In recent years, the number of patients with hepatic disease has been steadily increasing [1]. As a result of liver damage, free radical oxidation is activated, the concentration of peroxidation products and their metabolites that disrupt the processes of metabolism and energy in the body increases, processes leading to morphofunctional changes in hepatocytes development [2]. The free radical concept of liver damage reveals new possibilities for the use of medicines in the treatment of hepatitis in animals with antioxidant activity [3]. Currently, a number of substances with this activity have been proposed, but they are not enough and the search continues and the problem of using synthetic antioxidants as hepatoprotectors in animals remains poorly understood [3,4]. Promising in this direction is emoxypine succinate, which has a combined antioxidant and antihypoxic effects due to the presence of nitrogen-containing heterocyclic phenols, that are synthetic analogues of vitamin pyridoxine and succinic acid [5]. Emoxypine succinate is derived from 3-hydroxypyridine and succinic acid [6]. The presence of these two moieties allows to be a highly promising treatment approach in the management of metabolic diseases resulting from endothelial dysfunction. In addition to this, the membranotropic effects exerted by these molecules support the maintenance of the lipid bilayer viscosity [5]. Because hydrogen peroxide is produced as a byproduct of the mitochondrial oxidation of succinic acid and is known to play a role in triggering free-radical oxidation, the succinate ion in the structure of emoxypine succinate exhibits an indirect prooxidant action. [7]. Researchers have discovered several biological activities for emoxypine succinate realized on at least two neuronal and vascular levels. It has vegetotropic, neuroprotective, anticolvusant and anxiolytic effects. Emoxypine succinate also has antiatherosclerotic and cardioprotective properties, decreases cholesterol, enhances cerebral blood circulation and prevents thrombocyte aggregation [8]. The liver injury induced by carbon tetrachloride has been commonly used in animal’s models and characterized by inflammation, formation of trichloromethyl radicals and overproduction of Reactive Oxygen Species (ROS), which initiate lipid peroxidation and finally leading to hepatotoxicity [9]. The purpose of this work is evaluation the hepatoprotective and antioxidant effects of the emoxypine succinate on the liver of rats with hepatitis with toxic genesis.

The experiment was carried out at the animals’ house in college of pharmacy / University of Babylon, on mature males of white albino wistar rats (n = 40), with weights of 220-300 grams. The rats were kept in standard conditions with free access to standard balanced feed and drinking water. All procedures with animals were performed in accordance with the rules of The Animal Care and Protection Act 2001, which are used for experiments and other scientific purposes. 

In accordance with the set goal, the animals were divided into one intact group consists of 10 rats and two experimental groups with 15 rats for each one. Toxic liver damage in rats of the second and third groups was modeled by administration of 0.25 mL of single intravenous injection of carbon tetrachloride (CCl4) in the form of a 1 % solution in olive oil at a dose of 10mg /kg of animal weight [10]. Animals of the third group were injected intrapertonialy with solution of emoxypine succinate for 30 days at the dose of 25mg/kg of rat weight twice a day and placebo (isotonic sodium chloride solution) in appropriate volumes was administered to individuals of the second group during 30 days of the experiment.

During the whole experiment, the rats were clinically monitored. Before the start of the study, as well as on the 7th, the 14th, 21st and 30th days after exposure to the toxicant, the motor activity of rats was studied using the "open field" method and the number of deaths was noted. The "open field" method applied in the study of the motor component of the indicative reaction. The studies were carried out in a 60x70 cm chamber with a wall height of 35 cm. The floor of the chamber is marked with paint, which divides the field into 42 (6x7) equal squares. The rat was placed on a platform, after which the number of squares crossed by 4 paws was visually counted for 30 minutes [11].

The effect of emoxypine succinate on rats in carbontetrachloride induced hepatitis was assessed by the dynamics of the values of biochemical parameters of blood Aspartate Aminotransferase Enzyme (AST), Alanine Aminotransferase Enzyme (ALT) and Lactate Dehydrogenase Enzyme (LDH) on the automatic biochemical analyzer "Furuno CA-180" (Japan).

Chemiluminescence (CL) technique as described by Haklar and his colleagues [12], may be utilized as a direct non-invasive method for measuring reactive oxygen species. The intensity of iron-induced chemiluminescence of liver homogenates was recorded for 5 minutes on the hardware and software complex "Lum-100" (Russia) using the software "Power Graph version 3.3". For research CL of liver homogenate, its fragment weighing 0.2 g, was placed in a porcelain mortar and homogenized using a laboratory porcelain pestle at room temperature (25 ° C). 2.0 mL of salt buffer (pH 7.45) was added to the resulting tissue homogenate. The homogenate was centrifuged for 10 minutes at 3000 round/min on the Eva 200 centrifuge. The supernatant extempera was examined. To do this, liver homogenate was diluted 80 times (50 ml of homogenate was brought to 4 ml with a salt buffer). The intensity of CL was recorded in for 5 minutes and its glow was induced by adding 0.2 ml of 50 mM FeSO4 solution x 7 H2O.

Statistical processing of the data obtained was carried by using SPSS (statistical package for social sciences) software program version 25. Quantitative variables were expressed in the form of mean and standard deviation. The differences were regarded as significant at p <0.05.

Survival of Rats with Carbon Tetrachloride Induced Hepatitis

According to the results of the present study, it was found as in Table 1, that the modeling of toxic hepatitis in rats by a single intravenous injection of CCL4 leads to the fact that out of 15 experimental males of the second group on the first day, two animals died during the experiment and on the second day, other two were registered with a fatal outcome. Survival rate of individuals of the second experimental group was 73.3%.

