Antioxidant and radical scavenging properties of curcumin

Curcumin (diferuoyl methane) is a phenolic compound and a major component of Curcuma longa L. In the present paper, we determined the antioxidant activity of curcumin by employing various in vitro antioxidant assays such as 1,1-diphenyl-2-picryl-hydrazyl free radical (DPPH*) scavenging, 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radical scavenging activity, N,N-dimethyl-p-phenylenediamine dihydrochloride (DMPD) radical scavenging activity, total antioxidant activity determination by ferric thiocyanate, total reducing ability determination by the Fe(3+)-Fe(2+) transformation method, superoxide anion radical scavenging by the riboflavin/methionine/illuminate system, hydrogen peroxide scavenging and ferrous ions (Fe(2+)) chelating activities. Curcumin inhibited 97.3% lipid peroxidation of linoleic acid emulsion at 15 microg/mL concentration (20 mM). On the other hand, butylated hydroxyanisole (BHA, 123 mM), butylated hydroxytoluene (BHT, 102 mM), alpha-tocopherol (51 mM) and trolox (90 mM) as standard antioxidants indicated inhibition of 95.4, 99.7, 84.6 and 95.6% on peroxidation of linoleic acid emulsion at 45 microg/mL concentration, respectively. In addition, curcumin had an effective DPPH* scavenging, ABTS*(+) scavenging, DMPD*(+) scavenging, superoxide anion radical scavenging, hydrogen peroxide scavenging, ferric ions (Fe(3+)) reducing power and ferrous ions (Fe(2+)) chelating activities. Also, BHA, BHT, alpha-tocopherol and trolox, were used as the reference antioxidant and radical scavenger compounds. According to the present study, curcumin can be used in the pharmacological and food industry because of these properties.     

Optimisation of the azinobis-3-ethyl-benzothiazoline-6-sulphonic acid radical scavenging assay for physiological studies of total antioxidant activity in woody plant germplasm.

A robust spectroscopic method for determining total antioxidant activity in aqueous extractions has been applied to tissues from diverse woody plant species, including seeds of Coffea arabica and in vitro shoots from Ribes nigrum, Picea sitchensis and Shorea leprosula. The assay involves scavenging of an ABTS [2,2'-azinobis-(3-ethyl-benzothiazoline-6-sulphonic acid)] radical generated by the reaction of potassium persulphate with ABTS to produce an ABTS*(+) chromophore (lambda=734 nm). Antioxidants reduce ABTS*(+) back to ABTS with a concomitant decrease in absorbance. Aqueous extractions from C. arabica and S. leprosula had considerably higher (110-205 micromol Trolox eq. g(-1) FW) total antioxidant activities than P. sitchensis and R. nigrum (6-11 micromol Trolox eq. g(-1) FW). Further studies in two of these species showed that the inclusion of water-insoluble polyvinylpyrrolidone during aqueous tissue extraction enabled the combined phenolic and alkaloid antioxidant activity to be determined. These fractions accounted for 85% and 60% of total antioxidant activity for C. arabica seeds and R. nigrum shoots, respectively. The ABTS radical scavenging assay is presented herein as a robust method for determining total antioxidant activity in germplasm from diverse woody plant tissues and species. Its applicability to study oxidative stress in tissue cultures and germplasm employed in plant biotechnology, breeding and stress physiology programmes is discussed.     

Antioxidant activity: what do we measure?

Inhibition of oxidation of 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) by free radicals generated by decomposition of 2,2'-azobis(2-amidopropane) (ABAP) by antioxidants and biological material was studied. A correlation was found between the ability of various substances to delay the onset of ABTS oxidation and their rapid reduction of the ABTS+* cation radical, and between the ability to reduce the maximal rate of ABTS oxidation and slow reduction of ABTS+*. The length of the lag period of ABTS oxidation was found to be independent of ABTS concentration. Similar decrease of peroxynitrite-induced ABTS+* formation by antioxidants was observed when the antioxidants were added before and after peroxynitrite. All these findings indicate that the main effect of antioxidants in this system is reduction of ABTS+* and not prevention of its formation. Reduction of oxidation products rather than inhibition of their formation may be the predominant mode of action of antioxidants in various assays of antioxidant activity.     

