RU2177330C1 - Agent eliciting hepatoprotective effect - Google Patents

Abstract

FIELD: medicine, pharmacy. SUBSTANCE: invention relates to development of agents of vegetable origin as a hepatoprotective drug. Invention proposes viburnum crowns spirituous extract as a hepatoprotector. The proposed agent inhibits free-radical reactions and decreases formation of toxic products of lipid peroxidation processes, stabilizes lysosomal membranes and inhibits release of hepatocyte membranotropic hydrolases to cytoplasm, normalizes liver function that is characterized by synthesis of phospholipids from triacylglycerol and shows others effects. EFFECT: enhanced effectiveness of agent, valuable medicinal and biochemical properties. 1 cl, 5 tbl

Description

 The invention relates to biological compositions based on extracts of plant origin and can be used as a hepatoprotector, as well as a means of increasing the body's resistance to the effects of industrial chemicals, stressful situations, alcohol poisoning.

 In connection with the progressive deterioration of the environmental situation, a person is more and more actively exposed to various substances of technogenic origin. In our country, hygiene regulation currently covers about 2 thousand harmful chemicals, for which more than 3 thousand hygiene standards have been established for the maximum permissible content in various environmental objects. The impact of stressful situations (hypo- and hyperthermia, high physical exertion, breathing in hypoxic mixtures, being in the area of technological disasters, etc.), unbalanced nutrition, and alcoholism urgently require the search for unconventional approaches to the pharmacopracticities of emerging disorders in the body. It is known that the main barrier to neutralizing toxic pollution in humans and animals is the liver. Diseases of the liver and biliary system make up 40% in the group of nosological forms related to the pathology of the digestive system.

 Currently, an active search is underway for agents that increase the resistance of the liver to pathological effects, enhance its neutralizing functions by increasing the activity of the enzyme system, and also help restore its functions in various injuries, including alcohol intoxication.

 As a means of increasing the overall stability of the body, an extract of grape ridges is known (A.S. N 1473139, publ. 09/10/1998).

Known agent with anti-alcohol action obtained by extraction with alcohol of pre-dried and crushed grape ridges (and.with. N 1072309, publ. 10.09.1998)
Also known are drugs with a hepatoprotective effect, in which the active agent is a group of flavonoid compounds - Silimarin, isolated from extracts of the fruit of milk thistle (Sylybum marianum L). Based on these compounds, a number of silymarin, silibor, silibinin, karsil, and other drugs have been developed. All these drugs are foreign-made, so the relevance and need for searching and studying new sources of raw materials among natural, in particular plant wild non-food species.

 Closest to the claimed is the drug "Legalon", containing in its composition as an active agent a group of flavonoids from the extract of the fruit of milk thistle. Legalon has hepatoprotective and antitoxic effects. The mechanism of action is associated with the inhibition of lipid peroxidation, which prevents the destruction of cell membranes. In damaged hepatocytes, the drug stimulates the synthesis of proteins and phospholipids, as a result of which stabilization of cell membranes occurs, and the loss of cell components, such as transaminases, is prevented. The drug prevents the penetration of certain hepatotoxic substances into the cell. (Medicines of foreign firms in Russia, M. - Astrafarmservis, 1993, p. 350).

 An object of the invention is to expand the range of hepatoprotective agents through the use of new sources of polyphenolic compounds.

 The problem is solved by a means with hepatoprotective action, containing as an active principle alcohol extract of viburnum crests (Viburnum) in an effective amount.

The extract is obtained by the method usual for the preparation of extracts from plant materials. For this, the crests of viburnum (Viburnum) are dried at a temperature not exceeding 50 ^o C and crushed to accelerate and improve the extraction process. The crushed raw materials are extracted with 40% ethanol, as a rule, with a ratio of raw materials to extractant 1: 1.

 The composition of the obtained extract according to chromatography contains up to 50% polyphenolic compounds.

The finished extract has low toxicity (LD ₅₀ is 36 ml / kg) and does not have a harmful effect during prolonged injections into the stomach and parenterally, which allows experimental studies to show the pronounced hepatoprotective effect of the extract on the model of alcohol intoxication.

 The experiment is carried out according to the scheme given by A. Gajdos et al. ("Effet curatif de la (+) - catechine sur les troubles biochimigues du foie de rat intoxique par l'ethanol", CR Seances Soc. Biol.Fil, v.l66 No. 2, p. 277-279), and as a reference drug use Legalon.

The experiment was carried out on male Wistar rats weighing 130-180 g contained in a standard diet. First, for 7 days, ethanol was administered to rats at a dose of 7.5 ml per 1 kg of animal body weight intraperitoneally 2 times a day. Then, after the last day of ethanol administration, a viburnum extract is introduced in a dose of 0.2 ml per 100 g of mass for 7 days, while to exclude the influence of ethanol, the initial viburnum extract is pre-weathered in air or evaporated in a water bath at a temperature not exceeding 55 ^o C to 2/3 of the volume, and then adjusted with distilled water to the original volume. The viburnum extract treated in this way is administered orally to animals. Animals were divided into 5 groups: 1st — control (intact); 2nd - the introduction of 33% ethanol; 3rd - the introduction of 33% ethanol for 7 days, followed by cancellation (deprivation) within 7 days; 4th — introduction of Legalon after 7-day alcoholization; 5th - the introduction of the extract from the crests of viburnum after 7 days of alcoholization.

