Formation of free radicals in mitochondria plays a key role in the development of apoptosis, which includes formation of superoxide by the respiratory chain, formation of radicals by cytochrome c-cardiolipin complex in the presence of hydrogen peroxide or lipids, and chain lipid peroxidation resulting in cytochrome c release from mitochondria and initiation of the apoptotic cascade. In this work the effect of taxifolin (dihydroquercetin) and some other antioxidants on these three radical-producing reactions was studied. Peroxidase activity of the complex of cytochrome c with dioleyl cardiolipin estimated by chemiluminescence with luminol decreased by 50% with quercetin, taxifolin, rutin, Trolox, and ionol at concentrations 0.7, 0.7, 0.8, 3, and 10 microM, respectively. The lipid radical production detected by coumarin C-525-activated chemiluminescence decreased under the action of rutin and taxifolin in a dose-dependent manner, so that a 50% inhibition of chemiluminescence was observed at the antioxidant concentrations of 3.7 and 10 microM, respectively. Thus, these two radical-producing reactions responsible for apoptosis onset are inhibited by antioxidants at rather low concentrations. Experiments performed on liver slices and mash showed that taxifolin, quercetin, naringenin, and Trolox have low inhibitory effect on the lucigenin-dependent chemiluminescence in the tissue only at concentrations higher than 100 microM.

Free radical reactions play an important role in biological functions of living systems. The balance between oxidants and antioxidants is necessary for the normal homeostasis of cells and organisms. Experimental works demonstrate the role of oxidative stress that is caused by influenza virus as well as the toxic effects of some antiviral drugs. Therefore, antiviral drugs should be characterized by its pro-and antioxidant activity, because it can affect its therapeutic efficiency. The aim of the study was to quantify the antioxidant capacity and propose the mechanism of the antioxidant effect of the antiviral drug Umifenovir (Arbidol ® ). The kinetic chemiluminescence with the 2,2'-azobis (2-amidinopropane) dihydrochloride + luminol system was used to quantify the antioxidant capacity of Umifenovir relative to the standard compound Trolox. With computer simulation, the reaction scheme and rate constants were proposed. The antioxidant capacity of 0.9 µM Umifenovir (maximum concentration of Umifenovir in blood after oral administration of 200 mg) was as high as 1.65 ± 0.18 µM of Trolox. Thus, the total antioxidant capacity of Umifenovir is comparable to the antioxidant capacity of Trolox. Unlike Trolox, Umifenovir reacts with free radicals in two stages. For Trolox, the free radical scavenging rate constant was k = 2000 nM −1 min. −1 , for Umifenovir k 1 = 300 nM −1 min. −1 , k 2 = 4 nM −1 min. −1 . Slower kinetics of Umifenovir provides the prolonged antioxidant effect when compared to Trolox. This phenomenon can make a serious contribution to the compensation of oxidative stress that is caused by a viral disease and the therapeutic effect of the drug.

In this work, the effect of liposomes consisting of tetraoleyl cardiolipin and dioleyl phosphatidylcholine (1 : 1, mol/mol) on the rate of three more reactions of Cyt c heme with H2O2 was studied: (i) Cyt c (Fe2+) oxidation to Cyt c (Fe3+), (ii) Fe...S(Met80) bond breaking, and (iii) heme porphyrin ring decomposition. It was revealed that the rates of all those reactions increased greatly in the presence of liposomes containing cardiolipin and not of those consisting of only phosphatidylcholine, and approximately to the same extent as peroxidase activity. These data suggest that cardiolipin activates specifically Cyt c peroxidase activity not only because it promotes Fe...S(Met80) bond breaking but also facilitates H2O2 penetration to the reaction center.

In this work, the actions of bovine heart cardiolipin, synthetic tetraoleyl cardiolipin, and a nonspecific anionic detergent sodium dodecyl sulfate (SDS) on cytochrome c (Cyt c) peroxidase activity recorded by chemiluminescence in the presence of luminol and on the Fe...S(Met80) bond whose presence was estimated by a weak absorption band amplitude with peak at 695-700 nm (A(695)) were compared. A strict concurrency between Fe...S(Met80) breaking (A(695)) and cytochrome peroxidase activity enhancement was shown to exist at cardiolipin/Cyt c and SDS/Cyt c molar ratios of 0 : 1 to 50 : 1 (by chemiluminescence). Nevertheless, when A(695) completely disappeared, Cyt c peroxidase activity under the action of cardiolipin was 20 times more than that under the action of SDS, and at low ligand/protein molar ratios (=4), SDS failed to activate peroxidase activity while cardiolipin enhanced Cyt c peroxidase activity 16-20-fold. A(695) did not change on Cyt c binding with liposomes consisting of tetraoleyl cardiolipin and phosphatidylcholine (1 : 10 : 10), while peroxidase activity was enhanced by a factor of 8. Breaking of 70% of the Fe...S(Met80) bonds resulted in only threefold enhancement of peroxidase activity. Cardiolipin-activated Cyt c peroxidase activity was reduced by high ionic strength solution (1 M KCl). The aggregated data suggest that cardiolipin activating action is caused, first, by a nonspecific effect of Fe...S(Met80) breaking as the result of conformational changes in the protein globule caused by the protein surface electrostatic recharging by an anionic amphiphilic molecule, and second, by a specific acceleration of the peroxidation reaction which is most likely due to enhanced heme accessibility for H(2)O(2) as a result of the hydrophobic interaction between cardiolipin and cytochrome.

Chemiluminescence observed during LPO or reactions of nitric oxide and oxygen radicals and was named "ultraweak luminescence". In the presence of chemiluminescence activators (luminol, lucigenin, rhodamine G, or coumarine C-525) the appearance of radicals is associated with intensive fluorescence; the registration of this fluorescence is widely used in biomedical and clinical studies.

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The antioxidant potential (capacity and activity) of aqueous fullerene dispersions (AFD) of non-functionalized C60, C70, and Gd@C82 endofullerene (in micromolar concentration range) was estimated based on chemiluminescence measurements of the model of luminol and generation of organic radicals by 2,2′-azobis(2-amidinopropane) dihydrochloride (ABAP). The antioxidant capacity was estimated by the TRAP method, from the concentration of half-suppression, and from the suppression area in the initial period. All three approaches agree and show that the antioxidant capacity of AFDs increased in the order Gd@C82 < C70 < C60. Mathematical modeling of the long-term kinetics data was used for antioxidant activity estimation. The effect of C60 and C70 is found to be quenching of the excited product of luminol with ABAP-generated radical and not an actual antioxidant effect; quenching constants differ insignificantly. Apart from quenching with a similar constant, the AFD of Gd@C82 exhibits actual antioxidant action. The antioxidant activity in Gd@C82 is 300-fold higher than quenching constants.

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