RU2633590C1 - Drugs for toxic hepatitis prevention and treatment - Google Patents

Abstract

FIELD: pharmacology.

SUBSTANCE: dry hare's ear extract is administered into the patient's stomach at a dose of 10 mg per kilogram of the patient's weight during the period of liver exposure to toxic substances and within 5 days after this period, this extract contains phenolic compounds of aglucone flavonoids: luteolin - 0.3±0.01 mg/g, quercetin 13.3±0.50 mg/g, kaempferol - 1.1±0.10 mg/g, isoramnetin 9.1±0.30 mg/g; flavonoids: rutin - 6.2±0.10 mg/g, hyperoside 5.7±0.10 mg/g, astragaline - 0.4±0.01 mg/g; phenol carboxylic acids: ferulic acid - 657.4 mg/100, coffee acid - 83.9 mg/100, 4-caffeolic acid - 592.2 mg/100, rosmaric acid - 130.2 mg/100; tannins in terms of (+)- catechin - 7.2%±0.5%; saponins in terms of oleanolic acid - 8.4±0.3%; polysaccharides in terms of glucose - 1.80±0.15%; ascorbic acid - 0.4 mg/g; essential amino acids: valine - 0.12%, lysine 0.15%, threonine 0.07%, histidine 0.3%, arginine 0.14%; interchangeable amino acids: alanine 0.15%, cysteine 0.95%, glycine 0.05%, tyrosine 0.45%.

EFFECT: invention provides a pronounced hepatoprotective effect with respect to toxic hepatopathy.

3 dwg, 5 tbl, 1 ex

Description

One of the most important tasks of modern medicine is the search for new effective drugs for the prevention and treatment of liver diseases of various origins, such as toxic and viral hepatitis, cirrhosis, drug, radiation and ischemic injuries, etc.

The invention relates to the field of pharmacology, namely to hepatoprotective drugs, and can be used in the development of new herbal medicines, mainly for the prevention and treatment of toxic hepatopathy caused by mercuric chloride intoxication.

The nomenclature of drugs for the prevention and treatment of hepatitis is represented mainly by herbal preparations, the active substances of which are flavonoids. They have a wide range of pharmacological effects, the main of which is antioxidant, which allows inactivation of both free radicals and reactive oxygen species in the cell, which largely determines their high therapeutic efficacy in the treatment of liver diseases such as toxic and viral hepatitis, cirrhosis, and drug , radiation and ischemic injuries, etc. [Lutsenko SV, Feldman NB, Lutsenko EV, Bykov VA Plant flavolignans. Biological activity and therapeutic potential. M. 2006].

It is known, in particular, a tool for the treatment of chronic hepatitis of non-infectious etiology [RU 2139071, C1, A61K 35/78, 10.10.1999], which is a collection of medicinal plants, including chamomile, calendula grass, calendula flowers, sandwort inflorescences, mint leaves pepper, fennel fruits, corn stigmas, medicinal sweet clover, cowberry leaves, rhizomes with valerian roots, rhizomes with calamus roots, tansy flowers, blue cornflower, Canadian goldenrod, black elderberry flowers, linden flowers, bitter wormwood grass, nettle leaves s dioecious and rowan fruits in the following ratio of components in wt. %: immortelle of sandy inflorescence - 4-6, peppermint leaves - 9-11, fennel fruits - 7-9, calendula flowers - 8-10, corn stigmas - 9-11, grass strings 14-16, medicinal sweet clover - 10- 12, chamomile pharmacy - 4-6, lingonberry leaves - 9-11, rhizomes with valerian roots - 4-6, rhizomes with calamus roots - 5-7, tansy flowers - 2-4, cornflower blue flowers - 2-4, goldenrod Canadian - 4-6, elderberry black flowers - 7-9, linden flowers - 14-16, wormwood bitter grass - 2-4, nettle dioecious leaves - 4-6, mountain ash fruits - 6-8.

The disadvantage of this tool is its relatively high complexity of preparation.

