RU2405562C1 - Medication possessing hepatoprotective action - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to pharmaceutical industry, in particular to hepatoprotective medication. Medication, which has hepatoprotective action, represents product of extraction by ethanol of thalli of Cha-Kombu - Laminaria japonica Aresch., of laminarian family -Laminariaceae, which contains to 30 % of polyphenol compounds.

EFFECT: medication possesses efficient hepatoprotective action, favours accelerated restoration of metabolic reactions cascade, ensuring normalisation of biochemical characteristics of carbon and lipid exchange, as well as endogenous antioxidant protection system.

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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 chemicals of technogenic origin, poisoning by industrial poisons and drugs.

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 hygienic standards have been established for the maximum permissible content in various environmental objects. The influence of stressful situations (hypo- and hyperthermia, high physical exertion, breathing in hypoxic mixtures, being in the area of technological disasters, etc.), unbalanced nutrition, alcoholism urgently require the search for unconventional approaches to pharmacoprophylaxis 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 hepatoprotective agents that increase the resistance of the liver to pathological effects, enhance its neutralizing functions by increasing the activity of the enzyme system, as well as helping to restore its functions in various injuries, including poisoning with industrial poisons.

Known drugs with hepatoprotective action, belonging to the class of plant polyphenolic compounds. This is a number of drugs with similar composition and effect, which are produced in different countries under various names: "Silymarin", "Silibor", "Silibinin", "Karsil", etc.

Extracts from the crests of viburnum (Cl. RF No. 2177330, publ. December 27, 2001), Schisandra chinensis (Cl. RF No. 2179031, publ. 10.02.2002) and biomass extract are also known as agents with a hepatoprotective effect. Maackia amurensis Rupr.et Maxim (p. RF No. 2244553, publ. 01/20/2005), extract from the crests of Aralia Manchurian (p. RF No. 22399056, publ. 20.07.2008)

Closest to the claimed agent is the well-known drug "Legalon" widely used in medicine, containing as its active agent a group of plant polyphenols-flavonoids isolated from the fruits of milk thistle. The drug "Legalon" (hereinafter legalon) is a reference hepatoprotector, which is recommended by the official editions of the Pharmacopoeia Committee as a comparison drug when testing new drugs with hepatoprotective properties of plant nature (Vengerovsky A.I., Markova I.V. et al. Guidelines for the study of hepatoprotective activity of pharmacological substances // Guide to the experimental (preclinical) study of new pharmacological substances. Moscow. 2000. S.228-231). The drug is available in the form of dragees, capsules and suspensions.

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).

The objective of the invention is to expand the range of hepatoprotective agents.

The problem is solved by means with a hepatoprotective effect, based on the product of ethanol extraction of thalli of Japanese laminaria - Laminaria japonica Aresch., Of the laminaria family - Laminariaceae, containing up to 30% polyphenolic compounds.

The technical result consists in the manifestation of a pronounced hepatoprotective effect of the thallus extract of Japanese kelp - Laminaria japonica Aresch, the aralia family - Laminariaceae.

As raw materials for obtaining the claimed funds use the thallus of Japanese kelp. Japanese Laminaria Laminaria japonica Aresch (hereinafter referred to as kelp) belongs to the order of brown algae. It is traditionally widely used not only as a food product of high biological value, but as a raw material for the production of alginic acid preparations (polysaccharide complex), which are used as enterosorbents. It is known that brown algae contain significant amounts of polyphenolic compounds, the dominant group of which are florotannins. Florotannins are polymers of phloroglucinol (1,3,5-trihydroxybenzene) and can, according to published data, comprise up to 15% of the dry weight of brown algae (Ragan MA, Golombitza RW Phlorotannins, brow algal polyphenols. Progr. Phycol.Res. Vol. Vol. .4, P.129-241, 1986). However, it should be noted that the biological activity of the polyphenolic complex of kelp of the Japanese Laminaria japonica Aresch has not been studied in detail.

The authors believe that the use of kelp thalli is due to the fact that they are the part of the plant that, during the course of its life, is most exposed to the complex effects of light, oxygen and toxic substances dissolved in water, which inevitably leads to the formation of free radicals and other active oxidants. Moreover, the absence of obvious oxidative damage to structural components suggests the presence of antioxidant defense systems in their cells. According to the authors, one of the components of such protection are polyphenolic compounds contained in kelp. They act as acceptors of the active form of oxygen (ROS), metal chelators, modulators of enzymatic activity and lipid peroxidation inhibitors and are able to protect thallus cells from environmental factors.

