The antioxidant action of ergothioneine.

Ergothioneine is a product of plant origin that accumulates in animal tissues. Its suggested ability to act as an antioxidant has been evaluated. Ergothioneine is a powerful scavenger of hydroxyl radicals (.OH) and an inhibitor of iron or copper ion-dependent generation of .OH from hydrogen peroxide (H2O2). It is also an inhibitor of copper ion-dependent oxidation of oxyhaemoglobin, and of arachidonic acid peroxidation promoted by mixtures of myoglobin (or haemoglobin) and H2O2. Ergothioneine is a powerful scavenger of hypochlorous acid, being able to protect alpha 1-antiproteinase against inactivation by this molecule. By contrast, it does not react rapidly with superoxide (O2-) or hydrogen peroxide (H2O2) and it does not inhibit microsomal lipid peroxidation in the presence of iron ions. Overall, our results show that ergothioneine at the concentrations present in vivo could act as an antioxidant.     

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.     

Free radical induced damages to rat liver subcellular organelles: inhibition by Andrographis paniculata extract

Aqueous extract of Andrographis paniculata was examined for antioxidant activity using rat liver subcellular organelles as model systems. The study deals with two important biological oxidative agents, ascorbate-Fe(+2) and AAPH generating hydroxyl and peroxyl radical, respectively. Oxidative damage was examined against the inhibition of membrane peroxidation, protein oxidation and restoration in decreased SOD and catalase activity. The antimutagenic activity of Ap was examined following inhibition in AAPH induced strand breaks in plasmid pBR322 DNA. Extract was a potent scavenger of DPPH, ABTS radicals, exemplified by ESR signals, O2-*, *OH and H2O2, displayed excellent reducing power, FRAP potentials to reduce Fe (III) --> Fe (II) and had considerable amount of phenolics/ flavonoids contents, an effective antioxidant index. The observed antioxidant effect might be primarily due to its high scavenging ability for ROS. Effect was confirmed ex vivo following inhibition in peroxidation, restoration in SOD enzyme, SOD band intensity and protein degradation in Ap fed liver homogenate. Based on these results, it was concluded that the aqueous extract of Andrographis paniculata might emerge as a potent antiradical agent against various pathophysiological oxidants.     

Antioxidant activity of galloyl quinic derivatives isolated from P. lentiscus leaves

The antioxidant properties of galloyl quinic derivatives isolated from Pistacia lentiscus L. leaves have been investigated by means of Electron Paramagnetic Resonance spectroscopy (EPR) and UV-Vis spectrophotometry. Antioxidant properties have been also estimated using the biologically relevant LDL test. The scavenger activities of gallic acid, 5- O -galloyl, 3,5- O -digalloyl, 3,4,5- O -trigalloyl quinic acid derivatives, have been estimated against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, superoxide ( O 2 - ) radical, and hydroxyl (OH) radical. On the whole, the scavenger activity raised as the number of galloyl groups on the quinic acid skeleton increased. The half-inhibition concentrations (IC 50 ) of di- and tri-galloyl derivatives did not exceed 30 μM for all the tested free radicals. All the tested metabolites strongly reduced the oxidation of low-density lipoproteins (LDL), following a trend similar to that observed for the scavenger ability against OH radical.     

Protective effect of quercetin and luteolin in human melanoma HMB-2 cells

Multifunctional effects of flavonoids are reported to be markedly connected with their structure and the functional groups in the molecule. The important role in the activity play C2-C3 double bond, hydroxyl group at C3 and the number of hydroxyl groups at phenyl ring (B). In this paper, the DNA protective free radical scavenging potential of quercetin (QU) and luteolin (LU) against H2O2 and their clastogenic effect alone and in combination with melphalan (MH) were investigated in human melanoma HMB-2 cells. Elevated frequency of chromosomal aberrations induced by MH, that at high doses have shown a variety of toxic side effects, was statistically decreased by studied flavonoids regarding to control (QU at the concentration of 50 microM and LU already at the concentration of 20 microM). The results concerning DNA protective potential against free radicals in HMB-2 cells demonstrated that QU and LU have significant effect in dose dependent manner. The percentage of QU protective effect is 40% at the concentration 20 microM, resp. 80% at the concentration 100 microM. Comparable values were obtained with LU. Results are correlated to their structural arrangement and organization of the hydroxyl groups.     