In addition, it is known a tool for the treatment of patients with drug-induced hepatitis [RU 2419449, C1, A61K 36/288, 05/27/2011], which is a herbal collection, including: wild strawberry grass, medicinal dandelion grass, non-lingual inflorescences, dioecious nettle leaf, inflorescences tansy, calendula inflorescences, common juniper fruits, peppermint leaf, perforated hypericum grass, characterized in that it additionally contains linden flowers, tricolor violet grass, centaury grass, flax seed, dill seed , burdock root, common chicory grass, bird of highlander grass, hops common fruit, blue cornflower grass, cowberry leaf, bearberry leaf, corn stigmas, immortelle grass, three-leafed grass roots and rhizomes, angelica root, angelica root, medicinal melilot grass, , horsetail grass, in the following mass ratio of components:

Wild strawberry, grass 5-7 Medicinal dandelion, grass 4-6 Chamomile flowerless, inflorescences 10-12 Stinging nettle, leaf 6-8 Tansy, inflorescences 2-4 Calendula officinalis, inflorescences 1-3 Juniper, fruits 5-7 Peppermint leaf 8-10 Hypericum perforatum, grass 9-11 Linden flowers 20-22 Violet tricolor, grass 19-21 Centaury, grass 6-8 Flax seed 10-12 Dill seed 9-11 Burdock root 4-6 Common chicory, grass 7-9 Highlander bird, grass 6-8 Common hops, fertility 8-10 Cornflower blue, grass 3-5 Lingonberry leaf 6-8 Bearberry leaf 3-5 Corn stigmas 7-9 Immortelle, grass 4-6 Air, roots and rhizomes 5-7 Three-leaved watch, grass 7-9 Angelica root 7-9 Melilotus officinalis, grass 8-10 Elecampane root 7-9 Horsetail, grass 6-8

The disadvantage of this tool is its relatively high complexity of preparation.

The closest in technical essence and effect when applied is a therapeutic agent for the prevention and treatment of chronic liver diseases [RU 2526172, C2, A61K 36/21, 08/20/2014], which is amaranth oil obtained by cold pressing of the seeds and shells of amaranth seeds, which is used at a dose of 62.5-250 mg / kg body weight.

The disadvantage of the closest technical solution is the relatively narrow scope, which is expressed in the relatively low pronounced hepatoprotective properties in relation to toxic hepatopathy caused by intoxication with mercuric chloride.

The problem that is solved in the proposed invention is the expansion of the range of herbal medicines and the creation on this basis of a medicinal product for the prevention and treatment of toxic hepatopathy caused by mercuric chloride intoxication.

The required technical result is to expand the assortment of herbal medicines and increase the hepatoprotective effect against toxic hepatopathy caused by mercuric chloride intoxication.

The problem is solved, and the required technical result is achieved by a herbal medicine based on plants, according to the invention, dry extracts of a dry dose of 10 mg per kilogram of patient weight are used as a medicine.

In addition, the required technical result is achieved by the fact that dry extracts of hair are phenolic compounds of flavonoids-aglycones: luteolin 0.3 ± 0.01 mg / g, quercetin 13.3 ± 0.50 mg / g, campferol 1.1 ± 0.10 mg / g; isoramnetin 9.1 ± 0.30 mg / g; flavonoids: routine - 6.2 ± 0.10 mg / g, hyperoside 5.7 ± 0.10 mg / g, astragaline 0.4 ± 0.01 mg / g .; phenolcarboxylic acids: ferulic acid 657.4 mg / 100, caffeic acid 83.9 mg / 100, 4-caffeoylquinic 592.2 mg / 100, rosmarinic 130.2 mg / 100; tannins: on (+) - catechin 7.2% ± 0.5%; saponins in terms of oleanolic acid 8.4% ± 0.3%; polysaccharides in terms of glucose 1.80 ± 0.15%; ascorbic acid 0.4 mg / g; essential amino acids: valine - 0.12%, lysine - 0.15%, threonine - 0.07%, histidine - 0.3%, arginine - 0.14%; essential amino acids: alanine - 0.15%, cysteine - 0.95%, glycine - 0.05%, tyrosine - 0.45%.

The drawing shows:

in FIG. 1 - morphological picture of the liver of rats with mercuric chloride intoxication;

in FIG. 2 - morphological picture of the liver of rats treated with hair extracts at a dose of 10 mg / kg + mercuric chloride;

in FIG. 3 - morphological picture of the liver of rats treated with hair extracts at a dose of 100 mg / kg + mercuric chloride.

We present theoretical and experimental data confirming the effectiveness of the use of dry extract dryworm as a medicine in the prevention and treatment of toxic hepatitis.

Promising sources of biologically active substances are plants of the genus Volodushka, which has a wide distribution area in Russia, which provides a stable raw material base. In addition, work was carried out on the territory of the Republic of Bashkortostan to introduce golden beadwort into the culture and it was found that it is more technologically introduced into the culture than multicore, has a large (four times) land mass of plants, which is important when creating industrial stocks of plants. Experimental data on the biochemical study of extracts from goldenwort testify to a positive effect on the liver and biliary tract in experiments on animals.