Cytochrome c-catalyzed oxidation of organic molecules by hydrogen peroxide.

Cytochrome c catalyzed the oxidation of various electron donors in the presence of hydrogen peroxide (H2O2), including 2-2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), 4-aminoantipyrine (4-AP), and luminol. With ferrocytochrome c, oxidation reactions were preceded by a lag phase corresponding to the H2O2-mediated oxidation of cytochrome c to the ferric state; no lag phase was observed with ferricytochrome c. However, brief preincubation of ferricytochrome c with H2O2 increased its catalytic activity prior to progressive inactivation and degradation. Superoxide (O2-) and hydroxyl radical (.OH) were not involved in this catalytic activity, since it was not sensitive to superoxide dismutase (SOD) or mannitol. Free iron released from the heme did not play a role in the oxidative reactions as concluded from the lack of effect of diethylenetriaminepentaacetic acid. Uric acid and tryptophan inhibited the oxidation of ABTS, stimulation of luminol chemiluminescence, and inactivation of cytochrome c. Our results are consistent with an initial activation of cytochrome c by H2O2 to a catalytically more active species in which a high oxidation state of an oxo-heme complex mediates the oxidative reactions. The lack of SOD effect on cytochrome c-catalyzed, H2O2-dependent luminol chemiluminescence supports a mechanism of chemiexcitation whereby a luminol endoperoxide is formed by direct reaction of H2O2 with an oxidized luminol molecule, either luminol radical or luminol diazoquinone.     

Bicarbonate enhances the hydroxylation, nitration, and peroxidation reactions catalyzed by copper, zinc superoxide dismutase. Intermediacy of carbonate anion radical

The effect of bicarbonate anion (HCO(3)(-)) on the peroxidase activity of copper, zinc superoxide dismutase (SOD1) was investigated using three structurally different probes: 5, 5'-dimethyl-1-pyrroline N-oxide (DMPO), tyrosine, and 2, 2'-azino-bis-[3-ethylbenzothiazoline]-6-sulfonic acid (ABTS). Results indicate that HCO(3)(-) enhanced SOD/H(2)O(2)-dependent (i) hydroxylation of DMPO to DMPO-OH as measured by electron spin resonance, (ii) oxidation and nitration of tyrosine to dityrosine, nitrotyrosine, and nitrodityrosine as measured by high pressure liquid chromatography, and (iii) oxidation of ABTS to the ABTS cation radical as measured by UV-visible spectroscopy. Using oxygen-17-labeled water, it was determined that the oxygen atom present in the DMPO-OH adduct originated from H(2)O and not from H(2)O(2). This result proves that neither free hydroxyl radical nor enzyme-bound hydroxyl radical was involved in the hydroxylation of DMPO. We postulate that HCO(3)(-) enhances SOD1 peroxidase activity via formation of a putative carbonate radical anion. This new and different perspective on HCO(3)(-)-mediated oxidative reactions of SOD1 may help us understand the free radical mechanism of SOD1 and related mutants linked to amyotrophic lateral sclerosis.     

The iron chelator pyridoxal isonicotinoyl hydrazone (PIH) and its analogues prevent damage to 2-deoxyribose mediated by ferric iron plus ascorbate

