RU2406517C2 - Medication possessing immunostimulation action - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to chemical-pharmaceutical industry, namely to creation of medication, stimulating immunostimulating action, which represents liquid extract of marsh-mallow roots, obtained by method of repercolation with 40% ethanol.

EFFECT: medication extends arsenal of immunostimulating medications, amplifies humoral immune response, cell immune response and phagocytic activity of peripheral blood neutrophils.

3 tbl, 3 ex

Description

The invention relates to medicine, specifically to pharmacology, and relates to agents that affect the immune system.

Prescribing for therapeutic or prophylactic purposes in diseases associated with impaired immunity, drugs of a chemical or biological nature with immunotropic activity is called immunotherapy, and the drugs themselves can be divided into four large groups: immunomodulators, immunocorrectors, immunostimulants, immunosuppressants [9]. Immunostimulants - agents that enhance the immune response. Synthetic immunostimulants include levamisole, a number of peptide immunostimulants (thymogen, immunofan, etc.) [5]. Of the herbal preparations that stimulate the immune response, the most known are preparations based on purple coneflower (immunal, immunorm, Dr. Theiss tincture of coneflower) [8, 12]. Immunal and immunorm preparations contain juice obtained from freshly picked herbs of flowering Echinacea purpurea, Dr. Theiss - tincture of Echinacea leaves. The use of medicinal plants seems to be a promising direction for further development and improvement of immunorehabilitation methods, since drugs of natural origin do not possess toxic properties characteristic of synthetic drugs and do not cause an imbalance in the immune system [1, 2].

The aim of the invention is the expansion of the arsenal of immunostimulating agents.

This goal is achieved by using, as an immunostimulating agent, a liquid extract of the roots of Althea officinalis, obtained by repercolation using 40% ethanol.

The main biologically active substances of the roots of the marshmallow are polysaccharides, organic acids, tannins, and mineral salts. In modern medical practice, the roots of Althaea officinalis are used as anti-inflammatory, enveloping, expectorant drugs [7].

New in the invention is that the liquid extract of the roots of the marshmallow medicinal is first proposed for use as an immunostimulating agent.

The use of a liquid extract of the roots of Althaea officinalis, obtained by repercolation with 40% ethanol, as an immunostimulating agent is not described in the literature. The use for a new purpose was made possible thanks to the discovery of new properties in him, namely, stimulation of the humoral immune response, cellular immune response and nonspecific resistance. This property does not explicitly follow for a person skilled in the art. The liquid extract of Althea officinalis roots can be used in patients with immune deficiency associated with acute and chronic infectious diseases, against the background of radiation therapy and treatment with cytostatics.

Thus, this technical solution meets the criteria of the invention: "novelty", "inventive step", "industrial applicability".

New properties of the liquid extract of Althea officinalis roots were discovered through experimental studies.

To establish the immunostimulating properties of the liquid extract of the marshmallow roots, its effect on the humoral immune response, the cellular immune response, and also on the nonspecific resistance of the body was studied. A liquid extract of the roots of Althaea officinalis was obtained by repercolation using 40% ethanol. To obtain the extract: 0.450 kg of air-dried raw material of the marshmallow root is crushed on a grinder of the type of grass cutting to particles 3-4 mm in size. Then, the crushed raw material is freed from dust (particles less than 0.25 mm) using a sieve with an opening side of 0.25 mm and 0.400 kg of finished raw material is obtained. Extraction is carried out by repercolation in 4 percolators. For this, 0.100 kg of crushed and dust-free raw materials are placed in percolator 1 and 500 ml of 40% ethanol are poured to form a “mirror” and left for 12 hours at a temperature of 18-20 ° C. After 12 hours of infusion, 500 ml of extract is drained from percolator 1 and 0.100 kg of finished raw material is poured into percolator 2 until a “mirror” is formed, while an aliquot of fresh extractant is fed into percolator 1. After 12 hours of infusion, 500 ml of extract are drained sequentially from percolators 1 and 2. In this case, percolator 1 is replenished with fresh extractant, and percolator 2 is removed from percolator 1, percolator 3 is removed from percolator 2. Thus, the operation is repeated until the fourth percolator every 12 hours. After 12 hours of infusion, the extracts are sequentially transferred in a volume of 500 ml from percolator 1 to percolator 4, from percolator 4 is poured into a separate flask 100 ml of extraction - the finished product of the raw material extraction stage, and from percolator 1, the extraction residues (by-product) are poured into a separate container after its remaining ethanol in the feed is washed with twice the amount of water (recuperate). Percolator 1 is freed from waste materials. Then, in the same order, every 12 hours, the extracts are moved, excluding the raw materials from percolator 2, 3 from work, and the finished product (liquid extract) is drained from percolator 4. In the stopping period, 100 are drained from the last percolator (4) after 12 hours ml of extraction and combined with the extracts obtained during the working period, and receive 400 ml of a liquid extract, which is standing at a temperature of 3-5 ° C for 3-4 days and filtered through 3 layers of calico. Before administration, the extract is subjected to dealcoholation by incubation in a thermostat at a temperature of 37 ° C for 30 minutes. The standardization of the extract was carried out on a dry residue (GF X1, volume 2, p. 160).

