THERAPEUTIC EFFICACY OF A HEPATOPROTECTIVE DRUG BASED ON SILYMARIN AND SELENIUM NANOPARTICLES IN SHEEP LIVER DYSFUNCTION

THERAPEUTIC EFFICACY OF A HEPATOPROTECTIVE DRUG BASED ON SILYMARIN AND SELENIUM NANOPARTICLES IN SHEEP LIVER DYSFUNCTION

Mikhail Chekunov

2nd year postgraduate student, Saratov State University named after N.I.Vavilov,

Russia Saratov

Ekaterina Bykova

Veterinary doctor of Harmony LLC,

 Russia, Saratov

Sergey Kozlov

Doctor of Veterinary Sciences, Professor, Saratov State University named after N.I. Vavilov",

Russia, Saratov

ABSTRACT

This article is devoted to the design of a hepatoprotective drug based on silymarin and selenium nanoparticles and the study of its therapeutic in case of liver dysfunction in sheep.

Keywords: acute reactive hepatitis, silymarin, nanoparticles, polymer matrix, selenium, sheep.

Introduction.  Extracts of flowers and leaves of milk thistle (Silybum marianum (L.) Gaertn.) have been used for many centuries in the treatment of liver diseases. Janiak and Hänsel̈nsel (1960) [1, с.27] isolated the biologically active components of the plant. The researchers isolated a mixture of active ingredients isolated from milk thistle, which they called silymarin, and with which most clinical studies were conducted later.

Silymarin has a hepatoprotective effect, reducing the concentration of free radicals formed during the metabolism of toxic agents such as ethanol, acetaminophen or carbon tetrachloride, thereby reducing the degree of damage to cell membranes. Silymarin also inhibits the cyclooxygenase cycle in liver cells, reduces the synthesis of CP in the liver. It protects the liver from inflammation, which in turn reduces inflammation [2, с.59].

The most common liver diseases of an inflammatory nature in the target group of animals are: non-specific reactive hepatitis, chronic hepatitis and acute hepatitis. O ther common causes of liver disease include neoplasia, vascular abnormalities (i.e. portosystemic shunts), and biliary tract abnormalities.

Non-specific reactive hepatitis is a non-specific response to diseases of extrahepatic etiology. Histologically, the lesion is characterized by inflammatory infiltration in the area of the hepatic lobules and in the liver parenchyma, and necrosis of individual cells is noted [1, с.10]. The disease is a consequence of extrahepatic pathology; therapy should be aimed at eliminating the primary factor [1, с.22].

According to the WSAVA definition, acute hepatitis is morphologically characterized as an inflammatory process accompanied by hepatocellular apoptosis and necrosis. There are fewer cases of acute hepatitis than chronic hepatitis. The causes of the disease can be gross violations of feeding and maintenance, as well as the use of drugs that have a toxic effect on the liver, leptospirosis, Bacillus piliformis (disease Tizzer), Toxoplasma gondii, and a number of other septic bacterial diseases [2, с.30].

The aim of this study was to design a hepatoprotective drug based on silymarin and selenium nanoparticles and to study its therapeutic efficacy in comparison with a commercial drug пfor liver disorders in sheep

Materials and methods. A preparation of Selenium nanoparticles with silymarin was synthesized by the method of Nurul Kabir et al. [3, с.45] with our modifications.

Ingredients: Sili-5.85 g., NaOH-1.638 g., H2oSeO3-0. 645 g., distilled water up to 500 ml, organic acid up to pH9.2.

The study included

15 sheep under 10 years of age. The selection criterion for the study group was the presence of symptoms of damage to the hepatobiliary system. Animals were divided into groups according to the drug used.  Animals of all groups received the same food base according to the established norms.

Animals of the first control group (n=5) were prescribed a commercial reference drug as a hepatoprotective drug «Hepatoject» intramuscularly, 2-5 ml per animal depending on its weight, 2 times a day-7 days;

Animals of the 2nd experimental group (n=5) were injected intramuscularly daily for 7 days with a water-dioxysion solution of silymarin at a therapeutic dose of 100 mg / kg in dosage form;

Animals of the 3rd experimental group (n=5) were injected intramuscularly daily for 7 days with silymarin conjugated with selenium nanoparticles at a therapeutic dose of 100 mg / kg in dosage form;

The method of evaluating the results of treatment was based on determining the following indicators: body temperature, decreased appetite, decreased overall activity, mucosalictericity, and the results of biochemical studies of venous blood. The study of the drug was carried out on animals kept by MBU NMO "NSBIV" Natalia's Equestrian Club.