Iron chelating agents are essential for treating iron overload in diseases such as beta-thalassemia and are potentially useful for therapy in non-iron overload conditions, including free radical mediated tissue injury. Deferoxamine (DFO), the only drug available for iron chelation therapy, has a number of disadvantages (e.g., lack of intestinal absorption and high cost). The tridentate chelator pyridoxal isonicotinoyl hydrazone (PIH) has high iron chelation efficacy in vitro and in vivo with high selectivity and affinity for iron. It is relatively non-toxic, economical to synthesize and orally effective. We previously demonstrated that submillimolar levels of PIH and some of its analogues inhibit lipid peroxidation, ascorbate oxidation, 2-deoxyribose degradation, plasmid DNA strand breaks and 5,5-dimethylpyrroline-N-oxide (DMPO) hydroxylation mediated by either Fe(II) plus H(2)O(2) or Fe(III)-EDTA plus ascorbate. To further characterize the mechanism of PIH action, we studied the effects of PIH and some of its analogues on the degradation of 2-deoxyribose induced by Fe(III)-EDTA plus ascorbate. Compared with hydroxyl radical scavengers (DMSO, salicylate and mannitol), PIH was about two orders of magnitude more active in protecting 2-deoxyribose from degradation, which was comparable with some of its analogues and DFO. Competition experiments using two different concentrations of 2-deoxyribose (15 vs. 1.5 mM) revealed that hydroxyl radical scavengers (at 20 or 60 mM) were significantly less effective in preventing degradation of 2-deoxyribose at 15 mM than 2-deoxyribose at 1.5 mM. In contrast, 400 microM PIH was equally effective in preventing degradation of both 15 mM and 1.5 mM 2-deoxyribose. At a fixed Fe(III) concentration, increasing the concentration of ligands (either EDTA or NTA) caused a significant reduction in the protective effect of PIH towards 2-deoxyribose degradation. We also observed that PIH and DFO prevent 2-deoxyribose degradation induced by hypoxanthine, xanthine oxidase and Fe(III)-EDTA. The efficacy of PIH or DFO was inversely related to the EDTA concentration. Taken together, these results indicate that PIH (and its analogues) works by a mechanism different than the hydroxyl radical scavengers. It is likely that PIH removes Fe(III) from the chelates (either Fe(III)-EDTA or Fe(III)-NTA) and forms a Fe(III)-PIH(2) complex that does not catalyze oxyradical formation.     

An enzymatic colorimetric method for measuring naringin using 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid)(ABTS) in the presence of peroxidase

A sensitive colorimetric method for naringin estimation using 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) as peroxidase substrate is described. The method is based on the coupling reaction of an ABTS radical cation with an oxidation product of naringin formed by peroxidase. This coupling reaction leads to the formation of a purple-colored compound with a maximum absorption at 560 nm. A molar absorption coefficient at this wavelength was calculated to be 13,286 +/- 200 M-1 cm-1. The lowest amount of naringin that can be detected is 1 nmol. The application of this method to the quantification of naringin in grapefruit tissues is presented.     

Mechanism of reduction of bleomycin-Cu(II) by CO2- and oxidation of bleomycin-Cu(I) by H2O2 in the absence and presence of DNA

The reduction reaction of bleomycin-Cu(II) by CO2- has been studied by gamma and pulse radiolysis at pH7. The CO2- radical reduces bleomycin-Cu(II) at a rate of (6.7 +/- 0.7) X 10(8) dm3 mol-1 s-1. In the presence of calf thymus DNA the rate of the reduction decreased as the concentration of DNA increased, indicating that the reduction reaction proceeds through free bleomycin-Cu(II). The stoichiometry and the kinetics of the oxidation of bleomycin-Cu(I) by H2O2 in the presence and absence of DNA have been studied. Our observations suggest that the OH. radical is not produced during this reaction and the degradation of the drug occurs in the absence and presence of DNA. We assume that bleomycin-Cu(II) in the presence of a reducing agent and molecular oxygen or H2O2 does not cleave DNA since the oxidizing species, which are formed during the oxidation reaction by H2O2, attack the drug even in the presence of DNA.     

Laccase production by Phanerochaete chrysosporium– an artefact caused by Mn(III)?