Kelp extract is obtained in one of the following ways:

- extract fresh or frozen kelp thalli with 95% ethanol so that the concentration of ethyl alcohol in the final extract is about 70%, as a rule, when the ratio of raw material to extractant is 1: 2. For this, fresh or frozen thalli of kelp are crushed to particle sizes equal to 0.1-0.5 cm, and poured with ethanol. The extraction is carried out by maceration with stirring for 48 hours. The extract yield of 2 l per 1 kg of feedstock.

- extracted lyophilized and crushed thalli kelp 70% ethyl alcohol. The extraction is carried out by repercolation. The extract yield is 1 liter per 1 kg of feedstock.

- extract dry and crushed thalli of kelp with 70% ethyl alcohol. The extraction is carried out by repercolation. The extract yield is 1 liter per 1 kg of feedstock.

Such parameters are optimal, but not the only possible, since extraction can be carried out with ethanol with a different percentage concentration and at different ratios of extractant and raw material, obtaining the same high-quality set of extractives that determines the hepatoprotective effect of the agent. The hepatoprotective activity of the drug itself, in turn, is the combined effect of both polyphenolic compounds and other components formed during the extraction of the inventive plant materials. These parameters determine only the number of active components of the raw materials transferred to the extract.

The dry residue of the obtained extract according to the colorimetric method using Folin-Denis reagent contains up to 30% polyphenolic compounds. In addition, the extract contains polysaccharides: high molecular weight laminarin, mannitol, fructose, iodides, vitamins (B1, B2, B12, A, C, D, E, carotenoids), salts of potassium, sodium, magnesium, bromine, cobalt, iron, manganese, sulfur and phosphorus compounds, nitrogen-containing substances, proteins, carbohydrates, fats and a number of other organic compounds.

The finished product based on the product of extraction of kelp thalli has low toxicity (LD ₅₀ is 36 ml / kg) and does not have a harmful effect with prolonged injections into the stomach and parenterally, which allows experimental studies that have revealed a pronounced hepatoprotective effect of the extract on the model of carbon tetrachloride intoxication (CCl ₄ ). As a comparison, the plant-based polyphenolic preparation “Legalon” was used, selected as a prototype.

The model of experimental hepatitis caused by carbon tetrachloride, CCl ₄ , is considered the standard for liver damage with manifestations of organ dysfunction (Vengerovsky A.I., Markova I.V., Saratikov A.S. Preclinical study of hepatoprotective drugs // Bulletin of the Pharmacological Committee. 1999. No. 1. S.9-12.). The action of the poison is due to the defeat of the endoplasmic reticulum, lysosomes and other membrane structures by trichloromethine and chlorin radicals formed during the metabolism of xenobiotics in the P-450 cytochrome system. In addition, the functioning of this system produces superoxidion, which cause lipid peroxidation in the membranes (Golikov S.N., Sanotsky I.V., Tiunov L.A. General mechanisms of toxic effects. L .: Medicine, 1986. 280 p. )

The experiment was conducted on male Wistar rats weighing 130-180 g, contained in a standard diet. An experimental model of acute toxic hepatitis (CCl ₄ intoxication) in animals (rats) and drug administration were carried out according to the guidelines for preclinical trials (Vengerovsky A.I., Markova I.V., Saratikov A.S. Preclinical study of hepatoprotective drugs // Vedomosti Pharmacological Committee. 1999. No. 1. P.9-12.). Rats were injected subcutaneously (in the dorsal cervical fold) for 4 days with CCl ₄ (50% solution in olive oil) at a dose of 2 ml / kg. The kelp extract was administered orally to animals after the last day of CCl ₄ administration at a dose of 0.2 ml per 100 g of weight daily for 7 days.

The extract was obtained from the kelp thalli in the manner usual for the preparation of extracts from plant materials (IA Muravyov. Drug technology: 2-volume textbook. Moscow. Medicine. 1980. 704 p.) For this, 1 kg kelp thallus was dried at a temperature not exceeding 40 ° C, and crushed to accelerate and improve the extraction process. The crushed raw material was extracted by repercolation with 70% alcohol. The extract yield was 1 liter per 1 kg of feedstock.