Results

The results of biochemical studies showed a significant increase in the activity of liver cytolytic enzymes alanine and asparagin aminotransferases in relation to clinically healthy sheep (ALT-I-244.79±22.06, II-286.56±20.21, III-279.64±12, AST-I-239.91±16.46, II-258.72±9.63, III-250.56±18.48, (P ≤ 0.05 at t critical 2.10)).  Along with this, there is a significant increase in the activity of enzymes indirectly acting as markers of cholestasis –alkaline phosphatase (I-245.6±17.27, II-263.7±6.19, III-240.21±13.79,). However, the concentration of direct bilirubin in the blood serum is also significantly increased (I-19.21±0.97, II-22.6±1.26, III-29.22±0.91), which is a consequence of the insufficiency of the transport system for removing the sharply increased concentration of conjugated bilirubin from the cell through the bile ducts. When comparing the results of treatment of different groups, a significant decrease in the activity of cytolytic liver enzymes alanine and aspartic aminotransferases and the manifestation of cland other signs of liver dysfunction in sheep was found in the 3rd experimental group of animals that were prescribed silymarin conjugated with selenium nanoparticles as a hepatoprotective agent (ALT-I-220.79±22.06, II-240.56±20.21, III180.64±12, AST-I-219.91±16.46, II-228.72±9.63, III-150.56±18.48, (P ≤ 0.05 at t critical 2.10)).  Along with these changes, there is a significant increase in the activity of enzymes indirectly acting as markers of cholestasis – alkaline phosphatase (I-245.6±17.27, II-263.7±6.19, III-160.21±13.79,).

A significant decrease in cytolysis enzymes was observed in animals of all experimental groups 14 days after the appointment of therapy. Thus, the activity of alanine aminotransferase decreasedin the first experimental group to 200.71 (p≤0.05), in the second to 209.92 (p≤0.05), in the third to 108.43 (p≤0.05),The most intense decrease in the activity of this enzyme was also observedin the third experimental group of sheep, which were prescribed a preparation of silymarin conjugated with selenium nanoparticles. In this group, the activity of the enzyme was significantly lower than in other groups of animals. Although it is worth noting that in sheep of all groups, the activity of alanineнaminotransferase remained significantly higher than the reference values. Along with this, on the 14th day of the experiment, a significant decrease in another cytolytic enzyme, aspartate aminotransferase, was noted in the first group to 130.45 (p < 0.05), in the second –11.97, 97 (p < 0.05), in the third group the most intense decrease was observed to 75.43(p < 0.05) relative to the initial values.

Analysis of the dynamics of changes in the activity of cholestasis enzymes showed a significant decrease in the activity of alkaline phosphatase in all experimental groups of animals relative to the initial values.  In the first experimental group of sheep treated with the commercial hepatoprotective drug "Hepatoject", the activity of alkaline phosphatase in blood serum decreased to 150.4 (p < 0.05). In the second, up to 160.4 (p≤0.05), in the third to 70.3 (p≤0.05) relative to the initial values. It should be noted that the reference values for the activity of this enzyme were exceeded in the third experimental group of sheep, which were prescribed a preparation of silymarin conjugated with selenium nanoparticles as a hepatoprotector. 

Thus, 14 days after the appointment of treatment measures, the concentration of direct bilirubin decreased, respectively, in the first group of animals to 8.44 (p < 0.05), in the second group to 10.2 (p < 0.05), and in the third group to 3.2 (p < 0.05), relative to the values before the appointment of therapy. At the same time, it should be noted that the concentration of direct bilirubin in the blood serum of animals reached reference values only in the third experimental group. This fact is a consequence of a decrease in the mesenchymal-cellular response of the body in response to the action of the damaging agent. In the course of studying the indicators of the antioxidant system, a significant decrease in the concentration of malondialdehyde to background values was found in the third group of animals that were prescribed a preparation of silymarin conjugated with colloidal selenium. While in other groups of animals, although positive dynamics was observed, this indicator remained significantly higher than the background values. At the same time, the activity of glutathione peroxidase in all groups of animals did not significantly differ from the background animals, which, as already mentioned, is a consequence of the inclusion of compensatory mechanisms in the animal body. However, in the third experimental group, by the end of the experiment, the activity of this indicator was higher than both the experimental groups and the background values.  This indicates the inclusion of selenium contained in the preparation in the metabolic cycle of the antioxidant system of the animal body.

Table 1.

Indicators of lipid peroxidation and antioxidant system protection of sheep blood

Note: * The difference in this indicator is statistically significant between the experimental group and the background values, * * between the experimental groups of animals (P ≤ 0.05 at t critical 2.10),

Conclusion. Thus, it was reliably established that parenteral use of silymarin preparations based on colloidal particles (selenium) and polymer matrices for therapeutic purposes leads to restoration of functional activity of sheep liver. At the same time, the preparation of silymarin based on colloidal selenium, along with pronounced hepatoprotective properties, showed a pronounced antioxidant effect, as a result of which the regeneration processes and binding of toxic substances are more actively carried out.

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2. Colombo ML. An update on vitamin E, tocopherol and tocotrienol: Perspectives/M.L. Colombo //Molecules. – 2010. – N.15. – P.2103–2113.
3. Nurul Kabir, Hamid Ali, Muhammad Ateeq, Massimo F. Bertino, Muhammad Raza Shah,Louis Franzelc Silymarin coated gold nanoparticles ameliorates CCl4-induced hepatic injury and cirrhosis through down regulation of hepatic stellate cells and attenuation of Kupffer cells // RSC Adv., 2014,4, 9012-9020