AIMS: The possibility of laccase production by Phanerochaete chrysosporium was studied. METHODS AND RESULTS: A relatively high initial Mn(II) concentration (1-4 mM) in the growth medium leads to the development of reddish-brown coloration and intensive oxidation of 2.2'-azino-bis(3-etilbenz-tiazolin-6-sulfonate) (ABTS). The peak of ABTS oxidation was obtained approximately 1 day after the peak of MnP activity. CONCLUSION: ABTS oxidation was not caused by manganese peroxidase (MnP) nor by laccase but was the consequence of the action of Mn(III) which was stabilised in the growth medium. Decomposition of the complex took place after the biomass was removed from the growth medium and especially after the aeration of the culture was interrupted. Significance and Impact of the Study: Mn(III) seems to be the cause of false positive laccase reactions. More reliable data on MnP activity can be obtained if the complex is decomposed by the fungus before MnP activity is measured in the medium.     

Lipase-catalysed synthesis of esters of ferulic acid with natural compounds and evaluation of their antioxidant properties

Lipases from Candida antarctica (Novozyme 435^®), Candida rugosa, Chromobacterium viscosum and Pseudomonas sp. were used to perform transesterifications of vinyl ferulate with hydroxyl-steroids and p-arbutin. The antioxidant activity of the products was evaluated using the free radical 2,2′-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) free radical quenching antioxidant assays, and inhibition of the oxidation of low-density lipoprotein, LDL. Arbutin ferulate was found to possess a 19% higher antiradical activity against the ABTS free radical than its precursor ferulic acid, and it also inhibited the oxidation of LDL more efficiently (by 10%) than its precursors. All of the biocatalytically synthesised products exhibited higher antioxidant activity than Trolox, the well known commercial benchmark antioxidant, and their precursor, ferulic acid.     

A simple, post-additional antioxidant capacity assay using adenosine triphosphate-stabilized 2,2'-azinobis(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) radical cation in a G-quadruplex DNAzyme catalyzed ABTS-H2O2 system

The scavenging of 2,2'-azinobis(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) radical cation (ABTS(+)) by antioxidants has been widely used in antioxidant capacity assay. Because of ABTS(+) disproportionation, however, this radical cannot be prepared on a large scale and stored long-term, making it unsuitable for high-throughput detection and screening of antioxidants. We developed a modified "post-additional" antioxidant capacity assay. This method possessed two remarkable features: First, instead of natural peroxidases, an artificial enzyme, G-quadruplex DNAzyme, was used for the preparation of ABTS(+), thus greatly reducing the cost of the assay, and eliminating the strict demand for the storage of enzymes. Second, an ABTS(+) stabilizer, adenosine triphosphate (ATP), was used. In the presence of ATP, the disproportionation of ABTS(+) was effectively inhibited, and the lifetime of this radical cation was prolonged about 6-fold (12 days versus 2 days), making the large-scale preparation of ABTS(+) possible. Utilizing this method, the antioxidant capacities of individual antioxidants and real samples can be quantified and compared easily. In addition, this method can be developed as a high-throughput screening method for antioxidants. The screening results could even be judged by the naked eye, eliminating the need for expensive instruments.     

Oxidation of aromatic alcohols by laccase from Trametes versicolor mediated by the 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) cation radical and dication

Oxidation of aromatic alcohols, such as non-phenolic lignin model compounds, by oxidised species of 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) has been investigated. The cation radical and dication formed from ABTS were both capable of oxidising aromatic alcohols to aldehydes. The reactions terminated at the level of the aldehyde and no acids were formed. The cation radical and dication worked in a cycle as an electron-transfer compound between an oxidant and alcohol. In addition to the oxidation of the primary benzyl-hydroxyl group, an oxidation of the secondary α-hydroxyl group to the ketone by the dication was possible. All distinguishing features of these reactions corresponded to the results of the oxidation performed by the laccase of Trametes versicolor in the presence of ABTS. The decomposition products from the dication alone and ABTS with laccase confirmed the supposition that the dication was involved in the laccase mediator system. A reaction mechanism based on deprotonation of the alcohol cation radical was predicted to play a key role in the irreversible followup reaction and to be the driving force of the process. Received: 8 June 1998 / Received revision: 23 September 1998 / Accepted: 2 October 1998     

Inhibition of peroxidase activity and scavenging of reactive oxygen species by astilbin isolated from Dimorphandra mollis (Fabaceae, Caesalpinioideae)