To exclude the influence of ethanol, the initial extract of kelp thalli was evaporated in a vacuum evaporator at a temperature not exceeding 37 ° C to 1/3 of the volume, and then brought to the original volume with distilled water. The kelp thallus extract treated in this way was used in the experiment.

Legalon, a reference drug, was administered in the same manner as a suspension in 1% starch paste at a dose of 100 mg / kg body weight. The choice of dose for the comparison drug was carried out on the basis of literature data (Vengerovsky A.I., Markova I.V., Saratikov A.S. Preclinical study of hepatoprotective drugs // Vedomosti Pharmacological Committee. 1999. No. 1. P.9-12.) .

Animals were divided into the following groups: 1st — control (intact); 2nd — introduction of CCl ₄ for 4 days; 3rd — introduction of CCl ₄ followed by cancellation (deprivation) within 7 days; 4th — maintaining the extract from kelp during the deprivation period for 7 days; 5th - the introduction of legalon in the period of deprivation for 7 days.

Analysis of the obtained experimental data after 4-day administration of CCl ₄ shows that the weight of the animals decreased by 21% compared with intact animals (195.83 ± 4.91 g versus 246.67 ± 4.91 in the control, P <0.001), and the relative liver mass (g per 100 g of body weight) increased by 50% (4.72 ± 0.24 g versus 3.14 ± 0.15 in the control, P <0.001) (Table 1).

Upon external examination in animals, the hair was dull, sticking together into separate formations. Animals were poorly mobile, poorly ate food. In the liver, there was a continuous granularity of fatty inclusions, that is, pronounced fatty infiltration, characteristic of CCl ₄ intoxication, was manifested. The amount of total lipids in the liver was 142.09 mg / g of tissue, which was 3.5 times higher than the control level (42.17 mg / g) (P <0.001) (Table 2).

The development of toxic hepatitis in this experimental model is evidenced by a 7-fold increase in blood activity of the liver marker enzyme AlAT (307.71 ± 65.30 U / L versus 43.80 U / L in the control, P <0.001), due to the release of the enzyme from hepatocytes into the blood as a result of increased membrane permeability (table 3).

The activity of superoxide dismutase (SOD), a key enzyme of the antioxidant defense system, was 2.5 times lower than the control level (264.96 ± 4.5 Serv.units versus 669.17 ± 4.48 Serv.units in the control, P <0.001 ) (Table 4).

Glutathione peroxidase (GP) activity was also reduced by 22% (0.750 ± 0.062 μmol NADPH / min / ml plasma versus 0.961 ± 0.024 μmol NADPH / min / ml plasma in the control, P <0.01) and glutathione reductase (GR) by 38% (12.65 ± 1.15 nmol / min / ml plasma P <0.001) while reducing the concentration of reduced glutathione (G-SH) by 18% (4.76 ± 0.39 μmol / g Hb versus 5.82 ± 0.23 μmol / g Hb in the control , P <0.05). Such violations in the performance of the antioxidant defense system can be defined as its depletion. In addition, the depletion of the anti-radical defense of the body is expressed, which is confirmed by a decrease in the anti-radical activity of blood plasma (ARA) by 45% (6.49 ± 0.11 μmol of Trolox / L plasma versus 11.70 ± 0.16 μmol of Trolox / L plasma P <0.001),

Violations of the liver protective system were accompanied by a 2-fold increase in the amount of malondialdehyde (MDA) (7.27 nmol / ml of plasma versus 3.43 ± 0.3 nmol / ml of plasma in the control, P <0.001). This indicator characterizes the high activity of peroxidation of fatty acids that are part of membrane phospholipids, which is accompanied by an increase in the permeability of hepatocyte membranes.

In the study of carbohydrate metabolism after CCl ₄ intoxication, a significant decrease in blood glucose levels was observed, which was 2.954 ± 0.14 mmol / L, which is 36% lower than the intact control values (4.61 ± 0.25 mmol / L, P <0.001 ), due to glycogen depletion (Table 5). It is known that with toxic liver damage (chemical stress), glycogenolysis is activated to replenish substrates (glucuronic acid) of the 2nd phase of the xenobiotic detoxification system (Kostyuk V.A., Potapovich A.A. Bioradicals and bioantioxidants. - Minsk: BSU. 2004. - 174 s).