As a comparison drug used tincture of Echinacea purpurea ("GalenoPharm", St. Petersburg, is approved for use in Russia, reg. No. 000167.01-2000).

Experiments to study the effect of marshmallow root extract (EA) on the humoral immune response were performed on 60 CBA / CaLac male mice weighing 18-20 g. Animals were divided into 3 groups: 20 mice were injected for 5 days for 0.2 ml of distilled water intragastrically (control); 20 mice received dealcoholized tincture of Echinacea purpuric (EP), which was administered to animals for 5 days at a dose of 50 mg / kg in a volume of 0.2 ml intragastrically distilled water (experiment 1) - prototype; 20 mice received dealcoholized EA, which was administered to animals for 5 days at a dose of 50 mg / kg in a volume of 0.2 ml of intragastric distilled water (experiment 2) - the proposed tool. When studying the humoral immune response, mice of all groups were immunized with ram erythrocytes (EB) - 6 × 10 ⁸ / mouse (once intraperitoneally in a volume of 0.2 ml) at the end of a 5-day course of administration of EA, EP, or distilled water. On the 4th and 7th day after immunization, the total spleen cellularity (ACS) was determined by conventional methods [4], as well as the relative (%) and absolute (× 10 ⁶ ) number of antibody-forming cells (AOC) in the spleen of mice using the Cunningham method [ 11] and the titer of antibodies IgM and IgG in serum using a standard hemagglutination reaction (RGA) [3].

Experiments to study the effect of the extract of marshmallow roots on the cellular immune response were carried out on 18 male mice of the CBA / CaLac line weighing 18-20 g. Animals were divided into 3 groups: 6 ml of distilled water were administered to 6 mice for 5 days intragastric (control); 6 mice received dealcoholized tincture of Echinacea purpuric (EP), which was administered to animals for 5 days at a dose of 50 mg / kg in a volume of 0.2 ml intragastrically distilled water (experiment 1) - prototype; 6 mice received dealcoholized extract of the roots of Althea officinalis (EA), which was administered to animals for 5 days at a dose of 50 mg / kg in a volume of 0.2 ml of intragastric distilled water (experiment 2) - the proposed tool. To assess the effect of drugs on the cellular immune response, a delayed-type hypersensitivity reaction (HRT) was used [9]. 24 hours after the last administration of the extracts, the animals were sensitized with ram erythrocytes subcutaneously 1 × 10 ⁷ in a volume of 100 μl. A resolving dose of antigen (1 × 10 ⁸ in a volume of 20 μl) was administered on day 5 after sensitization under the aponeurotic plate of one of the hind limbs. In the contralateral paw, physiological saline in equal volume was injected as a control. The intensity of the inflammatory reaction was taken into account 24 hours after the administration of the resolving dose of the antigen. The inflammation index (VI) was determined by the mass difference between the experimental and control paws.

Experiments to study the effect of the extract of marshmallow roots on nonspecific resistance of the organism were carried out on 18 male mice of the CBA / CaLac line weighing 18-20 g. Animals were divided into 3 groups: 6 ml of distilled water were administered to 6 mice for 5 days intragastric (control); 6 mice received dealcoholized tincture of Echinacea purpurea (EP), which was administered to animals for 5 days at a dose of 50 mg / kg in a volume of 0.2 ml intragastrically distilled water (experiment 1) - prototype; 6 mice received dealcoholized extract of the roots of Althea officinalis (EA), which was administered to animals for 5 days at a dose of 50 mg / kg in a volume of 0.2 ml of intragastric distilled water (experiment 2) - the proposed tool. Non-specific resistance was assessed by studying the phagocytic activity of peripheral blood neutrophils in the latex particle absorption test [4].

Statistical processing of the results was carried out using the statistical software package Statistica for Windows (version 5.0) with a preliminary assessment of the normality of the distribution and using the Student t-test.

The results confirming the immunostimulating properties of the liquid extract of the roots of Althea officinalis.

Example 1. Control mice were injected within 5 days with 0.2 ml of distilled water intragastrically. The mice of the 1st experimental group were injected with a dealcoholized tincture of Echinacea purpurea (EP) for 5 days at a dose of 50 mg / kg in a volume of 0.2 ml of intragastric water. The mice of the 2nd experimental group were injected with a dealcoholized extract of the roots of Althea officinalis for 5 days at a dose of 50 mg / kg in a volume of 0.2 ml of intragastric water. Then, mice of all groups were immunized with sheep erythrocytes - 6 × 10 ⁸ / mouse (once intraperitoneally in a volume of 0.2 ml). On the 4th and 7th day after immunization, the total number of splenocytes (ACS) was determined, as well as the relative (%) and absolute (× 10 ⁶ ) number of antibody-forming cells (AOC) in the spleen of mice and the titer of IgM and IgG antibodies in blood serum . The course introduction of Echinacea purpurea tincture led to a significant increase in the relative number of antibody-forming cells on days 4 and 7 after immunization and the absolute number of antibody-producing cells on day 4 of the experiment compared with the control (Table 1). In addition, an increase in titer of class G immunoglobulins was noted for 4 days. An analysis of the results presented in Table 1 shows that a 5-day course administration of the extract of Althea officinalis roots increased the total cellularity of the lymphoid organ, the absolute number of antibody-forming cells and the IgM titer relative to the animals of the control group on the 4th day of the experiment. Compared to mice treated with echinacea tincture, the number of antibody producing cells did not statistically significantly increase, but their functional activity increased, which was expressed in an increase in titers of specific hemagglutinins. Thus, the titer of IgM antibodies in this experimental group was significantly higher at all stages of the study.

