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UDC 615.27-092.4
COMPARATIVE ASSESSMENT OF HISTOCHROME'S ANTIOXIDANT AND PROOXIDANT ACTIVITIES IN TERMS OF IN VITRO AND IN VIVO EXPERIMENTS
Altai State Medical University, Barnaul O.S. Talalayeva, Ya.F. Zverev, S.V. Zamyatina
The current study presents the results of comparison of antioxidant and pro-oxidant histochrome activity in terms
of in vitro and in vivo experiments. In vitro effect of two concentrations of the drug approximately corresponding
to the doses administered by animals was studied. In vivo experiments were performed on Wistar rats, randomized
into three groups: the 1st and 2nd groups received histochrome at a dose of 1 mg/kg (n = 15) and 10 mg/kg (n =
15), the 3rd one was the control group. After 14 days of drug injection, oxidative stress was induced and the total
antioxidant activity (TAA) and total pro-oxidant activity (TPA) in the rat blood were evaluated.
In terms of study it was revealed, that histochrome can have multidirectional effect on the processes of free-
radical oxidation in model systems in vitro and in vivo. The prevalence of antioxidant activity was registered
by the injection of the drug at a dose of 1 mg/mk.
Key words: histochrome, antioxidant and pro-oxidant properties.
Histochrome is a water-soluble dosage form of an individual substance, a natural quinoid pigment of marine invertebrates of echinochrome A (2,3,5,6,8-pentahydroxy-7-ethyl-1,4-naphthoqui-none) [1]. The basis for the development of drugs of the "Histochrome" series (state registration number 002363/01-2003 and 002363/02-2003) was the high pluripotent antioxidant activity of echino-chrome A [2,3,4].
The first data on the expressed reactivity of 2,3,5,6,8-pentahydroxy-7-ethyl-1,4-naphthoqui-none were obtained by the workers of the G.B. Elyakov Pacific Institute of Bioorganic Chemistry in in vitro experiments. In various model systems, it has been shown that the donor properties of echinochrome A allow it to restore lipid radicals, ensuring the structural and functional integrity of cytoplasmic membranes [5,6]. The ability of 2,3,5,6,8-pentahydroxy-7-ethyl-1,4-naphthoqui-none to neutralize superoxide aniline has been proved by competitive inhibition of the reduction of nitro blue tetrazolium [6]. The opinion was also expressed that the antioxidant potential of 2,3,5,6,8-pentahydroxy-7-ethyl-1,4-naphthoqui-none is largely due to its ability to chelate iron cations, which participate in the branching of chain radical reactions [5,7,8,9,10].
Thus, in numerous model systems and chemical tests, a high polytarget antiradical activity of echi-nochrome A was established.
At the same time, it is known that the antiox-idant activity of the substance detected in vitro should not be fully extrapolated to in vivo conditions, since under the conditions of a living organism, one and the same substances can simultaneously manifest both antioxidant and prooxidant activity [11,12]. In principle, the ability of histo-chrome to have a multidirectional effect in terms
of in vitro and in vivo experiments was previously shown by us in the study of the membrane stabilizing effect of the drug [13].
Research objective
Thus, the objective of the current study is a comparative evaluation of the antioxidant and proo-xidant activities of histochrome drug in in vitro and in vivo experiments.
Materials and methods
In the experiment, there was used the "Histochrome® solution for intravenous injection of 1% in 5 ml ampoules" (registration number P N002363 / 01-2003 dated 07.23.2008) (PIBOC FEB RAS, Vladivostok) [1].
Experiments in vivo were performed in out-bred Wistar rats of both genders aged 2-3 months and weighing 200-300 g grown in the breeding nursery of the Research Institute of Cytology and Genetics of the SB RAS (Novosibirsk). The animals were kept in standard conditions in a vivarium on a full balanced diet in accordance with the recommendations of the Institute of Nutrition, RAMS, in a well-ventilated room with a temperature of + 20 ± 2 °C and a moisture content of not more than 80%. The experiments were performed in the spring period from 9.00 to 15.00. Randomization of animals into three groups was performed by a block method. The first two groups of rats daily received histochrome at a dose of 1 mg/kg (experimental group 1, n = 15) and 10 mg/kg (experimental group 2, n = 15) for 14 days subcutaneously. The third group of animals was the control one and received equivolume subcutaneous injections of isotonic sodium chloride solution (n = 15).