 Table 1 shows the biochemical parameters of animals exposed to alcohol and the subsequent administration of Legalon and viburnum extract. As can be seen, alcohol intoxication caused by the introduction of 33% ethyl alcohol is accompanied by a significant decrease in the activity of membrane-bound lysosomal beta-glucosidase by 15% as a result of the membrane-damaging effect of ethanol. An increase in the activity of cytosolic beta-galactosidase by 13% (P <0.05) is due to an increase in the permeability of lysosome membranes and the release of the matrix enzyme.

 The ratio of the components of antioxidant protection is violated. A 3-fold increase in the activity of superoxide dismutase (SOD) (P <0.001) is associated with the formation of hydroxyethyl free radicals from ethanol, which activate microsomal enzymes of the monooxygenase system. A decrease in the activity of glutathione reductase (GR) by 61% (P <0.05) and the amount of reduced glutathione by 14% (P <0.05) indicates a decrease in the liver's ability to effectively detoxify. As a result, lipid peroxidation is activated, which is confirmed by an increase in the amount of malondialdehyde (MDA) by 35% (92.94 + 1.4 μmol / g versus 68.91 + 1.7 μmol / g in the control; P <0.05) .

 A decrease in the NAD + content by 49% (0.26 + 0.03 μmol / g versus 0.51 + 0.04 μmol / g in the control; P <0.05), which is a coenzyme of alcohol dehydrogenase, indicates a high oxidation rate ethanol, accumulation of the reduced form of NADH and, accordingly, disruption of the functioning of aerobic glycolysis. This is confirmed by an increase in the amount of lactate by 42% (P <0.05), which is the final product of anaerobic glycolysis.

 Table 2 shows the data on the effect of Legalon and the extract of viburnum crests on the composition of neutral liver lipids of rats when affected by ethanol (in% of total lipids). It is seen that in the content of neutral liver lipid fractions there is an increase in the amount of cholesterol (cholesterol) by 13% (P <0.001), triacylglycerol (TAG) by 4% (P <0.05), free fatty acids (FFA) by 18% ( P <0.001) and a decrease in cholesterol esters (ECS) by 14% (P <0.05). These changes are due to increased peripheral lipolysis (a stressful reaction to ethanol), as a result of which fatty acids and glycerin enter the liver from fat depots, followed by esterification to TAG. The increase in the amount of cholesterol (cholesterol) is associated with an increase in its synthesis from acetyl-CoA formed during the oxidation of ethanol. The decrease in the number of cholesterol esters is due to a violation of the esterifying function of the liver. Violation of this function is also manifested in a decrease in the amount of total phospholipids by 24% (P <0.05), as follows from the table. 3.

 Table 4 shows the data on the effect of Legalon and the extract from viburnum ridges on the phospholipid composition of rat liver when affected by ethyl alcohol. It is seen that among phospholipid fractions, the amount of phosphatidylcholine (FC) decreased by 11% (P <0.05) and phosphatidylserine (PS) by 19% (P <0.05). The amount of lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LFE) increased noticeably by an average of 30-40% (P <0.05), which is associated with an increase in the activity of phospholipase A2. The level of phosphatidylinositis (PI) increased by 28% (P <0.05).

 The obtained changes in the content of the fractional composition of phospholipids indicate an increase in the permeability of hepatocyte membranes.

 Thus, the obtained experimental data indicate that, under the influence of ethyl alcohol, a pronounced hepatotropic effect is observed, which manifests itself in the form of significant changes in the lipid and carbohydrate metabolism of the liver, as well as in the imbalance of enzyme systems.

 7 days after the cancellation of ethanol (Table 1, group 3), most of the studied biochemical parameters do not return to normal. Beta-glucosidase activity remains low (by 27%; P <0.05). The mismatch in the activity of the antioxidant defense system enzymes remains (a 2.9-fold increase in SOD, a 55% decrease in GR activity) and a reduced amount of reduced glutathione (25.2 + 0.16 μg / ml versus 28.2 + 0.15 μg / ml in control; P <0.05). The content of NAD + 7 days after the end of alcohol intoxication remains at the level of that indicator in the 2nd group (0.28 + 0.04 μmol / g and 0.26 + 0.03 μmol / g, respectively), which indicates the preservation of tissue hypoxia. In this regard, a significantly increased level of lactate (8.03 + 0.36 μmol / g) was also recorded. The content of triacylglycerol and free fatty acids remains elevated, that is, fatty infiltration is not eliminated (Table 2, group 3). The amount of total phospholipids (Table 3, group 3) is not restored to the control level (13% less; P <0.05). The content of cholesterol esters becomes even lower (Table 3, group 3) - by 24% compared with the control (P <0.001). Consequently, the esterifying function of the liver is not restored when ethanol is withdrawn within 7 days.