Experimental evidence that flavonoid metal complexes may act as mimics of superoxide dismutase

Radical scavenging activities of flavonoids rutin, taxifolin, (-)-epicatechin, luteolin, and their complexes with transition metal (Fe2+, Fe3+, and Cu2+) towards superoxide were determined using illumination of riboflavin as source and NBT as detector of O*2-. The scavenger potencies of flavonoid metal complexes were significantly higher than those of the parent flavonoids. To elucidate the mechanism of this phenomenon, the rates of superoxide-dependent oxidation of flavonoids and their metal complexes in photochemical system with riboflavin were examined. It was found for the first time that flavonoids bound to metal ions were much less subjected to oxidation compared with those of free compounds. The findings directly demonstrate superoxide scavenging activity of metal ions in complexes with flavonoids and support earlier suggestions that flavonoid metal complexes may exhibit superoxide dismuting activity.     

Chloroplast-located flavonoids can scavenge singlet oxygen

* The hypothesis was tested that flavonoids may scavenge singlet oxygen ((1)O(2)) in mesophyll cells of Phillyrea latifolia exposed to excess-light stress. * In cross-sections taken from leaves developed at 10% (shade) or 100% (sun) solar irradiance, we evaluated the excess photosynthetically active radiation (PAR)-induced accumulation of (1)O(2) in mesophyll cells by imaging the fluorescence quenching of the specific (1)O(2) probe N-[2-(diethylamino)ethyl]-N-[(2,5-dihydro-2,2,5,5-tetramethyl-1H-pyrrol-3-yl)methyl]-5-(dimethylamino)-1-naphthalenesulfonamide (DanePy). The intracellular location of flavonoids was also analyzed using three-dimensional deconvolution microscopy. * Photo-induced quenching of DanePy fluorescence was markedly greater in the mesophyll of shade leaves than in that of sun leaves, the former showing a negligible accumulation of mesophyll flavonoids. The photo-induced generation of (1)O(2) was inversely related to the content of flavonoids in the mesophyll cells of sun leaves. Flavonoids were located in the chloroplasts, and were likely associated with the chloroplast envelope. * Here we provide relevant evidence for the potential scavenger activity of chloroplast-located flavonoids against (1)O(2) and new insights into the photo-protective role of flavonoids in higher plants.     

Inhibition of prostaglandin delta 13 reductase activity in rabbit kidney cortex by glutathione disulfide.

t-Butyl hydroperoxide and H2O2-Fe(2+)-EDTA-glutathione system which produces hydroxyl radicals did not affect the 15-hydroxy prostaglandin dehydrogenase activity in rabbit kidney cortex. On the other hand, H2O2-Fe(2+)-EDTA-glutathione system inhibited the prostaglandin delta 13 reductase activity. Mannitol, a scavenger of hydroxyl radicals, had no effect on the inhibitory action of this system, indicating that the effect of H2O2-Fe(2+)-EDTA-glutathione system on the prostaglandin delta 13 reductase may not be due to produced hydroxyl radicals. As a result of further investigation, it was shown that glutathione disulfide, which is synthesized concomitantly with hydroxyl radicals from H2O2-Fe(2+)-EDTA-glutathione, inhibited the prostaglandin delta 13 reductase activity. These results suggest that hydroperoxides and hydroxyl radicals may not be likely candidates for the modulator of the catabolism of prostaglandins in the kidney cortex, and that glutathione disulfide has the potential to modulate the prostaglandin catabolism by affecting the prostaglandin delta 13 reductase activity.     

Structure-antioxidant activity relationships of flavonoids isolated from the resinous exudate of Heliotropium sinuatum

Relationships between the structural characteristics of flavonoids isolated from the resinous exudate of Heliotropium sinuatum and their antioxidant activity were studied. Radical formation energies, DeltaH of dehydrogenation and spin densities were calculated using DFT methods (B3LYP/6-31G*). Results show that studied flavonoids can be divided into two sets according to their activity. It has been found that antioxidant activity depends both on substitution pattern of hydroxyl groups of the flavonoid skeleton and the presence of an unsaturation at the C2-C3 bond. A good tendency between DeltaH of dehydrogenation and antioxidant activity was established.     