Astilbin (5,7,3',4'-tetrahydroxy-2,3-dihydroflavonol-3-?-o-rhamnoside), a flavonoid with a large range of biological activities, was isolated from Dimorphandra mollis, a shrub common to the Brazilian Cerrado. The purpose of this study is to verify the effects of astilbin on myeloperoxidase (MPO) and horseradish peroxidase (HRP), and its antioxidant activity against hypochlorous acid (HOCl) and total antioxidant activity (TAC) by the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS?+). Astilbin inhibited MPO and HRP activities in a concentration-dependent relationship and effectively scavenged HOCl. The TAC by ABTS?+ of astilbin (IC50 ~ 20 mM) was higher than that of uric acid, which was used as a positive control. These data demonstrate that astilbin is a potent antioxidant and that it inhibits MPO and HRP activities efficiently.     

Synthesis and antioxidant capacities of hydroxyl derivatives of cinnamoylphenethylamine in protecting DNA and scavenging radicals

Cinnamoylphenethylamine (CNPA) derivatives including feruloylphenethylamine (FRPA), caffeoylphenethylamine (CFPA), cinnamoyltyramine (CNTA), feruloyltyramine (FRTA) and caffeoyltyramine (CFTA) were synthesized in order to investigate the influence of the number and position of hydroxyl group on Cu(2+)/glutathione (GSH) and 2,2'-azobis(2-amidinopropane hydrochloride) (AAPH)-induced oxidation of DNA. The radical-scavenging properties of these CNPA derivatives were also evaluated by trapping 2,2'-azinobis(3-ethylbenzothiazoline-6-sulphonate) cationic radical (ABTS(+?)), 2,2'-diphenyl-1-picrylhydrazyl radical (DPPH) and galvinoxyl radical. In addition, these CNPA derivatives were tested by linoleic acid (LH)-β-carotene-bleaching experiment. The chemical kinetic was employed to treat the results from AAPH-induced oxidation of DNA and gave the order of antioxidant ability as CFTA > CFPA > FRTA > FRPA. CFTA and CFPA also possessed high abilities to inhibit Cu2(+)/GSH-mediated degradation of DNA, whereas FRPA and FRTA can protect LH against the auto-oxidation efficiently. Finally, CFPA and FRPA exhibited high activity in trapping ABTS(+?), DPPH and galvinoxyl radicals. Therefore, the cinnamoyl group bearing ortho-dihydroxyl or hydroxyl with ortho-methoxyl benefited for CNPA derivatives to protect DNA, while hydroxyl in tyramine cannot enhance the radical-scavenging abilities of CNPA derivatives.     

In vivo protection of synaptosomes by ferulic acid ethyl ester (FAEE) from oxidative stress mediated by 2,2-azobis(2-amidino-propane)dihydrochloride (AAPH) or Fe(2+)/H(2)O(2): insight into mechanisms of neuroprotection and relevance to oxidative stress-related neurodegenerative disorders

Ferulic acid ethyl ester (FAEE) is an ester derivative of ferulic acid, the latter known for its anti-inflammatory and antioxidant properties. Previous studies from our laboratory have shown that ferulic acid protects synaptosomal membrane system and neuronal cell culture systems against hydroxyl and peroxyl radical oxidation. FAEE is lipophilic and is able to penetrate lipid bilayer. Previous studies reported that FAEE reduces Alzheimer's amyloid beta peptide Abeta(1-42)-induced oxidative stress and cytotoxicity in neuronal cell culture by direct radical scavenging and by inducing certain antioxidant proteins. In the present study we tested the hypothesis that FAEE would provide neuroprotection against free radical oxidative stress in vivo. Synaptosomes were isolated from the gerbils that were previously injected intraperitoneally (i.p.) with FAEE or DMSO and were treated with oxidants, Fe(2+)/H(2)O(2) or 2,2-azobis(2-amidino-propane)dihydrochloride (AAPH). Synaptosomes isolated from the gerbil previously injected i.p. with FAEE and treated with Fe(2+)/H(2)O(2) and AAPH showed significant reduction in reactive oxygen species (ROS), levels of protein carbonyl, protein bound 4-hydroxynonenal (HNE, a lipid peroxidation product), and 3-nitrotyrosine (3-NT, another marker of protein oxidation formed by reaction of tyrosine residues with peroxynitrite) compared to Fe(2+)/H(2)O(2) or AAPH induced oxidative stress in synapotosomes isolated from the brain of gerbils that were previously injected with DMSO. The synaptosomes isolated from gerbil pre-injected with FAEE and subsequently treated with AAPH or Fe(2+)/H(2)O(2) showed induction of heme oxygenase (HO-1) and heat shock protein 70 (HSP-70) but reduced inducible nitric oxide synthase (iNOS) levels. These results are discussed with reference to potential use of this lipophilic antioxidant phenolic compound in the treatment of oxidative stress-related neurodegenerative disorders.     