A decrease in the content of the oxidized form of NAD ⁺ in the liver by 30% compared with the control (0.244 ± 0.018 μmol / g versus 0.348 ± 0.013 μmol / g in the control, P <0.001) indicates a disruption in the course of aerobic processes, in particular, reactions of the Krebs cycle. The observed twofold increase in the lactate content to 4.69 ± 0.22 μmol / g (2.46 ± 0.27 μmol / g in the control) while reducing the content of pyruvic acid by 28% (0.127 ± 0.0133 μmol / g against 0.176 ± 0.009 μmol / g in the control, P <0.01) is the result of high intensity anaerobic glycolysis. This phenomenon is due to the activation of the pyruvate-lactate shuttle mechanism to restore the pool of the oxidized form of NAD ⁺ from NADH. A decrease in the ratio of NAD ⁺ / NADH (according to the LDH reaction) to 244 (645 in the control) indicates a shift in the balance of the redox system towards the formation of reduced equivalents, which leads to a decrease in NAD ⁺ -dependent dehydrogenases, blocking the aerobic processes of glycolysis, and inhibition of gluconeogenesis and the development of tissue hypoxia in the body (Panin L.E. Biochemical mechanisms of stress. - Novosibirsk, 1983).

Thus, the results obtained on the effects of CCl ₄ on the body of experimental animals confirm the development of toxic hepatitis.

7 days after the abolition of CCl ₄ (deprivation period) in the liver of experimental animals (group 3), the majority of the studied parameters did not normalize, which indicated continuing toxic stress and insufficient body defenses to resist the development of toxic pathology.

The mass of animals during the deprivation period changed slightly relative to the 2nd group (CCl ₄ ) (increase by 8%), while relative to the control it remained significantly low (by 15%, P <0.001). The animal liver mass decreased by 8% (P <0.05) relative to the 2nd group (CCl ₄ ), but at the same time it still significantly exceeded the control level by 12% (P <0.05). At autopsy, there were granular lipid inclusions in the liver. Animals during external examination were poorly mobile, poorly ate food, hair was dull. The amount of total lipids (Table 2) in the liver relative to the 2nd group decreased by 15%, while relative to the control level, their value was exceeded by 3 times (P <0.001).

The persistence of toxic hepatitis in this group of animals is evidenced by a reliably high (by 32%, P <0.05) activity of the marker enzyme of the liver AlAT (Table 3).

When analyzing the indicators of the antioxidant defense system during the deprivation period (Table 4), a tendency to recovery should be noted, however, compared with those in the control, statistically significant differences were revealed: these are low SOD activity (by 34%, P <0.001), and GP (by 17%, P <0.05). There was an increase in the ARA value relative to the 2nd group (by 32%, P <0.001), however, when compared with the control, its value was significantly reduced by 27% (P <0.001). Also, a high level of MDA remained relative to the control (by 63%, P <0.001), which determines the continued high activity of lipid peroxidation. That is, during the deprivation period, toxic stress continues due to the presence of xenobiotic metabolic products, as well as due to the depletion of antioxidant protection.

The study of the parameters of carbohydrate metabolism during the deprivation period (Table 5) showed restoration to the control level of blood glucose and pyruvate. However, relative to the control, an increased level of lactate (by 41%, P <0.05) and a low level of NAD ⁺ (by 31%, P <0.001) remained. In this regard, the ratio ratio NAD ⁺ / NADH was lower than the control by 35% and amounted to 422 (in the control 645).

Thus, the obtained experimental data indicate continuing metabolic disturbances even in the absence of a toxic agent (during deprivation).

Studies on the use of kelp extract during the withdrawal period of CCl ₄ showed that the resulting biological effect was generally identical to that of the reference hepatoprotector legalon, but in some cases had a different degree of severity (Tables 1-5; 4th and 5th groups).

