RU2491949C2 - Method of obtaining immunotropic preparation as base for medication "immunoflan" - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to pharmaceutical industry, in particular to method of obtaining immunotropic preparation. Method of obtaining immunotropic preparation by extraction of air-dry leaves of Lophanthus anisatus Benth with aqueous ethyl alcohol, heating, cooling, filtering, solvent evaporation, residue dissolving and extraction with chloroform, filtering and drying of sediment, under specified conditions.

EFFECT: method makes it possible to obtain efficient immunotropic medication.

3 tbl, 5 ex

Description

The invention relates to the field of medicine, specifically to pharmacology, and for the development of agents that improve, and in some cases completely normalize, the immune status of the body.

Currently, immunotropic agents of synthetic, plant and animal origin are known: tyrolone, arbidol, kagocel, cycloferon, purple coneflower and a number of adaptogens with a pronounced immunotropic effect (Rhodiola rosea, ginseng, linden flowers, pantocrine, etc.). It is known to use Lofant anise as a hepato-, cardioprotective, dermatological, urological agent in traditional medicine.

Known pharmacopoeial method of producing flavonoids [1], in which the crushed raw materials of St. John's wort grass is poured with purified water at room temperature in a ratio of 1:10, taken into account the coefficient of water absorption, insist on a boiling water bath for 15 minutes, cooled for 45 minutes, filtered, squeezing the raw materials, and water is added to the desired recovery volume. In the infusion obtained by the pharmacopeia method, the flavonoid content is 0.2%. [2].

The known method (patent No. 2158740 RU), in which air-dried raw materials of St. John's wort grass, crushed to particle sizes of 2-3 mm, is poured with purified water at room temperature, taken into account the coefficient of water absorption in the ratio 1: 6.6 and insist on boiling water bathhouse. The obtained extract is filtered through a cotton filter, then, in order to maximize purification from the accompanying substances, the extract is placed on a 5 cm diameter column with 6 g of a polyamide sorbent (TU 6-09-10-822-73) with a particle size of 0.1-0.25 mm . After passing through the column of all the liquid, the sorbent is treated with 150 ml of purified water in three doses. The completeness of purification of the extract is controlled visually in visible and UV light, as well as by a qualitative reaction with a 5% alcohol solution of aluminum chloride (III). To elute the polyphenols, 70% ethanol in an amount of 50 ml is used, the passage time through the column is 7-8 minutes, and the passage speed is 70-80 drops in 1 minute. The amount of the eluting mixture necessary for the complete desorption of flavonoids is determined experimentally. Ethanol from the collected eluate is distilled off at a temperature above 70 ° C, the extract is dried to constant weight. The output of flavonoids in this method is 0.4-0.9%.

The problem solved by this invention is to expand the arsenal of funds with immunotropic activity, because the question of expanding the group of immunotropic drugs is extremely urgent, forming a social order for the development and reproduction of drugs used in various fields of practical medicine, as well as a more effective way to obtain funds with a high yield of the product (mixture of flavonoids).

This task is achieved due to the fact that in the present invention, it is proposed to use a mixture of flavonoids obtained by extracting them from an aqueous-alcoholic extract of the leaves of anise Lophanthus anisatus Benth (varieties Astrakhan 100 and Astrakhan 101) with particle size 1-3 mm collected during flowering. The proposed method allows to obtain the yield of the product (mixture of flavonoids) during extraction in the amount of 3%.

The properties of the obtained mixture of anisic Lofant flavonoids as an immunotropic agent were studied in an experiment on laboratory animals in the background, as well as during cyclophosphamide immunosuppression. The presence of immunotropic activity was judged by the following indicators: total leukocyte count, leukocyte population, cellular (delayed-type hypersensitivity reaction (RGT) with a reaction index), humoral (passive hemagglutination reaction (RPHA) with an antibody titer) and phagocytic (latex) a test to study the phagocytic activity of peripheral blood neutrophils with the determination of the phagocytic index and phagocytic number) immunogenesis links. When stating HRGT and RPHA, sheep erythrocytes were used as an antigenic stimulus.

The implementation of the proposed method for producing a mixture of flavonoids, their use as an immunotropic agent is illustrated in the following examples.

Example 1. The method for producing an immunotropic agent is as follows: 50 g of air-dried raw materials of anise Lofant leaves with a particle size of 1-3 mm, collected during flowering, pour 500 ml of 60% aqueous ethanol solution and heated in a water bath for 2.5 hours The solution is cooled to 50 ° C and filtered, and the solvent is evaporated on a rotary evaporator (residual pressure 1-2 mm). The residue was dissolved in 200 ml of water heated to a temperature of 50 ° C and extracted with 50 ml of chloroform. The aqueous layer is left to stand for 24 hours at a temperature of 5 ° C. The precipitate is filtered off and dried in air. Obtain 1.2 g of a mixture of flavonoids Lofant anise: the yield was 3% by weight of air-dried raw materials.

Example 2. We studied the effect of a mixture of flavonoids Lofant anisic when administered orally at a dose of 75 mg / kg on the total number of leukocytes and leukocyte formula.

The experiment was conducted on sexually mature Wistar rats. The animals were divided into the following groups: control — individuals who received distilled water in an equivalent volume and experimental — individuals received a mixture of anisic lofant flavonoids with a 10-day administration of per os, at a dose of 75 mg / kg. The results are shown in tab. No. 1.

