RU2259208C2 - Method for preparing agent eliciting hepatoprotective activity - Google Patents

Abstract

FIELD: medicine, phytotherapy, pharmacy.

SUBSTANCE: invention relates to medicinal agents and can be used in preparing agent eliciting hepatoprotective activity. Method involves extraction of hexapetalous dropwort (Filipendula) roots and rhizomes with water at heating in the ratio raw : extractant = 1:(10-25) for 30 min, filtration, evaporation up to 1/3-1/5 of the parent volume and the following purification by cooling the evaporated concentrate, precipitation of the end product from its by addition of three-fold volume of 96% followed by settling, filtering off the precipitate and its successive washing out with 96% and ether. Invention provides enhancing the specific activity and pharmacological effect of agent.

EFFECT: improved preparing method.

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Description

The invention relates to the pharmaceutical industry, and in particular to a method for producing biologically active compounds from plant materials.

There is a method of obtaining the sum of flavonoids from the fruits of milk thistle (Silibor preparation), which consists in extracting raw materials with ethyl alcohol, evaporating the extract, dissolving the residue in ethyl alcohol, treating with carbon tetrachloride and extracting with a mixture of chloroform-ethyl alcohol in a ratio of 2: 1 [1] .

However, flavonoids contribute to the accumulation of ascorbic acid in the liver and at the same time slow down its excretion from the body. Flavonoids enhance the enzymatic oxidation of ascorbic acid to dehydroascorbic acid and thus protect it from further oxidation and excretion from the body. Dehydroascorbic acid dissolves in fats better than ascorbic acid and, penetrating through membranes, accumulates more intensively in organs [2]. And, as a result, the drug obtained in a known manner has a low regenerative activity of hepatocytes. Side effects are also possible - skin allergic reactions [3, 4].

The purpose of the invention is the enhancement of specific activity.

This goal is achieved by the fact that in the method of obtaining funds with hepatoprotective activity, rhizomes and roots of meadowsweet are used as raw materials, extraction is carried out with water, a water-soluble polysaccharide complex is precipitated with ethanol.

A comparative analysis of the proposed solution with the prototype shows that the claimed method differs from the known one in that rhizomes and roots of meadowsweet are used as raw materials, extraction is carried out with distilled water when heated in the ratio of raw material: extractant 1: 10-1: 15 for 30 minutes, the extract is evaporated in vacuo to 1 / 3-1 / 5 volume, the water-soluble polysaccharide complex is precipitated with a three-fold volume of 96% ethanol, filtered off, purified and dried.

Thus, the claimed method meets the criteria of the invention of "novelty." Known technical solutions [1], in which obtaining hepatoprotective drugs consists in the use of other types of medicinal plants. However, with the above disadvantages, the goal that is specified in the claimed technical solution is not achieved. This allows us to conclude that it meets the criterion of "significant differences".

A method of obtaining funds with hepatoprotective activity, as follows. Dried and crushed powder with a particle size of 1-3 mm, the raw materials of the meadowsweet of sixfold are quantitatively placed in a round bottom flask, filled with hot distilled water (90-95 ° C) in a ratio of 1: 10-1: 15 and extracted in a boiling water bath under reflux for 30 minutes The extract is filtered through cotton, evaporated in vacuo to 1 / 3-1 / 5 volume, cooled. A triple volume of 96% ethyl alcohol is added to the obtained extract, sedimented for 24 hours, the precipitated water-soluble polysaccharide complex is filtered off, purified with 96% ethanol, ethyl ether and dried at room temperature.

It was experimentally established that the resulting product has a pronounced hepatoprotective activity.

Experimental studies were carried out on 38 Wistar rats weighing 120-160 g. Animals were divided into 4 groups.

Group 1 - intact.

In animals of the 2nd group, toxic liver damage was caused by 5-fold (with an interval of 24 hours) intramuscular injection of a 50% solution of carbon tetrachloride (CCl ₄ ) in peach oil at a rate of 0.3 ml per 100 g of weight.

Rats of the 3rd group were injected with Silibor at the rate of 10 mg per 100 g of mass orally 7 times with an interval of 24 hours against the background of CCl ₄ poisoning (5 times, every 24 hours). After the 5th administration of CCl _{4, the} animals received the drug 2 more times.

Animals of the 4th group received the remedy from the rhizomes and roots of the meadowsweet of Hexavail (1 ml per 100 g of mass) 7 times orally with an interval of 24 hours while CCl ₄ poisoning. The latter was administered 5 times with the drug, then 2 times the animals received only the drug.

All rats were euthanized by dissection of the jugular vein under ether anesthesia. Slaughter was carried out between 8-9 a.m. to exclude the influence of the daily periodicity of mitotic activity on the result of the experiment.

To study the regeneration processes, 1.0 × 1.0 × 1.5 cm pieces of the liver were taken, which were fixed in neutral formalin, after which they were embedded in paraffin to prepare sections 5–7 μm thick, stained with hematoxylin and eosin.

According to the preparations obtained, several morphological indicators characterizing the proliferative activity of hepatocytes were calculated (Sarkisov, 1977):

- the number of mitotically dividing cells and, on this basis, the mitotic index as the ratio of the number of mitoses to the total number of cells viewed;

- the number of binuclear cells, and the index of binuclear cells was determined;

- the diameter of the nuclei of hepatocytes.

All obtained results were processed using methods of variation statistics (Strelkov, 1966). The significance of differences in average values was evaluated by Student's criterion (Lakin, 1972).