At the end of the course, exudative inflammation was modeled. For this, 0.2 ml of a 3% forma-
lin solution was injected into the plantar aponeurosis of both posterior limbs of rats. Earlier in our laboratory, it was shown that subplantal injection of formalin is accompanied by the development of oxidative stress with a peak in two days after the injection of phlogistic [14]. On the third day of exudative inflammation, rats were decapitated under ether anesthesia and the indices of activity of free radical oxidation processes were determined in the blood. The results obtained were compared with the data specific for intact animals. The total prooxidant activity (TPA), the integra-tive index of the concentration of all prooxidants and the activity of the processes of lipid peroxi-dation were evaluated by the ability of the blood plasma to induce the oxidation of TWEEN-80 with further photocolorimetric determination of the content of products reacting with thiobarbituric acid (TBRP, the results are presented as a percentage of the value of the control sample) [15].
Antioxidant activity in erythrocyte hemolysate was assessed by the change in the integrative index of total antioxidant activity (TAA, determined by inhibition of the Fe2+/ascorbate induced accumulation of TBRP during the oxidation of TWEEN-80, the results are expressed as a percentage of the value of the control sample) [16,17,18]. The described system simulates the peroxidation of plasma lipids [19].
All in vivo experiments were carried out in accordance with the requirements of the European Convention for the Protection of Vertebrates Used for Experimental or Other Scientific Purposes (Strasbourg, 1986) and the Federal Law of the Russian Federation "On the Protection of Animals against Cruel Treatment" of 01.01.1997.
In two series of in vitro experiments, antioxidant and prooxidant activity of histochrome was evaluated [15,16,18]. To determine the total prooxidant (TPA) and total antioxidant activity (TAA), instead of the biological material, a corresponding volume of a solution of histochrome in the studied concentration was introduced into the test tubes (Table 1).
The peculiarity of the study was the use of patented model systems (ASMU, the Department of Biochemistry and Clinical Laboratory Diagnostics) maximally close to the conditions of the whole organism. Both in in vitro and in vivo experiments, evaluation of the an-tioxidant and prooxidant activity of the histo-chrome drug was performed using a single technique, which allowed to maximize the objectivity of the results of the comparative analysis. Analyzed drug concentrations were equivalent to histochrome doses used in two groups of rats. The methodologies were validated.
Table 1
Design of in vitro experiments
Test type Test number, n Initial concentrations of studied histochrome solutions
Prooxidant activity of histochrome
Control tests 20 Distilled water, 0,2 ml
Experimental tests 20 0,1 mg/ml, 0,2 ml 0,2 mg/ml, 0,2 ml
Antioxidant activities of histochrome
Control tests 20 Distilled water, 0,1 ml
Experimental tests 20 0,1 mg/ml, 0,1 ml 0,2 mg/ml, 0,1 ml
The results were processed by means of "Sta-tistica for Windows 6.0" software package. The results are presented in the form of M ± m, where M is the sample mean, m is the error of the mean, n is the sample. The dynamics of the indices in the dependent samples of pair measurements was carried out with the help of the Wilcoxon test. For the intergroup estimation of nonparametric indices of independent samples, the Mann-Whitney test was used. The level of statistical significance corresponded to p <0.05.
Results
The study of the antioxidant activity of histo-chrome in in vivo experiments was carried out
on the model of pathology accompanied by oxi-dative stress caused by the plantar injection of formalin followed by the formation of inflammatory edema of posterior limbs of the rats [14,20]. Preliminary, to determine the optimal time for the evaluation of antioxidant activity, the dynamics of oxi-dative stress development against the background of formalin edema was studied (Table 2).
Considering the dynamics of oxidative stress caused by the inflammatory process, the determination of antioxidant and prooxidant activity in in vivo experiments was performed at the peak of formalin edema reaching its maximum on the third day after the injection of the phlogogen.
Table 2
Effect of prolonged injection of various doses of histochrome on the activity of free radical oxidation processes in rat blood
Index Intact rats Control rats Histochrome
1 mg/kg 10 mg/kg
Total prooxidant activity, % 45,1 ± 1,06 60,1 ± 1,25* 23,7 ± 1,24*,# 61,1 ± 1,16*
Total antioxidant activity, % 73,7 ± 0,51 87,8 ± 0,86* 38,9 ± 2,01*,# 17,4 ± 0,70*,#
Note: * - statistically significant difference from the corresponding index in intact rats, # - significant difference from the corresponding index in control rats.
As in the case of in vitro experiments, the increase in free radical derivatives in the blood plasma of animals was characterized by a linear dependence on the amount of the active principle of histochrome (Tables 2, 3). It should be noted that in the group of animals receiving histo-chrome at a dose of 1 mg/kg, the TPA value was 2.5 times lower than the values characterizing ox-idative stress in animals with experimental edema, and half as much as in intact animals (Table 2). A tenfold increase in the dose of the drug neutralized the registered effect, and the concentration of free radical derivatives in the plasma of the experimental rats corresponded to that in the control group of animals under conditions of maximum activity of oxidative stress.
