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ACID PROPERTIES OF ECHINOCHROM-A AND ITS STRUCTURAL ANALOGUES, INTERACTION WITH SUPEROXIDE ANION-RADICAL
A.V. Lebedev, M.V. Ivanov, N.I. Krasnovid, E.A. Koltsov
Russian cardiological research-and-production complex, Ministry of Health of the Russian Federation, Moscow 121552, 3-rd Cherepkovskaya street, 15А
Pacific institute of organic chemistry of the Russian Academy of Science, Vladivostok, 100 years of Vladivostok’ avenue, 159
Acid properties polyhydroksy-1,4-naphthoqiunones (PHONQ), having cardio protective properties, spontaneous oxidation PHONQ and interaction with superoxide O2 have been investigated by methods of a polarography, potentiometric titration, ultraviolent and visible spectroscopy. Properties of the 6-ethyl -2,3,7-trihydroxynaphthazarin (echinochrom А), 3-acetyl -2,6,7- trihydroxynaphthazarin (spinochrom С), 2,3,6-trihydroxynaphthazarin (spinochrom D), 2,3,6,7-tetrahydroxynaphthazarin (spinochrom Е), 2,3-dihydroxy-6,7-dimetilnaphthazarin (А618) and 6-ethyl-2,3,7-trimetoxynaphthazarin (trimetoxyechinochrom А) have been investigated also.
Dissociation constants(O2-) of PHONQ in 40 % ethanol are determined. Enzymatic generation (O2-) (xanthine–xanthine oxidase, рН 8,5) resulted to spectrums’ change of echinochrom A, spinochroms D, Е, and А618. Transformation of these spectrums of PHONQ during reaction with O2 was characterized by expressed isosbestic point that specifies primary formation of one reaction’s product.
It is assumed that reaction product is 1,2,3,4-tetraketon, which is formed from 2,3,5,8-tetrahydroxy -1,4- naphthoqiunones (echinochrom A, spinochrom D, Е, and А618) as a result of oxidation hydroxygroups in 2-nd and 3-rd positions. The method of concurrent reactions with NBT (Nitro Blue Tetrazolium) determined specific reaction rates of PHONQ with O2. Constants have order 104 -105 M-1 c-1, decreasing in series: echinochrom А > spinochrom D > spinochrom С > NBT > trimetoxyechinochrom A. It is concluded that polyhydroksy-1,4-naphthoqiunones (PHONQ) which contain hydroxygroups in 2-nd and 3-rd positions can carry out a role of efficient interceptors of a superoxide anion-radical.
Key words: polyhydroksy-1,4-naphthoqiunones, echinochrom A, spinochroms with D and Е acid-base properties, dissociation constants, superoxide, specific reaction rates, spontaneous oxidation, a cardio protective drug.
Exuberant generation of active forms of Oxygen possesses the important role in damage of a myocardium at an ischemia-reperfusion with activation of free-radical processes [1-3]. Thereby the opportunities of wider application of antioxidants’ drugs in a cardiology are researched.
Expressed antioxidants’ properties of the polyhydroksy-1,4-naphthoqiunones (PHONQ) have formed a basis for development of a new medicinal agent “histochrom” (echinochrom А) . Cardio protective action of histochrom and its affined polyhydroksy-1,4-naphthoqiunones with absence of the inheritance of the intrinsic toxic properties [7, 8] was shown in experiments on models of an ischemia-reperfusion of a myocardium [4-6]. Successful clinical tests of histochrom were carried out at an acute infarct myocardium  and stenocardia . Physiological activity of PHONQ makes actual a problem of carrying out of researches of their physical and chemical properties, mechanisms of biochemical and pharmacological action.
Antioxidants’ properties of the polyhydroksy-1,4-naphthoqiunones (PHONQ) were investigated on models of the initiated oxidation of benzole-alkyls, thermal spontaneous oxidation of metillinoleat, oxidations of mineral and vegetable oils . Antiradical activity of echinochrom characterized by a constant k7 (a constant of interaction with peroxide radicals), is comparable with k7 for ionol — 3,5*104 and 2,2* 104 M-1 c-1 accordingly .
Most brightly antioxidant’s action of PHONQ is shown at inhibition of ascorbic iron within oxidations of liposomes from egg’s phosphatidylcholine, where antioxidants’ action of PHONQ surpasses significantly the action of phenolic antioxidants such as ionol and an alpha-tocopherol . In this model the ability of PHONQ to bind potent initiators of peroxide oxidation of lipids, cations Ferri lactas Fe +, in inactive complexes plays a main role in inhibition of radical reactions.
Antioxidants’ activity is provided with beta-hydroxyl substituents — in 2, 3 and 7 positions of moleculas of PHONQ. Changing of these hydroxyls in echinochrom A on metoxygroups reduces antioxidants’ activity of PHONQ to zero. In the given work subacid properties of echinochrom A, trimetoxyechinochrom, spinochroms D and Е, their spontaneous oxidation and interaction with superoxide an anion-radical are investigated.