In FGBNU VILAR, the optimal technology for the extract of dry goldenwort (longleaf) dry extract - Bupleurum aureum Fisch - has been developed. seu longifolium L., sem. Apiaceae are celery and standardized.

Dry hair extracts are phenolic compounds of flavonoids-aglycones: luteolin 0.3 ± 0.01 mg / g, quercetin 13.3 ± 0.50 mg / g, campferol 1.1 ± 0.10 mg / g, isoramnetin 9, 1 ± 0.30 mg / g; flavonoids: rutin - 6.2 ± 0.10 mg / g, hyperoside 5.7 ± 0.10 mg / g, astragaline 0.4 ± 0.01 mg / g; phenolcarboxylic acids: ferulic acid 657.4 mg / 100, caffeic acid 83.9 mg / 100, 4-caffeoylquinic 592.2 mg / 100, rosmarinic 130.2 mg / 100; tannins in terms of (+) - catechin 7.2% ± 0.5%; saponins in terms of oleanolic acid 8.4% ± 0.3%; polysaccharides in terms of glucose 1.80 ± 0.15%; ascorbic acid 0.4 mg / g; the essential amino acids valine - 0.12%, lysine - 0.15%, threonine - 0.07%, histidine - 0.3%, arginine - 0.14%; essential amino acids: alanine - 0.15%, cysteine - 0.95%, glycine - 0.05%, tyrosine - 0.45%.

A wide range of pharmacological activity of Goldenwax is due to its unique chemical composition.

An experimental model of toxic hepatitis caused by the introduction of mercuric chloride (mercury (II) chloride) was chosen as a pathology model. Mercury is one of the most powerful blockers of SH-groups, it is uniquely qualified as sulfhydryl poison. The connection of mercury with sulfur is almost irreversible. It is believed that this property of mercury is the basis of the mechanism of its toxic effect. Regardless of the chemical formula, mercury is a powerful cellular poison, since it can change the tertiary and quaternary structure of protein molecules, so mercury is potentially able to change the functional state of any cell, tissue, organ, leading to cellular apoptosis. Being a powerful thiol poison, mercuric chloride leads to damage to protein structures and inhibition of thiol-containing antioxidants, a decrease in the activity of antioxidant defense enzymes, and liver damage caused by the development of oxidative stress. In well-known works, it is shown that, under the influence of mercuric chloride, pronounced histological changes in the liver are formed. There is a pronounced picture of degeneration and continuous vacuolization of cell nuclei, focal centrolobar and periportal necrosis of hepatocytes, as well as signs of biliary stagnation. The hepatotoxic effect of mercuric chloride leads to the development of changes in blood biochemical parameters, namely an increase in the concentration (more than 6 times) of the activity of alanine aminotransferase, impaired lipid, protein and mineral metabolism, which in turn lead to the development of systemic pathophysiological conditions.

The aim of the study was to study the hepatoprotective action of the dry extract of the golden extract of gold on an experimental model of acute sublimate toxic intoxication of rats.

The studies were performed on 105 Wistar rats (males) with a body weight of 180-200 g. The experiments were carried out in accordance with the "Laboratory Practice Rules". Keeping, feeding and caring for animals was regulated by sanitary rules for the design, equipment and maintenance of experimental biological clinics - vivariums [Rules for the protection of vertebrate animals used for experimental and other purposes. European Convention, Stasburg, 1986].

The model of acute toxic damage to the liver of rats was caused by a single subcutaneous injection of mercuric chloride at a dose of 3 mg / kg (≈ LD ₅₀ when subcutaneous administration of mercuric chloride to white rats). Animals were divided into 4 groups of 15 animals each. Group I - intact control used to assess the toxic effects of mercuric chloride. Group II animals (model) were once injected subcutaneously (sc) sublimate at a dose of 3 mg / kg; Group III and IV animals received dry extract in doses of 10 and 100 mg / kg, respectively, 1 hour before the introduction of mercuric chloride and in the next 5 days. The choice of experimental doses of dry extract extract was determined by the fact that the maximum dose was 1/100 of the LD ₅₀ when administered intragastrically to rats.

In order to identify the severity of the hepatoprotective action of the dry extract extract, studies were conducted in comparison with the known hepatoprotector carlsil. The drugs were injected into the stomach of animals in the same dose of 10 mg / kg according to the same scheme as above. In this experiment, the animals were divided into 3 groups of 15 rats each: group I - subclimate sublimate at a dose of 3 mg / kg; Group II - extracts of hair 10 mg / kg + mercuric chloride; Group III - Carsil 10 mg / kg + mercuric chloride.