Radiation protection by the ocimum flavonoids orientin and vicenin: mechanisms of action

In previous studies, flavonoids, orientin and vicenin, that were isolated from the leaf extract of Ocimum sanctum, were found to protect mice against radiation injury. Several flavonoids are known to be good antioxidants. Therefore, the effect of orientin and vicenin on radiation-induced lipid peroxidation in vivo and their antioxidant activity in vitro were studied. Adult mice were injected intraperitoneally with 50 microgram/kg of orientin or vicenin and exposed whole-body to 3 Gy of gamma radiation. Lipid peroxidation was measured in the liver 15 min to 8 h postirradiation. The antioxidant activity of orientin/vicenin (10-500 microM) was studied by measuring inhibition of hydroxyl radicals generated by the Fenton reaction (Fe(3+)-EDTA-ascorbic acid-H(2)O(2)) in vitro. The compounds were also tested for possible pro-oxidant and iron chelation activities at the above concentrations in the in vitro system. Orientin and vicenin provided almost equal protection against radiation-induced lipid peroxidation in mouse liver. Both compounds showed a significantly greater free radical-inhibiting activity in vitro than DMSO. Neither orientin nor vicenin showed any pro-oxidant activity at the concentrations tested. Both compounds inhibited free radical formation in the absence of EDTA. Free radical scavenging appears to be a likely mechanism of radiation protection by these flavonoids.     

Biosynthesis and function of melanins in hepatic pigmentary system

In this report we show the most important results obtained from our study of the pigment liver cells of Amphibia and Reptilia. Contrary to the cutaneous pigment cells that derive from the neural crest, the liver pigment cells, instead, derive from the Kupffer cells: this can be seen from the results obtained from labelled precursor incorporation experiments, not only in vivo but also in vitro, using surviving liver slices or isolated melanosomes. Chemical analysis of both liver and cutaneous melanosomes reveal a great difference in their chemical composition, and this is in agreement with the different origins of these cells. We therefore propose that: liver pigment cells of Amphibia and Reptilia should be classified as "Extra Cutaneous Pigment Cells from Histocytic Origin". As regards the function of melanins, we show that O2 is trapped by these substances. Moreover, in my laboratory we have shown in several animal species, that the superoxide dismutase activity is inversely proportional to the quantity of melanin present; thus, we think that melanin could mime SOD activity.     

Identification of tyrosinase inhibitors from Marrubium velutinum and Marrubium cylleneum

Tyrosinase is a key enzyme in the production of melanins in plants and animals. Forty-five secondary metabolites isolated from Marrubium velutinum and Marrubium cylleneum belonging to the classes of flavonoids, phenylethanoid glycosides, phenolic acids and lignan glycosides were screened for their inhibitory activity against mushroom tyrosinase. Flavonoids and phenylethanoid glycosides showed moderate inhibitory activity, while phenolic acids were less active than phenylethanoid glycosides, suggesting that both phenolic groups are important for the activity.     

Scavenging of reactive oxygen species by the plant phenols genistein and oleuropein.

The plant-derived phenolic compounds genistein and oleuropein are known to exhibit several biological properties, many of which may result from their antioxidant and free radical scavenger activity. In this paper we report the results of a complex study of antioxidant activity of genistein and oleuropein, using electron spin resonance (ESR), chemiluminescence, fluorescence and spectrophotometric techniques. Different reaction systems were applied to study the inhibitory effect of the phenolic compounds studied: (a) the potassium superoxide/18-crown-6 dissolved in DMSO system, which generates superoxide radical (O(2).(-)) and hydrogen peroxide (H(2)O(2)); (b) the Co(II)-EDTA-H(2)O(2) system (the Fenton-like reaction), which generates hydroxyl radical (HO.); (c) 2,2'-azobis(2-amidino-propane)dichloride (AAPH) as the peroxyl radical (ROO.) generator, and the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical test. Results showed that genistein and oleuropein decreased the chemiluminescence sum from the O(2).(-) generating system, an inhibitory effect that was dependent on their concentration. These compounds also reacted with ROO radicals and they showed activity about two-fold greater than the standard Trolox. The antioxidant effects were studied at different concentrations and reflected in protection against the fluorescence decay of beta-phycoerythrin (beta-PE), due to ROO. attack on this protein. Using the Fenton-like reaction and the spin trap agent 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), the phenolic compounds examined were found to inhibit DMPO-.OH radical formation in the range 10-90% at concentrations of 0.1 mmol/L to 2 mmol/L. Furthermore, these compounds also inhibited HO.-dependent deoxyribose degradation; about 20% and 60% inhibitions were observed in the presence of 0.5 mmol/L genistein and oleuropein, respectively. It was also demonstrated that genistein had a weaker DPPH radical scavenging activity than oleuropein. Our results confirm good scavenging activity towards O(2).(-), HO. and ROO. and the antioxidant effect of genistein and oleuropein.     

Structure prerequisite for antioxidant activity of silybin in different biochemical systems in vitro.