Antioxidant activity applying an improved ABTS radical cation decolorization assay.

A method for the screening of antioxidant activity is reported as a decolorization assay applicable to both lipophilic and hydrophilic antioxidants, including flavonoids, hydroxycinnamates, carotenoids, and plasma antioxidants. The pre-formed radical monocation of 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS*+) is generated by oxidation of ABTS with potassium persulfate and is reduced in the presence of such hydrogen-donating antioxidants. The influences of both the concentration of antioxidant and duration of reaction on the inhibition of the radical cation absorption are taken into account when determining the antioxidant activity. This assay clearly improves the original TEAC assay (the ferryl myoglobin/ABTS assay) for the determination of antioxidant activity in a number of ways. First, the chemistry involves the direct generation of the ABTS radical monocation with no involvement of an intermediary radical. Second, it is a decolorization assay; thus the radical cation is pre-formed prior to addition of antioxidant test systems, rather than the generation of the radical taking place continually in the presence of the antioxidant. Hence the results obtained with the improved system may not always be directly comparable with those obtained using the original TEAC assay. Third, it is applicable to both aqueous and lipophilic systems.     

Reactions of the melatonin metabolite N1-acetyl-5-methoxykynuramine (AMK) with the tyrosine side-chain fragment, 4-ethylphenol

The melatonin metabolite N(1)-acetyl-5-methoxykynuramine (AMK) has previously been shown to interact with various free radicals. Using the ABTS cation radical [ABTS = 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid)] as an electron abstracting reactant, which does not destroy the aromate, we found that the reactive intermediate derived from AMK strongly interacts with the benzene rings of other AMK molecules to form di- and oligomers. Since oligomerization is rather unlikely at physiological concentrations, we investigated reactions with other putative reaction partners. The incubation of tyrosine or several of its structural analogs with AMK in the presence of the ABTS cation radical led to numerous products, amongst which were compounds not detected when one of the educts was incubated with the ABTS cation radical alone. With tyrosine and most of its analogs, the number of products formed in the presence of AMK and ABTS cation radical was relatively high and included numerous oligomers. To optimize the yield of products of interest as well as their separation from other compounds, especially oligomers, we investigated the interaction with 4-ethylphenol, which represents the side chain of tyrosine lacking the carboxyl and amino residues of the amino acid, which otherwise can undergo additional reactions. A prominent product was chromatographically separated and analyzed by mass spectrometry [(+)-ESI-MS, (-)-ESI-MS, (+)-HRESI-MS], (1)H-NMR, and H,H-COSY correlations. The substance was identified as N-{3-[2'-(5'-ethyl-2'-hydroxyphenylamino)-5'-methoxyphenyl]-3-oxopropyl} acetamide. This chemically novel compound represents an adduct in which the amino nitrogen of AMK is attached to the C-2 atom of 4-ethylphenol, which corresponds to the C-3 atom in the benzene ring of tyrosine. This finding suggests that, upon interaction of AMK with an electron-abstracting radical, the kynuric intermediate may modify proteins at superficially accessible tyrosine residues. In fact, protein modification by an unidentified melatonin metabolite has been observed in an earlier study. The possibility of protein AMKylation may be of interest with regard to an eventual interference with tyrosine nitration or, more importantly, with tyrosine phosphorylation.     

