Selective in vitro antioxidant properties of bisphosphonates

The aim of this study was to investigate the in vitro antioxidant profile of different bisphosphonates. Bisphosphonates were tested for their xanthine oxidase and microsomal lipid peroxidation inhibiting capacity. Furthermore, the effect of these different compounds on DPPH, a stable radical, was investigated. Clodronate, risedronate, and pyrophosphate were further tested for their hydroxyl radical scavenging activity. None of the tested compounds showed xanthine oxidase inhibiting activity or DPPH scavenging activity. All the tested bisphosphonates exhibited inhibiting capacities on the microsomal lipid peroxidation. The hydroxyl radical scavenging activity was dependent on the order of adding the different reagents and was highest for risedronate. Bisphosphonates possess an inhibiting activity on the microsomal lipid peroxidation and the Fenton reaction. In these reactions iron plays an important role suggesting that the selective in vitro antioxidant properties of the bisphosphonates are due to their iron chelating characteristics.     

Antioxidant activity and protective effect on DNA cleavage of extracts from Cistus incanus L. and Cistus monspeliensis L.

The genus Cistus includes many typical species of Mediterranean flora; Cistus species are used as antidiarrheics, as general remedies for treatment of various skin diseases in folk medicine and as anti-inflammatory agents. These species contain flavonoids that are considered to be chain-breaking antioxidants. In this work, we have investigated the effects of crude aqueous extracts from Cistus incanus and Cistus monspeliensis on DNA cleavage and their free-radical scavenging capacity. In addition, their effect on lipid peroxidation in rat liver microsomes was evaluated. These extracts showed a protective effect on DNA cleavage and a dose-dependent free-radical scavenging capacity; Cistus monspeliensis was more active than Cistus incanus; these results were confirmed by a significant inhibition of lipid peroxidation in rat liver microsomes. The experimental evidence, therefore, suggests that because of their antioxidant activity these extracts may offer excellent photoprotection for skin and may be useful in the treatment of human diseases where oxidative stress plays a key role. This revised version was published online in July 2006 with corrections to the Cover Date.     

L-Propionyl-carnitine as superoxide scavenger, antioxidant, and DNA cleavage protector

L-Propionylcarnitine, a propionyl ester of L-carnitine, increases the intracellular pool of L-carnitine. It exhibits a high affinity for the enzyme carnitine acetyltransferase (CAT) and, thus, is readily converted into propionyl-coenzyme A and free carnitine. It has been reported that L-propionylcarnitine possesses a protective action against heart ischemia–reperfusion injury; however, the antioxidant mechanism is not yet clear. L-Propionylcarnitine might reduce the hydroxyl radical production in the Fenton system, by chelating the iron required for the generation of hydroxyl radicals. To obtain a better insight into the antiradical mechanism of L-propionylcarnitine, the present research analyzed the superoxide scavenging capacity of L-propionylcarnitine and its effect on linoleic acid peroxidation. In addition, the effect of L-propionylcarnitine against DNA cleavage was estimated using pBR322 plasmid. We found that L-propionylcarnitine showed a dose-dependent free-radical scavenging activity. In fact, it was able to scavenge superoxide anion, to inhibit the lipoperoxidation of linoleic acid, and to protect pBR322 DNA from cleavage induced by H₂O₂ UV-photolysis. This revised version was published online in July 2006 with corrections to the Cover Date.     

Cyanidin and cyanidin 3-O-β-D-glucoside as DNA cleavage protectors and antioxidants

Anthocyanins, colored flavonoids, are water-soluble pigments present in the plant kingdom; in fact they are secondary plant metabolites responsible for the blue, purple, and red color of many plant tissues. Present in beans, fruits, vegetables and red wines, considerable amounts of anthocyanins are ingested as constituents of the human diet (180-215 mg daily). There is now increasing interest in the in vivo protective function of natural antioxidants contained in dietary plants against oxidative damage caused by free radical species. Recently, the antioxidant activity of phenolic phytochemicals, has been investigated. Since the antioxidant mechanism of anthocyanin pigments is still controversial, in the present study we evaluated the effects of cyanidin and cyanidin 3-O-beta-D-glucoside on DNA cleavage, on their free radical scavenging capacity and on xanthine oxidase activity. Cyanidin and cyanidin 3-O-beta-D-glucoside showed a protective effect on DNA cleavage, a dose-dependent free radical scavenging activity and significant inhibition of XO activity. These effects suggest that anthocyanins exhibit interesting antioxidant properties, and could therefore represent a promising class of compounds useful in the treatment of pathologies where free radical production plays a key role.     

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 beta-NADH/PMS system and xanthine/ xanthine oxidase system. Magnesium lithospermate B significantly inhibited the reduction of NBT induced by superoxide radicals with an IC(50) of 29.8 microg/mL and 4.06 microg/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.     