As can be seen from the table, the course introduction of a mixture of Lofant anisic flavonoids at a dose of 75 mg / kg is accompanied by an increase in the total number of leukocytes by more than 20%, changes in the leukocyte formula in the direction of the predominance of the segmented neutrophil fraction, which indicates an increase in non-specific immunoreactivity. The absence of an increase in the number of eosinophils suggests that the mixture of flavonoids of anise lofant does not show a sensitizing effect on the body of laboratory animals.

Example 3. We studied the effect of a mixture of flavonoids Lofant anisic when administered orally at a dose of 75 mg / kg on the cellular, humoral and phagocytic units of immunogenesis.

The experiment was conducted on sexually mature Wistar rats. The animals were divided into the following groups: control — individuals who received distilled water in an equivalent volume and experimental — individuals received a mixture of anisic Lofant flavonoids at 10 days per os, at a dose of 75 mg / kg. When stating HRGT and RPHA, sheep erythrocytes were used as an antigenic stimulus. The results are shown in tab. No. 2.

The data presented demonstrate that, under the influence of Lofant anisic flavonoids, the cellular response intensifies - the local HRT reaction rate increased by more than 35%. In addition, activation of phagocytosis processes was noted: the phagocytic index and phagocytic number significantly exceed the indices of intact animals, which, as in example 1, confirms the increase in nonspecific resistance. The introduction of a mixture of flavonoids of lofant anise to laboratory animals does not contribute to a change in the processes of antibody formation (tab. No. 2).

Example 4. We studied the ability of a mixture of flavonoids Lofant anisic when administered orally at a dose of 75 mg / kg to eliminate violations in the processes of leukopoiesis that develop with cyclophosphamide immunosuppression.

The experiment was conducted on sexually mature Wistar rats. The animals were divided into the following groups: control No. 1 (individuals that received distilled water in an equivalent volume), control No. 2 (animals with immunosuppression caused by a single intraperitoneal administration of cyclophosphamide at a dose of 150 mg / kg) and experimental (individuals with immunosuppression received per os a mixture of flavonoids Lofant anisic in a dose of 75 mg / kg in a course of 10 days). The results are shown in table No. 3.

As can be seen from the table, a single injection of cyclophosphamide at a dose of 150 mg / kg is accompanied by the development of severe cytopenia (the total number of leukocytes decreases by 90%), there is also a sharp shift of the leukocyte formula to the left due to an increase in the percentage of stab neutrophils. The introduction of a mixture of flavonoids in animals with experimental immunosuppression is accompanied by a restoration of the total number of leukocytes to 36% of the initial level and a tendency to normalize the leukocyte formula (tab. No. 3).

Example 5. We studied the ability of a mixture of flavonoids Lofant anisic when administered orally at a dose of 75 mg / kg to eliminate immune disorders that develop with cyclophosphamide immunosuppression.

The experiment was conducted on sexually mature Wistar rats. The animals were divided into the following groups: control No. 1 (individuals that received distilled water in an equivalent volume), control No. 2 (animals with immunosuppression caused by a single intraperitoneal administration of cyclophosphamide at a dose of 150 mg / kg) and experimental (individuals with immunosuppression received per os a mixture of flavonoids of Lofant anisic at a dose of 75 mg / kg in a course of 10 days). The results are shown in table No. 4.

The data presented in table No. 4 demonstrate that the induction of immunosuppression by a single administration of cyclophosphamide at a dose of 150 mg / kg is accompanied by an increase in the response to the corpuscular antigen (the HRT reaction rate increased by more than 5 times) with a simultaneous decrease in the titer of anti-erythrocyte antibodies (RPHA decreased by more than 2 times). The activation in this case of the RGTT index while suppressing antibody formation is due to increased activity of the T-suppressor immunity, which ultimately led to inhibition of the anti-erythrocyte immune response. When a mixture of Lofant anisic flavonoids was introduced against the background of the induced pathology, the local reaction was almost restored (more than 3 times) and the process of formation of anti-erythrocyte antibodies (more than 1.5 times) was strengthened compared to the group of animals with immunosuppression (tab. No. 4) .

Thus, the data obtained during preclinical trials, data on the immunotropic and immunotherapeutic properties of the Lofant anisic flavonoids mixture give grounds for using this product as a basis for the development of the new Immunoflan preparation, which can be produced in the form of dried raw materials, tinctures and tablets.

Literature.

1. The State Pharmacopoeia of the USSR XI, issue 2, p. 147.

2. Pharmacy, 1994., N1, p.30-34.

Claims (1)

A method of producing an immunotropic agent by extracting plant materials with ethyl alcohol, characterized in that the air-dried leaves of Lophanthus anisatus Benth (cultivars Astrakhan 100 or Astrakhan 101) with a particle size of 1-3 mm, collected during flowering, are poured 60 % aqueous ethanol solution based on 500 ml of 60% ethanol per 50 g of air-dried leaves of Lophanthus anisatus Benth, heated in a water bath for 2.5 hours, then the solution was cooled to 50 ° C and filtered, and the solvent was evaporated on a rotary evaporator to residual pressure of 1-2 mm Hg ., the residue is dissolved in 200 ml of water heated to a temperature of 50 ° C, and extracted with chloroform in an amount of 50 ml, the aqueous layer is left to stand for 24 hours at 5 ° C, the precipitate is filtered off and dried in air.