The determination of total concentration and activity of antioxidants in foodstuffs

We developed a new method for the analysis of active antioxidants that is based on their reactions with the ABTS+. cation radical obtained by oxidation of ABTS, 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt. The feasibility of this method was confirmed by electrochemical and kinetic studies of model antioxidants. ABTS+. was shown to react rapidly with active and slowly with weak antioxidants, which allows it to be used as a model radical for the quantitative determination of the total content of natural antioxidants (antioxidant equivalent) in natural extracts and wines. Another analytical method based on the competitive oxidation of Pyrogallol Red (a detecting molecule) and the examined antioxidants by radicals derived from peroxynitrite was used for measuring the relative activity of antioxidants. A combination of both methods helped measure the total concentration of antioxidants and their average specific activities (per molecule of active compound) in extracts from grape, olive, and tomato and concentrates of various popular beverages (wines, beers, and juices), as well as in the commercial concentrated food product Kréto-A, made from grape, red wine, tomato, and olive. Red wine and red grape juice were shown to be the most rich in antioxidants (up to 20 mM), with their activity being similar to that of polyphenols. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2002, vol. 28, no. 6; see also http://www.maik.ru.     

In vitro antioxidant studies of Sphaeranthus indicus (Linn)

The free radical scavenging potential of the plant S. indicus was studied by using different antioxidant models of screening. The ethanolic extract at 1000 microg/ml showed maximum scavenging of the radical cation, 2,2-azinobis-(3-ethylbenzothiazoline-6-sulphonate) (ABTS) observed upto 41.99% followed by the scavenging of the stable radical 1,1-diphenyl, 2-picryl hydrazyl (DPPH) (33.27%), superoxide dismutase (25.14%) and nitric oxide radical (22.36%) at the same concentration. However, the extract showed only moderate scavenging activity of iron chelation (14.2%). Total antioxidant capacity of the extract was found to be 160.85 nmol/g ascorbic acid. The results justify the therapeutic applications of the plant in the indigenous system of medicine, augmenting its therapeutic value.     

Oxidative degradation of cardiotoxic anticancer anthracyclines to phthalic acids. Novel function or ferrylmyoglobin

We show that the pseudoperoxidase activity of ferrylmyoglobin (MbIV) promotes oxidative degradation of doxorubicin (DOX), an anticancer anthracycline known to induce severe cardiotoxicity. MbIV, formed in vitro by reacting horse heart MbIII with H2O2, caused disappearance of the spectrum of DOX at 477 nm and appearance of UV-absorbing chromophores that indicated opening and degradation of its tetracyclic ring. Electron spray ionization mass spectrometry analyses of DOX/MbIV ultrafiltrates showed that DOX degradation resulted in formation of 3-methoxyphthalic acid, the product of oxidative modifications of its methoxy-substituted ring D. Other methoxy-substituted anthracyclines similarly released 3-methoxyphthalic acid after oxidation by MbIV, whereas demethoxy analogs released simple phthalic acid. Kinetic and stoichiometric analyses of reactions between DOX and MbIII/H2O2 or hemin/H2O2 showed that the porphyrin radical of MbIV-compound I and the iron-oxo moiety of MbIV-compound II were sequentially involved in oxidizing DOX; however, oxidation by compound I formed more 3-methoxyphthalic acid than oxidation by compound II. Sizeable amounts of 3-methoxyphthalic acid were formed in the heart of mice treated with DOX, in human myocardial biopsies exposed to DOX in vitro, and in human cardiac cytosol that oxidized DOX after activation of its endogenous myoglobin by H2O2. Importantly, H9c2 cardiomyocytes were damaged by low concentrations of DOX but could tolerate concentrations of 3-methoxyphthalic acid higher than those measured in murine or human myocardium. These results unravel a novel function for MbIV in the oxidative degradation of anthracyclines to phthalic acids and suggest that this may serve a salvage pathway against cardiotoxicity.