Role of cellular superoxide dismutase against reactive oxygen metabolite injury in cultured bovine aortic endothelial cells.

We examined the protective effect of cellular superoxide dismutase against extracellular hydrogen peroxide in cultured bovine aortic endothelial cells. 51Cr-labeled cells were exposed to hydrogen peroxide generated by glucose oxidase/glucose. Glucose oxidase caused a dose-dependent increase of 51Cr release. Pretreatment with diethyldithiocarbamate enhanced injury induced by glucose oxidase, corresponding with the degree of inhibition of endogenous superoxide dismutase activity. Inhibition of cellular superoxide dismutase by diethyldithiocarbamate was not associated either with alteration of other antioxidant defenses or with potentiation of nonoxidant injury. Enhanced glucose oxidase damage by diethyldithiocarbamate was prevented by chelating cellular iron. Inhibition of cellular xanthine oxidase neither prevented lysis by hydrogen peroxide nor diminished enhanced susceptibility by diethyldithiocarbamate. These results suggest that, in cultured endothelial cells: 1) cellular superoxide is involved in mediating hydrogen peroxide-induced damage; 2) superoxide, which would be generated upon exposure to excess hydrogen peroxide independently of cellular xanthine oxidase, promotes the Haber-Weiss reaction by initiating reduction of stored iron (Fe3+) to Fe2+; 3) cellular iron catalyzes the production of a more toxic species from these two oxygen metabolites; 4) cellular superoxide dismutase plays a critical role in preventing hydrogen peroxide damage by scavenging superoxide and consequently by inhibiting the generation of the toxic species.     

Antioxidant and radioprotective properties of an Ocimum sanctum polysaccharide

The antioxidant activity of two polysaccharides isolated from the Indian medicinal plants, Ocimum sanctum and Tinospora malabarica, was studied. Only the O. sanctum polysaccharide (OSP) showed significant activity. OSP could prevent oxidative damage to liposomal lipids and plasmid DNA induced by various oxidants such as iron, AAPH and gamma-radiation, besides scavenging important ROS such as the superoxide radical and hydrogen peroxide and inhibiting xanthine oxidase. In addition, OSP could prevent gamma-radiation-mediated cell deaths in mouse splenocytes.     

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.     

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.     

Effects of Ceruloplasmin on Superoxide-Dependent Iron Release from Ferritin and Lipid Peroxidation

Ceruloplasmin (CP) effectively inhibited superoxide and ferritin-dependent peroxidation of phospholipid liposomes, using xanthine oxidase or gamma irradiation of water as sources of superoxide. In addition, CP inhibited superoxide-dependent mobilization of iron from ferritin. suggesting that CP inhibited lipid peroxidation by decreasing the availability of iron from ferritin. CP also exhibited some superoxide scavenging activity as evidenced by its inhibition of superoxide-dependent cytochrome c reduction. However, superoxide scavenging by CP did not quantitatively account for its inhibitory effects on iron release. The effects of CP on iron-catalyzed lipid peroxidation in systems containing exogenously added ferrous iron was also investigated. CP exhibited prooxidant and antioxidant effects; CP stimulated at lower concentrations, reached a maximum. and inhibited at higher concentrations. However. the addition of apoferritin inhibited CP and Fe(II)-catalyzed lipid peroxidation at all concentrations of CP. In addition, CP catalyzed the incorporation of Fe(II) into apoferritin. Collectively these data suggest that CP inhibits superoxide and ferritin-dependent lipid peroxidation via its ability to incorporate reductively-mobilized iron into ferritin.     

Novel 3-O-acyl mesquitol analogues as free-radical scavengers and enzyme inhibitors: synthesis,biological evaluation and structure-activity relationship

A new isomer of mesquitol (2,3-trans-3',4',7,8-tetrahydroxyflavan-3-ol) was isolated from Dichrostachys cinerea in excellent yields. It has shown free-radical scavenging property and alpha-glucosidase inhibitory activities but, it could not display xanthine oxidase inhibitory property. However, it was observed that acylation of 3-OH group significantly enhanced the alpha-glucosidase inhibition and displayed xanthine oxidase inhibitory potential. The structure activity relationship revealed that the degree of lipophilicity played a major role in improving enzyme inhibitory activities. A positive correlation was observed between enzyme inhibitory potential and acyl chain length (upto C-16) of aliphatic esters.     