A high level of lysophosphatidylcholine remains (25% increase compared with the control; P <0.05) (Table 4; group 3), which indicates an increased activity of phospholipases. It should be noted that the amount of diphosphatidylglycerol (DGF) decreased by 19% (P <0.05) compared with the control, which was not observed after 7 days of ethanol intoxication,
Thus, the abolition of ethanol within 7 days is accompanied by an incomplete restoration of the functional state of the rat liver. In our opinion, the period of withdrawal of a toxic agent is stressful for the body, since the magnitude of the changes in some of the studied biochemical parameters has increased (the number of free fatty acids has increased and the number of esters of cholesterol, diphosphatidylglycerol, phosphatidylserine has decreased).

 Studies on the use of the hepatoprotector of Legalon and the extract from viburnum ridges during the period of ethanol withdrawal show that the obtained biological effect was generally identical, but in some cases has a different degree of severity (tables 1-5; 4th and 5th groups). So, under the action of both drugs, the activity of lysosomal glycosidases (beta-glucosidase and beta-galactosidase), the level of NAD +, the content of triacylglycerol and free fatty acids are normalized. That is, both drugs have a pronounced hepatoprotective effect, manifested in the removal of fatty degeneration of the liver.

 At the same time, under the action of Legalon, the amount of lactate and the activity of superoxide dismutase remain increased, the activity of glutathione reductase and the level of reduced glutathione are reduced. Under the action of the extract from the crests of viburnum, these values correspond to the control level.

 It should be noted that the higher content of NAD + in the liver of rats receiving extract from the crests of viburnum (0.6 + 0.02 μmol / g). With the introduction of Legalon, this value is 0.52 + 0.03 μmol / g, which is 18% less.

 You should also pay attention to the level of cholesterol esters in the liver of rats of groups 4 and 5. So, with the introduction of Legalon, the level of cholesterol esters corresponds to the control value (respectively 19.0 + 1.49% and 19.42 + 0.61%). When the extract from the crests of viburnum is introduced, the content of cholesterol esters is 24.27 + 0.92%, which is 25% (P <0.001) higher than the control level. This phenomenon is probably due to the influx into the hepatocyte of an increased flow of cholesterol in an esterified form in the composition of high density lipoproteins. That is, the introduction of the extract from the crests of viburnum is accompanied by a hypocholesterolemic effect.

 Both drugs have the same restoring ability of the biochemical parameters of phospholipid metabolism, which indicates the normalization of the esterifying function of the liver.

 In addition to ethyl alcohol, thiopental anesthesia was chosen to test the effect of a chemical nature. Thiopental was administered to rats intraperitoneally at a dose of 60 mg per kg of body weight 2 hours after the intragastric administration of Legalon (100 mg per kg of body weight) or extract from viburnum ridges (0.4 ml per 100 g of weight). In the control group, 2 rats died. In the 2nd and 3rd groups, there was no death (table 5). It is seen that the preliminary administration of drugs is accompanied by a decrease in the lateral position time, which indicates the activation of the monooxygenase system, which carries out the biotransformation of xenobiotics. However, the action of the extract from the crests of viburnum was more effective than that with the introduction of Legalon.

Thus, based on the obtained experimental data, it follows:
1. The extract from the crests of viburnum has a pronounced hepatoprotective effect in alcoholic liver damage and the introduction of thiopental.

2. The mechanism of the therapeutic effect of the extract from the crests of viburnum is due to the beneficial effect on metabolic toxicity and liver function:
* Inhibits free radical reactions and reduces the formation of toxic lipoperoxidation products;
* Stabilizes lysosome membranes and inhibits the release of membranotropic hydrolases into the cytoplasm of hepatocytes;
* Normalizes the structure of hepatocytes due to the regulation of cholesterol synthesis, its esterification and inclusion in the structure of membranes;
* Normalizes the esterifying function of the liver, characterized by the synthesis of phospholipids from triacylglycerol;
* Restores the pool of oxidized NAD +, reducing acidosis and increasing the activity of energy supply processes due to the activation of erobic glycolysis.

 3. Extract from the crests of viburnum is an effective stimulant of the antitoxic function of the liver against the background of its inhibition by ethyl alcohol and thiopental.

 4. The investigated extract from the crests of viburnum under conditions of intoxication with ethyl alcohol exceeds the reference hepatoprotector Legalon in its ability to increase the formation of the oxidized form of NAD + and reduced glutathione, cholesterol esters; to a greater extent reduce the time of thiopental anesthesia. According to other indicators, the extract from the crests of viburnum shows a biological activity equal to that of Legalon.

Claims (1)

 An agent with a hepatoprotective effect, containing as an active principle an extract of plant origin, characterized in that it contains an alcoholic extract of viburnum as an active principle.

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