Materials and methods. Reagents.
Hepes, Tris, EDTA, Nitro Blue Tetrazolium (NBT), a xanthine, xanthine oxidase, a superoxide scavenger (Sigma, the USA) were used in this work. A series of the polyhydroksy-1,4-naphthoqiunones (PHONQ) was kindly given by E.A. Koltsova, N.K. Utkina and V.F. Anufriev (Pacific institute of organic chemistry of the Russian Academy of Science, Vladivostok). The 6-ethyl -2,3,7-trihydroxynaphthazarin (echinochrom А), 2,3,6,7-tetrahydroxynaphthazarin (spinochrom Е), 3-acetyl-2,6,7-trihydroxynaphthazarin (spinochrom С), 2,3,6-trihydroxynaphthazarin (spinochrom D) were discharged from testas of sea hedgehogs Strongylocentrotus intermedius and Strongylocentrotus nudus in Pacific institute of organic chemistry ; 2,3-dihydroxy-6,7-dimetilnaphthazarin (А618) and 6-ethyl-2,3,7-trimetoxynaphthazarin (trimetoxyechinochrom А) were synthesized as it was described earlier .
Generation of a superoxidic anion-radical
The xanthine-xanthine oxidase system  was used for generation of O2. Experiments carried out at ambient temperature in a buffer solution keeping 50 mM of Hepes-Tris, 100 microM of EDTA, 100 microM of a xanthine at рН 8,5. Reaction started with addition of xanthine oxidase — 5-40 mcg / ml.
Spectrophotometric registration of hydroxynaphthazarins
Measuring of parameters was carried out on spectrophotometer Shimadzu UV-260 (Japan) in standard quartz pans with depth of 1 cm. A hydroxynaphthazarin’s spectrum (25 microns) was written down in long-wavelength range of absorption — 300-600 nanometers. Reaction of formation of O2 was started with adding xanthine oxidase (40 mcg / ml) timing since this moment. Changes of a spectrum were recorded through identical time terms — 4 minutes.
Calculation of a kinetic constant
Kinetic constants of reactions of PHONQ with O2 were determined by a method of concurrent reactions with Nitro Blue Tetrazolium (NBT) . Concentration of NBT was 75-90 microM and concentration of naphthazarin was 1-25 microM. Regeneration of NBT was recorded based on absorption lambda 560 nanometer, epsilon NBT = 2,78*104M-1c-1 . Rate of generating O2 was measured based on regeneration of cytochrome C with selecting in such way that O2 was significantly less then [NBT] and concentration of naphthazarin. In these conditions spontaneous dismutationO2 can be neglected .
The interrelation of recovery rates NBT in absence (Vnbt) and at presence (V*nbt) of naphthazarin is equal:
The method of competitive inhibition of regeneration NBT could not be applied for definition of a specific reaction rate spinochrom Е with O2 since during spontaneous oxidation spinochrom Е there was a fast decrease of concentration spinochrom and formation additional O2 and regeneration of NBT has resulted from that.
Measuring of an oxygen absorption
Oxygen absorption was measured by a polarographic method using Clark-type electrode (YSI Model 5331) and Oxygen monitor -5300 Yellow Spring instrument Co., Inc. (USA).
Potentiometric titration of PHONQ
The ranges of buffer capacity describing of рH-parameter for PHONQ were determined based on acid-base titration 0,2 — 0,5 mM of PHONQ’ solutions in 40 % ethanol in a range рН from 4 up to 10. Spontaneous oxidation of echinochrom A, spinochroms Е and D at рН > 9, 5 limited range of titration of these PHONQ. The glass electrode and a calomel electrode of comparison were used (рН-meter Radelkis, Hungary).
Calculation рH was carried out by approximating experimental dependence of buffer capacity
from рН theoretical curve
Results and discussion
Acid properties of polyhydroxylated derivants of naphthazarin. The results of acid-base titration of PHONQ in aqueous-alcoholic solutions are submitted in Table 1. The greatest values рH characterize hydroxyls in 5-th and 8-th positions of naphthol rings of trimetoxyechinochrom. It is necessary to take into account that values рH obtained in 40 % aqueous-alcoholic solutions are approximately 0, 5 units рН higher than values of рH in water solutions. Hence, in water solutions the dissociation of OH — groups in 2 and 3 positions of echinochrom A, spinochroms D and Е descends at рН from 5 up to 7,5.
Thus, polyhydroksy-1,4-naphthoqiunones at physiological values рН are one or divalent anions that can influence essentially on their reactions with ions and radicals.
Spontaneous oxidation of polyhydroxylated derivants of naphthazarin. In protonic form the investigated PHONQ are steady in alcohol and acidic water solutions. Rate of spontaneous oxidation PHONQ increased with augmentation рН and depended on the chemical nature of 2,3,6,7-substitutes PHONQ and cleanliness of solutions. Additive EDTA retarded or completely intercepted spontaneous oxidation. It specifies on essential influence of extrinsic cations of heavy metals on fastness of PHONQ in the anionic form.