Consequence of the antioxidant activities and tyrosinase inhibitory effects of various extracts from the fruiting bodies of Pleurotus ferulae

This study was initiated to screen the antioxidant activities, tyrosinase inhibitory effects on the fruiting bodies of Pleurotus ferulae extracted with acetone, methanol and hot water. The antioxidant activities were performed on β-carotene–linoleic acid, reducing power, DPPH, ferrous ions chelating abilities, and xanthine oxidase. In addition to this, phenolic compounds were also analyzed. The methanolic extract showed the strongest β-carotene–linoleic acid inhibition and high reducing power as compared to other extracts. The scavenging effects on DPPH radicals, the acetonic and methanolic extracts were more effective than hot water extracts. The strongest chelating effect was obtained from the methanolic extract as compared to the tested synthetic antioxidant. Gallic acid, protocatechuic acid, caffeic acid, vanillin, ferulic acid, naringin, resveratrol, naringenin, hesperetin, formononetin and biochanin-A were detected from acetonitrile and hydrochloric acid (5:1) solvent extract. Xanthine oxidase and tyrosinase inhibitory activities of acetonic, methanolic, and hot water extracts of P. ferulae increased with increasing concentration. The results suggested that consumption of P. ferulae might be beneficial to the antioxidant, xanthine oxidase, and tyrosinase protection system of the human body against oxidative damage and others complications.     

The effect of some tannins on trout erythrocytes exposed to oxidative stress

In order to gain more knowledge on the role of tannins as antioxidants, their ability to protect (Salmo irideus) erythrocytes against oxidative stress was investigated. Antioxidant activity of different tannins (tannic, gallic and ellagic acid) was evaluated by chemiluminescence (CL) techniques using lucigenin and luminol as chemiluminogenic probes for the superoxide radical generated by the xanthine/xanthine oxidase system and hydrogen peroxide, respectively. The superoxide-scavenging activity of these tannins was shown for all the compounds; however, it is not clear if this is due to their ability of scavenging the superoxide radical or to their inhibitory activity on xanthine oxidase. Tannic and ellagic acid showed a marked effect on the reduction of H2O2-luminol chemiluminescence. The influence of these tannins on the rate of hemolysis in stressed trout erythrocytes was investigated and the results indicate that tannic acid accelerates the hemolytic event while gallic and ellagic acid have no significant effect. The possible protective action of these compounds against oxidative DNA damage was assessed using the comet assay, a rapid and sensitive single-cell gel electrophoresis technique, used to detect primary DNA damage in individual cells. The results here reported show that tannins under study are capable at low concentrations of protecting DNA breakage, while at high concentrations they can be genotoxic.     

ortho-dihydroxyisoflavone derivatives from aged Doenjang (Korean fermented soypaste) and its radical scavenging activity

One new ortho-dihydroxyisoflavone, 7,3',4'-trihydroxyisoflavone (2), and two known ortho-dihydroxyisoflavone derivatives were isolated from 5-year-old Doenjang (Korean fermented soypaste), and evaluated as potent antioxidant by comparing with other known isoflavones. 7,8,4'-Trihydroxyisoflavone (1), 7,3',4'-trihydroxyisoflavone (2), and 6,7,4'-trihydroxyisoflavone (3) inhibited DPPH (Diphenyl-1-picryl hydrazyl) formation by 50% at a concentration of 21.5+/-0.2, 28.7+/-0.4 and 32.6+/-0.6 (IC(50)), respectively, whereas three isoflavones showed weak DPPH radical scavenging activity. In xanthine oxidase (XO) system, in which both inhibition of xanthine oxidase and superoxide scavenging effect were measured in one assay. Compound 1 (IC(50)= 6.6+/-0.4 microM) and 2 (IC(50)=16.8+/-1.2 microM) show significant inhibitory activity and greater effect than allopurinol. But, compound 3 and other isoflavones showed lower inhibition activity. This study shows that the position of hydroxyl substituent at the aromatic ring of isoflavone plays an important role in radical scavenging effect.     

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.     

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.     

Hydroxyl radical is not a product of the reaction of xanthine oxidase and xanthine. The confounding problem of adventitious iron bound to xanthine oxidase