During the first hours after intravenous administration of CCL4, depression and heavy breathing were observed in rats. After a few hours, the condition of the animals stabilized, activity increased on the second day, appetite and moderate thirst appeared. It is known that when ingested, CCL4, being absorbed, enters the portal vein system and the liver. Already at the first passage through it, it undergoes biotransformation [10]. On the background introduction of CCL4 and correction with an antioxidant emoxypine succinate, one rat died for 2 days, the survival rate of rats after exposure to the toxicant and correction with the emoxypine increased by 20%, compared with animals without correction and amounted to 93.3%.

Table 1: Distribution of Animals into Groups Depending on the Experimental Conditions and Their Survival Rates

Horizontal Activity of Rats in the "Open Field" Method

Throughout the experiment as explained in Table 2, the number of locomotions was reduced in animals of all experimental groups after exposure to the toxicant, which led to a decrease in the number of crossed squares (p≤0.05), compared with rats of the control group. In rats of the second experimental group, the number of crossed squares in the "open field" test increased by 394.3% on day 14 (p<0.05) and on day 30 it significantly increased by 35.7%. In animals treated with a 5% solution of emoxypine succinate, by the 14th day of the study, horizontal activity increased, compared with rats of the second experimental group, by 142 % and by the 30th day by 107% (p≤0.05).

Table 2: Horizontal Activity of Rats in the "Open Field" Method

* p<0.05 comparison with the data of the control group, ** p<0.05 comparison with the data of the 2nd experimental group

Dynamics of Biochemical Parameters in Rats with Carbon Tetrachloride Induced Hepatitis

From the data presented in Table 3, it can be seen that in rats under the action of a toxicant in the blood throughout the experiment increased the activity of AST, ALT and LDH (p≤0.05). By the 30th day of the experiment in individuals of the second experimental group, compared with the values of the intact group, the activity of enzymes increased as follows: AST by 1.95 times; ALT by 2.45 times and LDH by 2.7 times.

Table 3: Dynamics of Biochemical Parameters in Rats with Hepatitis of Toxic Genesis

* p<0.05 comparison with the data of the control group, ** p<0.05 comparison with the data of the 2nd experimental group

By the 14th day of the experiment, hyperproteinemia was recorded in the blood (p<0.05). Parenteral administration of a 5% solution of emoxypine succinate to animals for 30 days contributed to a significant decrease in AST activity in the blood by 47.5%, ALT by 54.8%, LDH by 47.7%, in comparison with the data of rats of the second experimental group (p<0.05).

Indicators of CL in Rats with Carbon Tetrachloride Induced Hepatitis

From the data obtained in the experiment, as explained in Table 4, the value of the light sum in iron - induced hepatic homogenate (CL) was increased by 165% in rats after exposure to a toxicant against the background of placebo administration by the 30th day of the experiment. The addition of a 5% solution of emoxypine succinate contributed to a 56% decrease in the luminous sum of the glow, compared with the value in rats of the second experimental group.

In the model of carbon tetrachloride hepatitis on mature male rats treated parenterally with placebo, it was found that after intravenous administration of carbon tetrachloride, a fatal outcome was recorded in 26.7% of individuals, while in the surviving individuals, a significant violation of protein and carbohydrate metabolism were revealed and an increase in the value of the light sum CL of liver homogenates was noted. Consequently, intoxication of experimental animals with CCL4 causes structural and functional changes in hepatocytes. Its hepatotoxic effects are due to the fact that under the influence of the cytochrome P-450 enzyme monooxygenase system, free radicals CCL and CCL3 are formed, which activate the processes of peroxidation and reduce the activity of the antioxidant system. As a result of these processes, metabolism is disrupted in hepatocytes, hypoxia develops in them and the transmembrane potential decreases, which increases the permeability of their membrane. As a result of cytolysis, the blood levels of AST, ALT and LDH, were increased and the kinetics of free radical formation was elevated [13,14].

Intravenous administration to rats with hepatitis of emoxypine solution ocontaining ethylmethyl-hydroxypyridine succinate in its composition contributes to the development of a positive trend in reducing the values of AST, ALT, LDH indicators, increasing animal survival by 20%, adaptive capabilities and locomotor activity by day 14 by 142% and by the 30th day by 107%, compared with the indicators of rats of the second experimental group. Against the background of the use of emoxypine succinate reduces the generation of free radicals. It is obvious that the limiting effects of emoxypine succinate on the destructive effect of free radicals on the biological membrane of the hepatocyte is due not only to the interaction of the easily mobile hydrogen atom of the phenolic group in the molecule with the peroxy and alkoxyradials formed during lipid peroxidation, the ability of the active substance molecule to increase the activity of superoxide dismutase and other antioxidant enzymes [15], but also by the antihypoxic property of succinic acid and its specific effect on energy metabolism by strengthening the transition to preferential oxidation of succinate as one of the mechanisms for increasing the resistance of hepatocytes to hypoxia [16].The catecholaminomimetic property of succinate [17], which causes an increase in reactivity, a decrease in anxiety syndrome and an increase in the number of locomotions in the "open field" test, is of no small importance when the CCL4 toxicant acts on rats.

In an experiment on male rats, the antioxidant effect of emoxypine succinate was confirmed and its hepatoprotective effect was established for the first time. Parenteral administration of emoxypine succinate solution in rats at a dose of 5 mg/kg for 30 days with toxic carbon tetrachloride induced hepatitis contributes to increased reactivity and adaptive capabilities, correction of carbohydrate (LDH) and protein metabolism total protein, AST and ALT. The ability to inhibit the processes of free radical oxidation and the development of oxidative stress throughout the course of therapy is also important.

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