As shown in Table 2, an inverse relationship was recorded for antioxidant activity and dose of the drug. In conditions of prolonged injection of histochrome in rats with experimental inflammation, a dose-dependent but multidirectional drug effect was detected. Thus, the injection of 1 mg/kg histochrome in animals with experimental inflammation led to suppression of the processes of lipid peroxidation and a tenfold increase in the dose to activation.
The data of the in vitro experiments presented in Table 3 show a concentration-dependent increase in both prooxidant and antioxidant activities. At the same time, the increase in TPA prevailed at all concentrations used.
Table 3
Indexes of prooxidant and antioxidant activities of various concentrations of histochrome in in vitro experiments
Index Studied histochrome concentrations
0,1 mg/ml, n = 20 0,2 mg/ml, n = 20
TPA (%) 15,5 ±1,45 20,3 ±1,78, p = 0,053
TAA (%) 9,4 ± 1,20, p* = 0,003 12,2 ±1,24, p* = 0,000
Note: p - statistically significant differences in effects between the tween the corresponding concentrations of TAA and TPA.
Discussion
The results of the study detected differences in the nature of the changes in processes of free radical oxidation in in vivo and in vitro experiments.
The analysis of the prooxidant activity of histo-chrome in in vivo experiments showed that when the dose equivalent to the therapeutic dose of a human was injected into the animals for a prolonged period, the drug, by lowering the level of free-radical derivatives in the rat blood plasma, manifested itself as an antioxidant. At a dose of 10 mg/kg, according to the TPA data, histochrome did not possess such an effect. In parallel, the evaluation of an-tioxidant activity in in vivo experiments showed that the increase of the dose of the drug is accompanied by a decrease in its protective properties. At the same time, in in vitro experiments, the in-
concentrations studied, p* - statistically significant differences be-
crease in prooxidant activity prevailed in all analyzed concentrations of the drug.
The most logical explanation of the differences in the results obtained in in vivo and in vitro experiments is the involvement of certain additional mechanisms in the action of histochrome, induced in a holistic organism. Modern studies have shown fundamental differences in the activity of phenolic antioxidants in chemical tests and in the cellular system resulting from the involvement of endogenous mechanisms [12,21]. It is known, for example, that many polyphenolic compounds are capable of modifying the activity of endogenous antiox-idant systems [20, 21, 22], secondary intracellular messengers and possess genomic effects [21, 23]. Specifically, the presence of genomic effects was established in echinochrome A [24, 25, 26]. Among
the latter, special attention is paid to the ability of this naphthoquinone to modulate the activity of the p53 gene [26].
At the same time, it should be noted that, in spite of the significant antioxidant capacity, under certain conditions, polyphenols can exhibit prooxidant properties [12, 27]. It is natural that with the increase of the amount of active substance, the prooxidant activity of the drug increases. The obtained results to a certain extent explain the data of the quantum-chemical analysis of the structural basis of histochrome-2,3,5,6,8-pen-tahydroxy-7-ethyl-1,4-naphthoquinone. It can be assumed that prolonged injection of high doses in animals with experimental inflammation was accompanied by excessive production of the reactive radical naphthoquinone and hydrogen peroxide, which in turn supported the activity of lipid peroxidation at a high level [27,28]. In this case, it becomes clear why the increase in the dose of histochrome to 10 mg/kg was accompanied by the activation of the free-radical oxidation processes. In turn, the antioxidant effect of histo-chrome manifested itself only in the absence of its prooxidant activity, which was recorded by the use of smaller doses of the drug.
The protective effect of histochrome in relation to the processes of free radical oxidation is most likely determined not by the minor mechanism, but by the ratio of the various antioxidant effects of the drug. Histochrome realizes antioxidant activity in doses equivalent to the therapeutic dose of a human, and a tenfold increase in the latter is accompanied by an increase in the prooxidant activity of the drug.
Conclusions
The results of the study showed a predominance of prooxidant activity of histochrome in in vitro experiments.
The antioxidant/prooxidant activity ratio of the drug in in vivo experiments was positive when a dose equivalent to the therapeutic dose of a human was injected. A ten-fold increase in the dose of histochrome transformed the ratio toward the activation of free radical oxidation processes.
The multidirectional action of Histochrome® drugs on biological processes in model systems in vitro and in vivo indicates the presence of endogenous mechanisms in echinochrome.
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Contacts
Corresponding author: Talalayeva Olga Sergeyev-na, Candidate of Medical Sciences, Associate Professor of the Department of pathological physiology of ASMU, Barnaul. 656038, Barnaul, Lenina Prospekt, 40, Tel: 8 (3852) 24-18-56. E-mail: talalaeva_olga@mail.ru