The reaction of xanthine and xanthine oxidase generates superoxide and hydrogen peroxide. In contrast to earlier works, recent spin trapping data (Kuppusamy, P., and Zweier, J.L. (1989) J. Biol. Chem. 264, 9880-9884) suggested that hydroxyl radical may also be a product of this reaction. Determining if hydroxyl radical results directly from the xanthine/xanthine oxidase reaction is important for 1) interpreting experimental data in which this reaction is used as a model of oxidant stress, and 2) understanding the pathogenesis of ischemia/reperfusion injury. Consequently, we evaluated the conditions required for hydroxyl radical generation during the oxidation of xanthine by xanthine oxidase. Following the addition of some, but not all, commercial preparations of xanthine oxidase to a mixture of xanthine, deferoxamine, and either 5,5-dimethyl-1-pyrroline-N-oxide or a combination of alpha-phenyl-N-tert-butyl-nitrone and dimethyl sulfoxide, hydroxyl radical-derived spin adducts were detected. With other preparations, no evidence of hydroxyl radical formation was noted. Xanthine oxidase preparations that generated hydroxyl radical had greater iron associated with them, suggesting that adventitious iron was a possible contributing factor. Consistent with this hypothesis, addition of H2O2, in the absence of xanthine, to "high iron" xanthine oxidase preparations generated hydroxyl radical. Substitution of a different iron chelator, diethylenetriaminepentaacetic acid for deferoxamine, or preincubation of high iron xanthine oxidase preparations with chelating resin, or overnight dialysis of the enzyme against deferoxamine decreased or eliminated hydroxyl radical generation without altering the rate of superoxide production. Therefore, hydroxyl radical does not appear to be a product of the oxidation of xanthine by xanthine oxidase. However, commercial xanthine oxidase preparations may contain adventitious iron bound to the enzyme, which can catalyze hydroxyl radical formation from hydrogen peroxide.     

Amplification of antioxidant activity and xanthine oxidase inhibition of catechin by enzymatic polymerization

To amplify the antioxidant activity, we synthesized poly(catechin) by the enzyme-catalyzed oxidative coupling using horseradish peroxidase as a catalyst. The poly(catechin) showed great improvement in antioxidant activity such as radical scavenging activity against the superoxide anion and inhibition effects against free radical induced-oxidation of low-density lipoprotein, compared with a catechin monomer. In addition, poly(catechin) showed very high inhibition effects on xanthine oxidase activity, whereas the catechin monomer showed very less inhibition effects. The amplified activities might offer a high potential as a therapeutic agent for prevention of various free radicals and/or enzyme-related diseases.     

The role of cellular oxidases and catalytic iron in the pathogenesis of ethanol-induced liver injury

Free radical generation and catalytic iron have been implicated in the pathogenesis of alcohol-induced liver injury but the source of free radicals is a subject of controversy. The mechanism of ethanol-induced liver injury was investigated in isolated hepatocytes from a rodent model of iron loading in which free radical generation was measured by the determination of alkane production (ethane and pentane). Iron loading (125mg/kg i.p.) increased hepatic non-heme iron 3-fold, increased the prooxidant activity of cytosolic ultrafiltrates 2-fold and doubled ethanol-induced alkane production. The addition of desferrioxamine (20μM), a tight chelator of iron, completely abolished alkane production indicating the importance of catalytic iron. The role of cellular oxidases as a source of ethanol induced free radicals was studied through the use of selective inhibitors. In both the presence and absence of iron loading, selective inhibition of xanthine oxidase with oxipurinol(20μM) diminished ethanol-induced alkane production 0–40%, inhibition of aldehyde oxidase with menadione (20μM) diminished alkane production 36–75%, while the inhibition of aldehyde and xanthine oxidase by feeding tungstate (100mg/kg/day) virtually abolished alkane production. Addition of acetaldehyde(50μM) to hepatocytes generated alkanes at rates comparable to those achieved with ethanol indicating the importance of acetaldehyde metabolism in free radical generation. The cellular oxidases (aldehyde and xanthine oxidase) along with catalytic iron play a fundamental role in the pathogenesis of free radical injury due to ethanol.     

Effect of chelating agents and superoxide on human neutrophil NAD(P)H oxidation

NAD(P)H oxidation is frequently measured to assay the activity of the neutrophil O-2-generating oxidase. It was found that 10(-4) M ethylene glycol bis (beta-aminoethyl ether)-N-N'-tetraacetic acid (EGTA) increased NAD(P)H oxidation by the 27,000 g granule fraction of resting and stimulated human neutrophils without altering net O-2 production. The commonly used chelating agents EDTA and diethylene triamine pentaacetic acid had similar effects. The addition of superoxide dismutase eliminated the effect of the chelating agents and thus demonstrated that the stimulated reaction was dependent upon O-2. KCN and bathophenanthroline disulfonate, an iron-chelating agent, prevented O-2-dependent NADPH oxidation by neutrophil granule fractions in the presence of EGTA. In contrast, bathocuproine disulfonate, a copper-chelating agent, mimicked the EGTA effect. The effects of both bathophenanthroline disulfonate and bathocuproine disulfonate were completely abolished when the agents were saturated with iron and copper, respectively. All the chelating agents studied, except bathophenonthroline disulfonate, also promoted O-2-dependent NADPH oxidation in a system wherein O-2 was generated by xanthine oxidase. Thus, commonly used chelating agents, by interacting with available iron and copper, may alter the apparent stoichiometry of the neutrophil O-2-generating oxidase and artifactually increase NADPH oxidation in other systems where O-2 is present.