So, under the action of both drugs, there was a tendency to restore the weight characteristics of animals, which was more pronounced than in animals in the group with pure deprivation. For animals from the group that received kelp, the average weight of animals was 226.66 ± 4.43 g; in the group that received legalon, this indicator was 225.00 ± 4.40 g, which on average exceeded that in the 3rd group (210.83 ± 4.90 g) by 8% (P <0.05) (Table 1). It is noteworthy that the relative liver mass (WMD) decreased to the level of control values. In animals of the 4th (kelp) and 5th (legalon) OMP groups amounted to 3.40 ± 0.18 and 3.54 ± 0.16 g / 100 g of body weight, respectively, which was 8-12% less ( P <0.05) than in animals from the 3rd group (4.10 ± 0.13 g / 100 g body weight). Moreover, the indicators of the 4th and 5th groups did not significantly differ from the intact control (3.14 ± 0.15 g / 100 g body weight). In animals, the hair became smooth, shiny, they began to eat well and move actively. The amount of total lipids in the liver also completely normalized with respect to the control and amounted to 45.95 ± 4.26 mg / g for the 4th group (kelp), 46.16 ± 3.44 mg / g for the 5th (legalon) (Table 2), in the control 42.17 ± 1.97 mg / g of liver. Moreover, in the deprivation group, this indicator was almost 3 times higher, amounting to 120.46 ± 12.33 mg / g of liver. That is, both drugs have a pronounced hepatoprotective effect, manifested in the removal of fatty degeneration of the liver.

The activity of the marker enzyme of toxic hepatitis AlAT in the blood of the 4th and 5th groups of rats did not significantly differ from the control values and amounted to 47.77 ± 2.51 and 43.79 ± 2.54 U / L, respectively (in control 43, 80 ± 4.25 U / L), which indicates the membrane-stabilizing properties of the studied drugs (Table 3). This phenomenon, according to the authors, is due to the localization of monomers and low molecular weight oligomers of florotannins within the lipid bilayer of plasma membranes [Afanasyeva Yu.G., Fakhretdinova ER, Spirikhin LV, Nasibullin RS About the mechanism of interaction of some flavonoids with phosphatidylcholine of cell membranes // Chem. Journal. 2007. No7. S.12-14.], The consequence of which is a decrease in its permeability.

The study of the values of the anti-radical activity of blood plasma and the biochemical parameters of the endogenous antioxidant defense system with the introduction of kelp extract showed that relative to the 3rd group (deprivation), a significant change in biochemical parameters towards normalization occurred (Table 4). The activity of SOD in the 4th group (kelp) was 649.65 ± 6.80 conv., Which is 48% higher than that in the third group (438.40 ± 1.76 conv., P <0.001) and There were no significant differences from the animals in the control group (669.17 ± 4.48 conventional units). The activity of the enzymes of the system for the production of the pool of reduced glutathione of GR and GP in the liver of animals of the 4th group (kelp) was 21.08 ± 1.01 nmol / min / ml of plasma and 0.974 ± 0.66 mmol of NADPH / min / ml of plasma, which corresponded to similar indicators in animals of the control group (20.50 ± 1.16 nmol / min / ml plasma and 0.961 ± 0.024 μmol NADPH / min / ml plasma for GR and GP, respectively). In animals of the 3rd group (pure deprivation), GR activity was 20% (17.53 ± 2.24 nmol / min / ml plasma), and GP was 22% (0.797 ± 0.062 μmol NADPH / min / ml plasma) lower than the corresponding indicators in animals treated with kelp extract. The lower activity of glutathione regeneration enzymes was accompanied by a tendency to its lower content in the liver of animals of the 3rd group (5.45 ± 0.25 μmol / g Hb) compared to the same indicator in animals of the 4th group (5.85 ± 0 , 46 μmol / g Hb), in which it did not differ from the control indicators (5.82 ± 0.23 μmol / g Hb). In the 5th group (legalon), all the above indicators did not significantly differ from those in animals in the control group.