During the experiment, the basic integral indicators of experimental animals were recorded: general condition, behavior, dynamics of body weight, consumption of dry food and water, death. Twice, on the 5th and 10th day of the experiment, hematological parameters were studied (the number of red blood cells, white blood cells, platelets, the level of hemoglobin, hematocrit, the average content and concentration of hemoglobin in the red blood cell, the average volume of red blood cells, the distribution of red blood cells by volume) on a semi-automatic hematological analyzer "BC-2300", MINDRAY (China). Clinical biochemistry parameters were determined at the same time: total protein (OB), total cholesterol (OH), triglycerides (TP), glucose (Hl), alkaline phosphatase (ALP) activity, alanine and aspartate aminotransferases (AlTA, AsTA), lactate dehydrogenase ( LDH) on an automatic biochemical blood analyzer URIT-8030 from Urit Medical Electronic (China), using kits from Human, Germany. At the end of the experiment, the animals were euthanized in a CO ₂ chamber, their internal organ coefficients (liver, spleen, thymus) were determined with their histopathological evaluation when stained with hematoxylin and eosin and examined by light microscopy at magnifications of × 100 and × 200.

After the completion of the experimental studies, a statistical processing of the obtained results was carried out by the method of variation statistics using Student t-test. The significance of differences with control was considered at P <0.05. Statistical data was processed using Office Excel.

Under the influence of mercuric chloride, the animals developed toxic liver damage characteristic of acute poisoning of rats with this hepatotropic poison, a sharp violation of its functional state, a decrease in the main types of interstitial metabolism, damage to the membranes of hepatocytes and their death. The indicated effects of the hepatotoxic effect of mercuric chloride were accompanied by a violation of the general condition of the animals (decrease in feed and water consumption, dynamics of body weight by 39-51%, lethargy, physical inactivity, softening of feces), the death of 4 animals, which amounted to 26.6%, as well as leukocytosis. Sublimate intoxication led to a statistically significant increase in rat serum levels of total protein, glucose, total cholesterol and triglycerides, the activity of "liver" enzymes: AcTA and ALTA, LDH, alkaline phosphatase.

In animals of this group, a decrease in the weight coefficient of the liver and immunocompetent organs — spleen and thymus — was noted. The histological picture of the liver showed (Fig. 1) the presence of hepatocyte dystrophy, characterized by hyaline droplet and vacuole dystrophy.

The introduction of dry extract extracts at doses of 10 and 100 mg / kg improved the general condition of the animals, prevented a sharp decrease in body weight and motor activity. On the 5th day of the experiment, the decrease in the body weight of rats receiving the extract in both tested doses did not exceed 12-15% as compared with the pathology model, and on the 10th day the body weight of the animals of these groups corresponded to the intact control (Table 1).

In the group of animals treated with dry extracts of dry hair at a dose of 10 mg / kg, the death of 2 animals was recorded, which amounted to 13%, and in the group of animals that received dry extracts of dry extract at a dose of 100 mg / kg, no deaths were observed. Rats in these groups had a neat appearance, decorated feces, willingly ate food.

Under the influence of dry extract drywort at the indicated doses, both hematological and biochemical parameters were normalized in both follow-up periods: leukocytosis was not observed, there was a decrease in the level of total protein, total cholesterol, triglycerides and glucose (tables 3, 4). The introduction of dry extract of dry extract in the indicated doses also prevented a sharp increase in the activity of a number of blood serum enzymes characterizing the functional state of the liver, and contributed to their faster normalization, and these changes were somewhat more pronounced in animals receiving the maximum extract dose of 100 mg / kg (tables 2 and 3).

However, a direct correlation dependence of the level of hepatoprotective action of extract extractwort according to biochemical parameters and the activity of blood serum enzymes and their normalization rate from the tested doses of the extract has not been established. The drug shows pronounced hepatoprotective properties already at a dose of 10 mg / kg and in the future there is only a slight increase in this effect even with an increase in the dose of the extract by 10 times. This is confirmed by the results of histopathological studies of the liver of experimental animals. The introduction of dry extract of dry extract at a dose of 10 mg / kg against the background of mercuric chloride intoxication showed the absence of dystrophic changes in hepatocytes, and in the group of animals treated with the drug at a dose of 100 mg / kg, in addition, hepatocyte regeneration processes and the absence of signs of proliferation of intralobular connective tissue were observed ( Fig. 2, 3).