Spinochrom Е, keeping hydroxyls in 2, 3, 6 and 7-th positions, is least steady in neutral and alkaline water solutions. 25 microM of spinochrom Е in Hepes-Tris buffer are almost completely oxidized during 20 minutes at рН 8,5 at presence EDTA. Echinochrom A and spinochroms D and C in similar conditions (Hepes-Tris buffer, 100 microM of EDTA) are steady, and that has allowed to use them in experiences at research of interaction PHONQ with a superoxidic radical at рН 8,5. In alkaline conditions at рН> 9,5 echinochrom A, spinochroms D and C are unstable. At spontaneous oxidation an oxygen absorption was observed together with change of spectrums, formation of O2-, which was recorded on SOD (superoxide dismutase) -inhibited regeneration NBT it (is not shown). The superoxide dismutase retarded spontaneous oxidation of PHONQ.
Table 1. Acid properties polyhydroksy-1,4-naphthoqiunones (PHONQ)
Apparently spontaneous oxidation of the investigated hydroxynaphthazarin is similar to spontaneous oxidation of 1,4-naphthohydroqiunones and it is accompanied by formation of seven-quinones and a superoxidic anion — radical [7, 8].
Interaction of polyhydroxylated derivants of naphthazarin with O2. In concentrations up to 40 microM the tested materials did not change saturating rate of Oxygen in a xanthine-xanthine oxidase reaction and, therefore, did not influence activity of an enzyme.
The changes of spectrums of echinochrom A and spinochrom D in superoxide-generating system are given on Figure 1. In additional experiments it was shown, that hydrogen dioxide did not influence on the changes of spectrums of investigated materials. Hence, observable change of spectrums on Figure 1 is not connected to the formation of H2O2 in a xanthine-xanthine oxidase reaction. Spectrums of spinochrom C after adding of xanthine oxidase was varied weakly (is not shown). In the same conditions spectrums of trimetoxyechinochrom were not varied (is not shown).
Changes of spectrums of echinochrom A and spinochrom D in superoxide-generating system. 1 — initial spectrums of echinochrom A and spinochrom D. 2, 3, etc. are obtained after addition of xanthine oxidase (40 mcg / ml) at regular intervals (4 minutes).
In each series of spectrums on Figure 1 the points 427 and 350 nanometers accordingly are visible izobestical. Similar changes of spectrums were observed for interaction of spinochrom Е and synthetic polyhydroksy-1,4-naphthoqiunone А618 with O2, (izobestical points of 435 and 417 nanometers accordingly). Presence of izobestical points at transformation of spectrums during reaction specifies formation of unique reaction product. Izobestical point in spectrums of spinochrom C was not possible to register in range of lengths of waves in 300 nanometers and more; this range is accessible to gauging at the presence of a xanthine. The prospective two-stage schema of reactions of superoxide with echinochrom is shown below (Figure 2). The intermediate product is seven-quinone of echinochrom, and a stable end-product is tetraketon.
There is regeneration of NBT in superoxide-generating system at various concentrations of echinochrom A. Concentrations of NBT is 80 microM.NBT (rec) is the concentration of the restored NBT. Timing is started from the moment of the addition of xanthine oxidase (5 mcg / ml). Concentrations of echinochrom A are specified in the Figure 2.
The average value was calculated based on results of 3-4 measurements.
Similar reactions with a superoxidic anion — radical can be assumed for spinochroms D andЕ which also contain hydroxyls in 2-nd and 3-rd positions. For spinochrom C and trimetoxyechinochrom in reaction with O2 it is possible to assume participation of the OH – groups of a benzole ring.
Specific reaction rates of interaction PHONQ with O2 were determined by a method of competitive inhibition of regeneration NBT (Figure 2) and it has order (Table 2). Echinochrom A and spinochrom D have the greatest values among the tested materials. The constant for 2,3,7-trimetoxyechinochrom is lower by one order, for spinochrom C has mediate value.
It is possible to draw a conclusion, that hydroxyl groups in 2-nd and 3-rd positions of qiunone rings (echinochrom A and spinochrom D) are more reactive at interaction with O2. Polyhydroxy-1,4- naphthoqiunones, keeping hydroxyls only in a benzole ring, interreact with O2 apparently more slowly (spinochrom C and trimetoxyechinochrom А).
Thus, the obtained results show that the investigated natural polyhydroxy-1,4-naphthoqiunones interact with a superoxidic anion — radical. Echinochrom A is most effective among them.
Physiological efficacy of echinochrom A [4-6, 9, 10] can be defined by its antioxidants’ properties. Thus breakage of radical reactions of natural PHONQ is determined, as shown earlier, by chelation of cations Ferri lactas, intercepting of radicals phospholipids moleculas  and, as shown in this work, intercepting of a superoxidic anion — radical.
We are grateful to O.B. Maksimov and his colleagues in Pacific institute of organic chemistry of the Russian Academy of Science (Vladivostok) for the given materials and fruitful discussions.
Work was supported by the grant of the Russian Federal Property Fund 98-04-48661
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