Structural analogues (flavanone: 2-4 and flavone: 5 and 6, respectively) of silybin (1a) were synthesized and tested for inhibitory activity on O(2)(-) release and PKC translocation in PMA-stimulated neutrophils as well as xanthine oxidase activity in order to identify the molecular structures responsible for the antioxidant property of silybin. Concerning the prevention of hem-mediated oxidative modification of LDL by silybin, the hydroxyl radical scavenging activity of its structural analogues was also determined. We demonstrated that the basic skeleton of 1a (4) is responsible for its inhibitory activity on O(2)(-) release in PMA-stimulated neutrophils via inhibition of PKC translocation, since introduction of a double bound and hydroxyl groups at C-5 and C-7 position (5 and 6) did not result in further increase in inhibition of O(2)(-) release. It has been shown that the presence of the phenolic hydroxyl group at C-5 and C-7 of 1a is essential for the inhibition of xanthine oxidase activity. Moreover, introduction of a double bond into the C-ring of 2 and 3, resulting in flavone derivatives (5 and 6), markedly enhanced the antioxidant effect in all the tested systems. Finally, silybin (1a) and its flavon derivatives (5 and 6) directly scavenged hydroxyl radicals as well. On the basis of these results it might be concluded that different moiety of silybin is responsible for inhibition of overproduction of O(2)(-) in stimulated neutrophils, xanthine oxidase activity, and for prevention of hem-mediated oxidative modification of LDL.     

Free radical generation during the activation of hemolymph prepared from the homopteran Dactylopius coccus

Superoxide anion (O(-) (2)) and nitric oxide (NO) generation in Dactylopius coccus hemolymph obtained by perfusion and activated with zymosan was studied. Activated hemolymph reduced 3-[4,5 dimethylthiazolil-2]-2,5-diphenyl tetrazolium bromide. This reduction was prevented by superoxide dismutase (SOD) indicating O(-) (2) generation. This activity was dependent on temperature, and hemolymph incubated at 75 degrees C lost its activity. Chromatocytes incubated with zymosan released their content and produced O(-) (2). Activated hemolymph also produced NO and this activity was prevented in the presence of NG-nitro-L-arginine methyl ester, suggesting that nitric oxide synthase (NOS) might be present in D. coccus hemolymph. The probable source of O(-) (2) in the D. coccus hemolymph is the anthraquinone oxidation, since commercial carminic dye produced O(-) (2) during its oxidation by Agaricus bisporus tyrosinase. Gram+ Micrococcus luteus exposed to activated hemolymph were killed in vitro, and addition of NG-nitro-L-arginine methyl ester and D-Mannitol (a hydroxyl radical scavenger) prevented their killing. The cytotoxic effect produced by the activated hemolymph was not observed with the Gram- bacteria Serratia marcescens. These results suggest that D. coccus activated hemolymph generates reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI) that may limit M. luteus growth.     

Co-oxidation of luminol by hypochlorite and hydrogen peroxide implications for neutrophil chemiluminescence

Stimulated neutrophils produce several potent oxidants including H2O2, O2- and HOCl. Previous studies have revealed all of these compounds to be capable of oxidizing luminol, a reagent often used to indicate, by its chemiluminescence, the oxidative burst of neutrophils. Data presented in this paper indicate that H2O2 and HOCl spontaneously react at physiologic pH to produce luminol-dependent chemiluminescence 100 times the sum of the chemiluminescence of either reagent alone. This enhancement is due to a co-oxidation by HOCl and H2O2, or to a novel oxidant generated by the interaction of HOCl and H2O2. The HOCl scavenger, taurine, inhibits the chemiluminescence. Evidence is presented against the participation of hydroxyl radical, O2- or singlet oxygen in the oxidation of luminol by HOCl and H2O2. These findings have implications for potential anti-inflammatory compounds.     

Structure–Activity Relationships for Antioxidant Activities of a Series of Flavonoids in a Liposomal System

Structurally diverse plant phenolics were examined for their abilities to inhibit lipid peroxidation induced either by Fe(II) and Fe(III) metal ions or by azo-derived peroxyl radicals in a liposomal membrane system. The antioxidant abilities of flavonoids were compared with those of coumarin and tert-butylhydroquinone (TBHQ). The antioxidant efficacies of these compounds were evaluated on the basis of their abilities to inhibit the fluorescence intensity decay of an extrinsic probe, 3-(p-(6-phenyl)-1,3,5-hexatrienyl)phenylpropionic acid (DPH-PA), caused by the free radicals generated during lipid peroxidation. All the flavonoids tested exhibited higher antioxidant efficacies against metal-ion-induced peroxidations than peroxyl-radical-induced peroxidation, suggesting that metal chelation may play a larger role in determining the antioxidant activities of these compounds than has previously been believed. Distinct structure–activity relationships were also revealed for the antioxidant abilities of the flavonoids. Presence of hydroxyl substituents on the flavonoid nucleus enhanced activity, whereas substitution by methoxy groups diminished antioxidant activity. Substitution patterns on the B-ring especially affected antioxidant potencies of the flavonoids. In cases where the B-ring could not contribute to the antioxidant activities of flavonoids, hydroxyl substituents in an catechol structure on the A-ring were able to compensate and become a larger determinant of flavonoid antioxidant activity.     