The introduction of CCl ₄ caused a significant increase in the processes of reparative regeneration, as evidenced by the increase:

- the number of mitoses in hepatocytes from 0.36 to 0.86%;

- the diameter of the nuclei of hepatocytes from 5.35 to 7.02 microns;

- the index of binuclear cells from 18.9 to 22.1 (table. 1).

Simultaneous administration of Silibor to experimental rats leads to a significant increase in the number of mitoses in hepatocytes, but practically does not affect other indicators of regeneration (Table 1).

Table 1
Comparative pharmacological evaluation of herbal preparations No. gr. A drug Count alive. Dose Mitotic index,% Bin Cell Index Hepatocyte core diameter, microns 1 - 10 - 0.36 ± 0.036 18.9 ± 1.82 5.35 ± 0.36 2 - nine - 0.86 ± 0.062 ^{* 1} 22.1 ± 0.83 7.02 ± 0.14 3 Silibor nine one hundred
mg / kg 1.05 ± 0.176 ^{* 1.2} 22.4 ± 2.54 ^{* 1} 7.82 ± 0.36 ^{* 1} 4 Meadowsweet polysaccharides 10 10
ml / kg 1.63 ± 0.192 ^{* 1-3} 25.1 ± 1.08 ^{* 1-3} 8.29 ± 0.27 ^{* 1-3} Note: asterisk indicates significant differences; the numbers next to the asterisk indicate in relation to which group these differences are significant.

The use of a water-soluble polysaccharide complex from the rhizomes and roots of the meadowsweet of the six-petals significantly accelerates the processes of reparative regeneration at the cellular and intracellular levels, surpassing the official preparation Silibor in this regard.

Example 1. 100 g of pre-dried and pulverized powder with a particle size of 1-3 mm raw six-planted meadowsweet is placed in a round bottom flask, pour 1000 ml of hot distilled water (90-95 ° C) and extracted in a boiling water bath under reflux for 30 minutes. The extract is filtered, evaporated in vacuo to 330 ml, cooled. 1000 ml of 96% ethanol are poured onto the cooled extract, the resulting mixture is shaken vigorously and left to stand at room temperature for 24 hours. The precipitated water-soluble polysaccharide complex is filtered off through a paper filter, washed with 200 ml of 96% ethanol and 100 ml of ethyl ether, and dried in air. .

Pharmacological activity:

- mitotic index - 1.57 ± 0.188%;

- index of binuclear cells - 24.7 ± 2.03;

- the diameter of the hepatocyte nucleus is 8.01 ± 0.32 μm.

Example 2. 100 g of pre-dried and pulverized powder with a particle size of 1-3 mm raw six-petaled meadowsweet is placed in a round bottom flask, pour 1200 ml of hot distilled water (90-95 ° C) and extracted in a boiling water bath under reflux for 30 minutes. The extract is filtered, evaporated in vacuo to 300 ml, cooled. 900 ml of 96% ethanol are added to the cooled extract, the resulting liquid is vigorously shaken and left to stand at room temperature for 24 hours. The precipitated water-soluble polysaccharide complex is filtered off through a paper filter, washed with 200 ml of 96% ethanol and 100 ml of ethyl ether, and dried in air. .

Pharmacological activity:

- mitotic index - 1.52 ± 0.143%;

- index of binuclear cells - 25.4 ± 1.63;

- the diameter of the hepatocyte nucleus is 8.08 ± 0.22 μm.

Example 3. 100 g of pre-dried and pulverized powder with a particle size of 1-3 mm raw six-petaled meadowsweet is placed in a round bottom flask, filled with 1500 ml of hot distilled water (90-95 ° C) and extracted in a boiling water bath under reflux for 30 minutes. The extract is filtered, evaporated in vacuo to 300 ml, cooled. 900 ml of 96% ethanol are added to the cooled extract, the resulting liquid is vigorously shaken and left to stand at room temperature for 24 hours. The precipitated water-soluble polysaccharide complex is filtered off through a paper filter, washed with 200 ml of 96% ethanol and 100 ml of ethyl ether, and dried in air. .

Pharmacological activity:

- mitotic index - 1.65 ± 0.188%;

- index of binuclear cells - 24.9 ± 2.13;

- the diameter of the hepatocyte nucleus is 8.31 ± 0.29 microns.

Thus, the increase in the specific activity of the agent obtained by the above method is expressed in an 1.6-fold increase in the mitotic index; binuclear cell index 1.2 times; the diameter of the hepatocyte nuclei is 1.2 times compared with the prototype.

LITERATURE

1. A.S. USSR No. 603386, A 61 K 35/78, 1978.

2. Herbal medicines / Ed. N.P. Maksyutina. - Kiev: 3 health, 1985, p. 85-102.

3. Mashkovsky M.D. Medicines: In 2 volumes. T. 1, M .: Medicine, 1987, p. 516.

4. Handbook of Clinical Pharmacology and Pharmacotherapy / Ed. I.S. Chekman, A.P. Peleschuk, O.A. Pyatak. Kiev: Health, 1987, p.309.

Claims (1)

A method of obtaining a agent having hepatoprotective activity by extracting plant materials with a solvent by heating, evaporation, cleaning and drying, characterized in that, in order to increase specific activity, rhizomes and roots of six-petalled meadowsweet (Filipendula hexapetala G) are used as raw materials, extraction is carried out with water when the ratio of raw materials: extractant 1: 10-15 for 30 min, the resulting extract is filtered, evaporated to 1 / 3-1 / 5 volume, and purification is carried out by cooling the evaporated concentrate, precipitating from it spruce product by adding three times the volume of 96% ethanol, sedimenting, filtering the precipitate, washing it successively with 96% ethanol and ether.