The introduction of dry extract of dry extract in both doses contributed to the preservation of the relative mass of the liver, spleen and thymus at the level of indicators of intact control (table 4).

Thus, a pronounced hepatoprotective effect of dry extract of dry extract was established on the model of acute mercuric chloride intoxication, and the effect of the drug was already apparent at a dose of 10 mg / kg.

A study on a comparative evaluation of the hepatoprotective effect of two drugs in acute mercuric chloride intoxication showed a slightly superior effect of the dry extract powder compared with carlsil. This was expressed by the fact that this effect in the extract occurred already in the initial period of the experiment (5 days) and persisted until its end, while carlsil prevented the toxic effect of mercuric chloride only by 10 days of the experiment.

The introduction of the studied drugs into the stomach of rats with acute toxic hepatitis completely prevented the death of animals, while in the control group of rats, only with mercuric chloride intoxication, the death rate was 26.9%. The studied drugs slowed down a sharp decrease in the body weight of animals, prevented a violation of protein, lipid and carbohydrate metabolism in the liver. At the same time, the level of total protein in the blood serum of rats receiving dry extract and carlsil against the background of intoxication of sublimate volodushka was not higher than 78.2 g / l compared with animals receiving sublimate - 86.4 g / l. The concentration of total cholesterol in the blood serum of animals (mercuric chloride) increased to 4.20-4.80 mmol / l (respectively on days 5 and 10 of the experiment) in rats treated with extracts and carlsilus, the corresponding indicator was at the level of 3.38-2 , 53 mmol / L. Similar results were obtained when analyzing the level of triglycerides. The introduction of extract extract and carcil at a dose of 10 mg / kg reduced the hypertriglyceridemia that was observed in animals receiving mercuric chloride. Under the influence of the studied drugs, the level of activity of serum enzymes, such as AcTA, AlTA, and alkaline phosphatase, decreased. LDH activity did not change significantly (table 5).

The introduction of dry extracts of dry carlsil and helped to maintain the relative mass of the liver at the level of intact control - 3.82 ± 0.32; pathology model (mercuric chloride 3 mg / kg) - 2.96 ± 0.18; dry extracts of dry + mercuric chloride - 3.61 ± 0.10; Karsil + mercuric chloride - 3.45 ± 0.10 (P <0.05).

Thus, the results of the studies confirmed the previously established pronounced hepatoprotective properties of dry golden extracts of the dry extract on the model of toxic hepatopathy caused by mercuric chloride intoxication, and indicate that the dry extract at a dose of 10 mg / kg is slightly higher in terms of the effectiveness of the hepatoprotective action of the above-mentioned liver pathology model. exceeded karsil (at a dose of 10 mg / kg) - the most widely used hepatoprotector currently used in medical practice.

The results obtained indicate the promise of a further more in-depth study of dry golden extracts of dry gold as a medicine for the treatment of liver diseases of various etiologies.

Claims (1)

A method of treating toxic hepatitis, characterized in that dry extracts of whey are injected into the patient’s stomach at a dose of 10 mg per kilogram of the patient’s weight during the period of exposure to toxic substances on the liver and for 5 days after this period, said extract containing phenolic aglycone flavonoids : luteolin - 0.3 ± 0.01 mg / g, quercetin - 13.3 ± 0.50 mg / g, camperol - 1.1 ± 0.10 mg / g, isoramnetin - 9.1 ± 0.30 mg / g; flavonoids: rutin - 6.2 ± 0.10 mg / g, hyperoside - 5.7 ± 0.10 mg / g, astragaline - 0.4 ± 0.01 mg / g; phenol carboxylic acids: ferulic acid - 657.4 mg / 100, caffeic acid - 83.9 mg / 100, 4-caffeoylquinic acid - 592.2 mg / 100, rosmarinic acid - 130.2 mg / 100; tannins in terms of (+) - catechin - 7.2% ± 0.5%; saponins in terms of oleanolic acid - 8.4% ± 0.3%; polysaccharides in terms of glucose - 1.80% ± 0.15%; ascorbic acid - 0.4 mg / g; essential amino acids: valine - 0.12%, lysine - 0.15%, threonine - 0.07%, histidine - 0.3%, arginine - 0.14%; essential amino acids: alanine - 0.15%, cysteine - 0.95%, glycine - 0.05%, tyrosine - 0.45%.

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