The antioxidant action of taurine, hypotaurine and their metabolic precursors.

It has been suggested that taurine, hypotaurine and their metabolic precursors (cysteic acid, cysteamine and cysteinesulphinic acid) might act as antioxidants in vivo. The rates of their reactions with the biologically important oxidants hydroxyl radical (.OH), superoxide radical (O2.-), hydrogen peroxide (H2O2) and hypochlorous acid (HOCl) were studied. Their ability to inhibit iron-ion-dependent formation of .OH from H2O2 by chelating iron ions was also tested. Taurine does not react rapidly with O2.-, H2O2 or .OH, and the product of its reaction with HOCl is still sufficiently oxidizing to inactivate alpha 1-antiproteinase. Thus it seems unlikely that taurine functions as an antioxidant in vivo. Cysteic acid is also poorly reactive to the above oxidizing species. By contrast, hypotaurine is an excellent scavenger of .OH and HOCl and can interfere with iron-ion-dependent formation of .OH, although no reaction with O2.- or H2O2 could be detected within the limits of our assay techniques. Cysteamine is an excellent scavenger of .OH and HOCl; it also reacts with H2O2, but no reaction with O2.- could be measured within the limits of our assay techniques. It is concluded that cysteamine and hypotaurine are far more likely to act as antioxidants in vivo than is taurine, provided that they are present in sufficient concentration at sites of oxidant generation.     

Structure-activity relationships and molecular docking of thirteen synthesized flavonoids as horseradish peroxidase inhibitors

For the first time, the structure–activity relationships of thirteen synthesized flavonoids have been investigated by evaluating their ability to modulate horseradish peroxidase (HRP) catalytic activity. Indeed, a modified spectrophotometrically method was carried out and optimized using 4-methylcatechol (4-MC) as peroxidase co-substrate.The results show that these flavonoids exhibit a great capacity to inhibit peroxidase with Ki values ranged from 0.14 ± 0.01 to 65 ± 0.04 mM. Molecular docking has been achieved using Auto Dock Vina program to discuss the nature of interactions and the mechanism of inhibition. According to the docking results, all the flavonoids have shown great binding affinity to peroxidase. These molecular modeling studies suggested that pyran-4-one cycle acts as an inhibition key for peroxidase. Therefore, potent peroxidase inhibitors are flavonoids with these structural requirements: the presence of the hydroxyl (OH) group in 7, 5 and 4′ positions and the absence of the methoxy (O–CH₃) group. Apigenin contributed better in HRP inhibitory activity.The present study has shown that the studied flavonoids could be promising HRP inhibitors, which can help in developing new molecules to control thyroid diseases.     

Hydroxyl radical and hypochlorous acid scavenging activity of small Centaury (Centaurium erythraea) infusion. A comparative study with green tea (Camellia sinensis)

Small centaury (Centaurium erythraea Rafin.) is a herbal species with a long use in traditional medicine due to its digestive, stomachic, tonic, depurative, sedative and antipyretic properties. This species is reported to contain considerable amounts of polyphenolic compounds, namely xanthones and phenolic acids as the main constituents. Although the antiradicalar activity of some pure polyphenolic compounds is already known, it remains unclear how a complex mixture obtained from plant extracts functions against reactive oxygen species. Thus, the ability of small centaury infusion to act as a scavenger of the reactive oxygen species hydroxyl radical and hypochlorous acid was studied and compared with that of green tea (Camellia sinensis L.). Hydroxyl radical was generated in the presence of Fe3+-EDTA, ascorbate and H2O2 (Fenton system) and monitored by evaluating hydroxyl radical-induced deoxyribose degradation. The reactivity towards hypochlorous acid was determined by measuring the inhibition of hypochlorous acid-induced 5-thio-2-nitrobenzoic acid oxidation to 5,5'-dithiobis(2-nitrobenzoic acid). The obtained results demonstrate that small centaury infusion exhibits interesting antioxidant properties, expressed both by its capacity to effectively scavenge hydroxyl radical and hypochlorous acid, although with a lower activity against the second than that observed for green tea. Green tea exhibited a dual effect at the hydroxyl radical scavenging assay, stimulating deoxyribose degradation at lower dosages.