Indicators of the level of free radical processes (ARA) and lipid peroxidation (MDA) in the blood plasma of animals of the 4th group (kelp) had a pronounced tendency to normalize, in contrast to similar indicators of animals of the 3rd group (pure deprivation). So ARA activity was 11.23 μmol of Trolox / L of plasma, which was 31% (8.56 ± 0.04 μmol of Trolox / L of plasma, P <0.001) than in animals of the 3rd group. The MDA content was 29% (3.96 ± 0.16 nmol / ml plasma, P <0.001) of the corresponding blood plasma index in animals of the 3rd group (5.61 ± 0.19 nmol / ml plasma). At the same time, the values of the indicated biochemical parameters in animals of the 4th group did not significantly differ from the control (11.70 ± 0.16 μmol of Trolox / L plasma and 3.43 ± 0.3 nmol / ml of plasma) and indicators in animals treated with the reference hepatoprotector legalon (11.42 ± 0.12 μmol Trolox / L plasma and 3.43 ± 0.11 nmol / ml plasma). According to the authors, the antioxidant and antiradical function is assumed by polyphenolic compounds that are part of herbal preparations as “traps” of free radicals (Skottova, N., L. Kazdova, et al. (2004). "Phenolics-rich extracts from Silybum marianum and Prunella vulgaris reduce a high-sucrose diet induced oxidative stress in hereditary hypertriglyceridemic rats. " Pharmacol Res 50 (2): 123-30).

The introduction of kelp extract after CCl4 intoxication contributed to the normalization of the biochemical parameters of carbohydrate metabolism (Table 5). A significant decrease in the level of lactate to 2.60 ± 77 μmol / g was observed against that in animals of the 3rd group (3.46 ± 0.29 μmol / g, P <0.05) and an increase in the content of NAD ⁺ to 0.304 ± 0.019 μmol / g (in the 3rd group, 0.241 ± 0.017 μmol / g, P <0.05). Such a change in biochemical parameters was accompanied by an increase in the NAD ⁺ / NADH (LDH reaction) index, which was 613, which is 45% higher than the corresponding indicator of animals of the 3rd group (422), which indicates the normalization of aerobic glycolysis and the removal of tissue hypoxia. The indicated values of biochemical parameters in animals of the 4th group did not significantly differ from those in animals from the intact control group.

The fact of the restoration of the level of antiradical activity of the liver, the activity of enzymes of the endogenous antioxidant defense system (SOD, GR, GP), the pool of reduced glutathione, the concentration of lactate and a higher NAD + content indicates the regulation of the redox balance in the body under the influence of kelp polyphenols, which effectively inactivate free radicals.

The experimental results obtained by the authors definitely indicate that the extract from kelp containing the polyphenolic complex exhibits a pronounced antitoxic effect in case of CCl ₄ damage, promotes the accelerated restoration of cascades of metabolic reactions, ensuring the normalization of biochemical parameters of carbohydrate and lipid metabolism, as well as the endogenous antioxidant defense system.

The therapeutic effect of the claimed drug is due to the beneficial effect of polyphenols on toxic metabolic disorders and liver functions, which is reflected in the restoration of the activity of antioxidant defense system enzymes (SOD, GR, GP), inhibition of free radical reactions and a decrease in the formation of toxic lipid peroxidation products; maintaining the level of restored glutathione and antiradical activity of the liver; restoration of the NAD + pool, reduction of acidosis and tissue hypoxia; stabilization of hepatocyte membranes.

In addition to CCl ₄ , thiopental anesthesia was chosen as factors of a chemical nature. Thiopental was administered to rats intraperitoneally at a dose of 60 mg per kg body weight 2 hours after the intragastric administration of legalon (100 mg per kg body weight) or kelp extract (100 mg OPF per kg body weight). There was no death in any of the groups. The lateral position time in rats after administration of thiopental was 56.2 ± 2.21 min. Preliminary administration of drugs is accompanied by a decrease in lateral position time, which amounted to 43.8 ± 2.45 min (P <0.05) for animals treated with Japanese kelp extract, and 45.2 ± 2.35 min (P <0, 05), which indicates the activation of a monooxygenase system that carries out biotransformation of xenobiotics.

It should be noted that the claimed tool is not only inferior to the reference hepatoprotector legalon in its ability to increase the antiradical activity of the liver, the formation of NAD ⁺ and normalization of the pool of reduced glutathione, but also more effectively helps to restore the integrity of cell membranes. So, if the AlAt activity in the blood plasma of animals treated with kelp extract did not differ from the intact control, then that in the group with the introduction of legalon was 9% higher (Table 3). This fact is important, since it is in case of toxic damage to CCl _{4 that the} main target is membranes, in particular, the endoplasmic reticulum - the localization site of P450 cytochromes. P450 cytochromes are integral membrane proteins of the endoplasmic reticulum, responsible in mammals for the oxidation of drugs, carcinogens, and toxic substances. Therefore, a more efficient restoration of cell and subcellular membranes provides a higher activity of the xenobiotic detoxification system, which was observed in the model of thiopental anesthesia. The duration of thiopental sleep in animals treated with kelp extract was shorter than that in the group with the introduction of legalon (table 6).

