be useful in the treatment of various diseases whose pathogenesis is active oxygen-related.