Thus, based on the obtained experimental data, it follows:

1. The extract from kelp has a pronounced hepatoprotective effect both in liver damage CCl ₄ , and with the introduction of thiopental.

2. The mechanism of the therapeutic effect of the extract from kelp is due to its beneficial effect on metabolic and liver dysfunction under the influence of a toxicant:

- restores the activity of enzymes of the endogenous antioxidant defense system SOD, GP, GR;

- saves the pool of reduced glutathione in the antioxidant defense system of the liver due to the protonophore properties of the flotannins contained in it;

- inhibits free radical reactions, increases the integral anti-radical activity of the liver and reduces the formation of toxic lipoperoxidation products;

- stabilizes hepatocyte membranes by incorporating kelp flotannins within the lipid bi-layer of the plasma membrane, normalizing their structure, and inhibits the release of hepatic enzymes (AlAT) into the blood plasma;

- removes fatty liver.

- restores the pool of oxidized NAD *, reducing acidosis and increasing the activity of energy supply processes due to the activation of aerobic glycolysis.

3. The investigated extract from kelp is not inferior to the legalon hepatoprotector selected as a comparison in its ability to restore the activity of the antioxidant defense system, increase the formation of NAD ⁺ , normalize the pool of reduced glutathione, increase the antiradical activity of the liver, and even surpasses it in its ability to restore the integrity of cell membranes .

Thus, the claimed tool allows you to expand the range of hepatoprotective agents, while finding new sources of polyphenolic compounds not previously used for these purposes, in particular algae for food purposes (Japanese kelp) from the seas of the Far East.

Table 1 The effect of plant prodrugs on the mass of animals and the liver after damage with carbon tetrachloride and the introduction of extract from kelp (M ± m) Groups of animals The mass of animals (g) Liver mass (g) 1 group Control 246.67 ± 4.91 7.72 ± 0.35 2 group Carbon tetrachloride 195.83 ± 4.36 ^*** 9.21 ± 0.44 ^* Group 3 Deprivation (CCl ₄ ) 210.83 ± 4.90 ^*** 8.62 ± 0.23 ^* 4 group Deprivation + kelp extract ^* 226.66 ± 4.43 " ^* 7.68 ± 0.28 5 group Deprivation + legalon ^* 225.00 ± 4.4 ^{** *} 7.97 ± 0.16 Note: the differences are statistically significant at ^* - P <0.05; ^** - P <0.01; ^*** - P <0.001. Asterisks on the right - comparison with the control group, asterisks on the left - comparison with the 3rd group (deprivation).

table 2 The effect of herbal preparations on the content of total lipids in rat liver (M ± m) Groups of animals Total lipids (mg / g tissue) Group 1 Control (intact) 42.17 ± 1.97 2 group CCl ₄ 142.09 ± 7.22 ^*** Group 3 Deprivation (CCl ₄ ) 120.46 ± 12.33 ^*** Group 4 Deprivation (CCl ₄ ) + kelp extract ^*** 45.95 ± 4.26 5 group Deprivation (CCl ₄ ) + Legalon ^*** 46.16 ± 3.44 Note: the differences are statistically significant at ^* - P <0.05; ^** - P <0.01; ^*** - P <0.001, Stars on the right - comparison with the control group, stars on the left - comparison with the 3rd group (deprivation).

Table 3 The effect of herbal preparations on the activity of alanine aminotransferase (AlAT) in the blood of rats after damage with carbon tetrachloride (M ± m) Groups of animals ALAT activity (Unit / L) Group 1 Control (intact) 43.80 ± 4.25 2 group CCl ₄ 307.71 ± 65.30 ^*** Group 3 Deprivation (CCl ₄ ) 58.03 ± 1.75 ^* Group 4 Deprivation (CCl ₄ + Legalon ^*** 47.77 ± 2.51 5 group Deprivation (CCl ₄ + kelp extract ^*** 43.79 ± 2.54 Note: the differences are statistically significant at ^* - P <0.05; ^** - P <0.01; ^*** - P <0.001. Asterisks on the right - comparison with the control group, asterisks on the left - comparison with the 3rd group, (deprivation).

Table 4 The effect of herbal preparations on the performance of the system “Lipid peroxidation-antioxidant defense” in rat liver after exposure to carbon tetrachloride (M ± m) Groups of animals ARA (Unit trolox / ml plasma) MDA (Nmol / ml plasma) SOD (Services Unit) GPO (μmol NADPH / min / ml plasma) GR (nmol / min / ml plasma G-SH (μmol / g Hg) 1 group Control 11.70 ± 0.16 3.43 ± 0.3 669.17 ± 4.48 0.961 ± 0.024 20.50 ± 1.16 5.82 ± 0.23 2 group CCl ₄ 6.49 ± 0.11 ^*** 7.27 ± 0.15 ^*** 264.96 ± 4.5 ^*** 0.750 ± 0.062 ^** 12.65 ± 10.15 ^*** 4.76 ± 0.39 ^* Group 3 Deprivation (CCl ₄ ) 8.56 ± 0.04 ^*** 5.61 ± 0.19 ^*** 438.40 ± 1.76 ^*** 0.797 ± 0.062 ^* 17.53 ± 2.24 5.45 ± 0.25 4 group Deprivation + kelp extract ^*** 11.23 ± 0.19 ^*** 3.96 ± 0.16 ^*** 649.65 ± 6.80 0.974 ± 0.66 21.08 ± 1.01 5.85 ± 0.46 5 group Deprivation + legalon ^*** 11.42 ± 0.12 ^*** 3.43 ± 0.11 653.47 ± 4.74 0.979 ± 0.044 20.29 ± 2.01 5.96 ± 0.53 Note: the differences are statistically significant at ^* - P <0.05; ^** - P <0.01; ^*** - P <0.001. Asterisks on the right - comparison with the control group, asterisks on the left - comparison with the 3rd group (deprivation).

Table 5 The effect of herbal preparations on the content of metabolites of carbohydrate metabolism in the liver and blood of rats after damage with carbon tetrachloride (M ± m) Blood glucose (mmol / l) Pyruvate (μmol / g) Lactate (μmol / g) NAD ^* (μmol / g) NAD ⁺ / NADH (LDH reaction) Group 1 Control (intact) 4.61 ± 0.25 0.176 ± 0.009 2.46 ± 0.27 0.348 ± 0.013 645 2 group CCl ₄ 2.95 ± 0.14 ^*** 0.127 ± 0.013 ^** 4.69 ± 0.22 ^*** 0.244 ± 0.018 ^*** 244 Group 3 Deprivation (CCl ₄ ) 4.21 ± 0.34 0.162 ± 0.008 3.46 ± 0.29 ^* 0.241 ± 0.017 ^*** 422 Group 4 Deprivation (CCl ₄ ) + kelp extract 4.36 ± 0.12 0.177 ± 0.011 ^* 2.60 ± 0.17 ^* 0.304 ± 0.019 613 5 group Deprivation (CCl ₄ ) + legalon 4.67 ± 0.19 0.180 ± 0.008 ^* 2.60 ± 0.21 ^*** 0.353 ± 0.008 624 Note: the differences are statistically significant at ^* - P <0.05; ^** - P <0.01; ^*** - P <0.001. Asterisks on the right - comparison with the control group, asterisks on the left - comparison with the 3rd group (deprivation).

Table 6 The influence of the studied drugs on the time of thiopental anesthesia (min; M ± m). 1st (control) 2nd (legalon) 3rd (extract from thalli of Japanese kelp) 56.2 ± 2.21 43.8 ± 2.44 ^* 45.2 ± 2.35 ^* Note. Significant differences with control: ^* - P <0.05;

Claims (1)

An agent with a hepatoprotective effect based on an extract of plant origin, characterized in that the agent is an ethanol extraction product containing up to 30% polyphenolic compounds of Japanese laminaria thalli - Laminaria japonica Aresch., Laminaria family - Laminariaceae.