Table 1 The effect of the extract of the roots of Althea officinalis (EA) and tincture of Echinacea purpurea (EP) on the humoral immune response of CBA / Ca Lac mice, (X ± m) Group of mice Indicators Study Dates 4 days 7 days Control (EB) ACS, 10 ⁶ / organ 159.20 ± 16.50 149.8 ± 7.71 AOK,% 7.36 ± 0.10 7 ± 0.21 AOK, 10 ⁶ / organ 11.72 ± 1.23 10.47 ± 0.57 IgM, log ₂ T 2 ± 0.32 2.2 ± 0.32 IgG, log ₂ T 8.4 ± 0.75 7.30 ± 0.50 Experience 1 (EP + EB) ACS, 10 ⁶ / organ 190.00 ± 22.67 163.00 ± 10.85 AOK,% 11.44 ± 0.49 * 5.64 ± 0.19 * AOK, 10 ⁶ / organ 21.89 ± 3.20 * 9.22 ± 0.77 IgM, log ₂ T 2 ± 0.32 1.2 ± 0.49 IgG, log ₂ T 10.4 ± 0.25 * 10 ± 0.32 * Experience 2 (EA + EB) ACS, 10 ⁶ / organ 193.40 ± 8.47 * 171.50 ± 25.47 AOK,% 7.44 ± 0.76 # 7.00 ± 0.41 # AOK, 10 ⁶ / organ 14.26 ± 1.19 * # 11.98 ± 1.73 # IgM, log ₂ T 4.00 ± 0.32 * # 4.25 ± 0.25 # IgG, log ₂ T 7.60 ± 0.40 # 7.50 ± 0.50 # Notes: here and in Tables 2, 3 * - differences with the corresponding control values are significant; # - differences with the corresponding values of the group “experience I” are significant.

Example 2. Control mice were injected within 5 days with 0.2 ml of distilled water intragastrically (control); the animals of the first experimental group received dealcoholized tincture of Echinacea purpurea (EP), which was administered to the animals for 5 days at a dose of 50 mg / kg in a volume of 0.2 ml of distilled water intragastrically; mice of the 2nd experimental group received a dealcoholized extract of the roots of Althea officinalis (EA), which was administered to the animals for 5 days at a dose of 50 mg / kg in a volume of 0.2 ml of intragastrically distilled water. 24 hours after the last administration of the extracts, the animals were sensitized with ram erythrocytes subcutaneously 1 × 10 ⁷ in a volume of 100 μl. A resolving dose of antigen (1 × 10 ⁸ in a volume of 20 μl) was administered on day 5 after sensitization under the aponeurotic plate of one of the hind limbs. In the contralateral paw, physiological saline in equal volume was injected as a control. The intensity of the inflammatory reaction was taken into account 24 hours after the administration of the resolving dose of the antigen. The inflammation index (VI) was determined by the mass difference between the experimental and control paws. The experimental results shown in Table 2 indicate a significant stimulating effect of the extract of the roots of Althea officinalis on the formation of a clone of antigen-specific T-lymphocytes.

table 2 The effect of the extract of the marshmallow roots medicinal and tincture of Echinacea purpurea (EP) on the cellular immune response of CBA / Ca Lac mice, (X ± m) Groups of mice The control Experience 1 (EP) Experience 2 (EA) Inflammation index 18.05 = 1.04 18.53 = 1.96 28.63 = 2.85 * #

Example 3. Control mice were injected within 5 days with 0.2 ml of distilled water intragastrically. The mice of the 1st experimental group were injected with a dealcoholized tincture of Echinacea purpurea (EP) for 5 days at a dose of 50 mg / kg in a volume of 0.2 ml of intragastric water. The mice of the 2nd experimental group were injected with a dealcoholized extract of the roots of Althea officinalis (EA) for 5 days at a dose of 50 mg / kg in a volume of 0.2 ml of intragastrically distilled water. After 24 hours, the phagocytic activity of peripheral blood neutrophils of mice of all groups was evaluated by the ability of these cells to absorb latex particles (particle suspension concentration of 60 million / ml); the percentage of neutrophils that absorbed the particles (phagocytic index - PH) and the average number of particles absorbed by one cell (phagocytic number - PS) were taken into account.

The results of these experiments presented in Table 3 show that marshmallow root extract significantly stimulates the absorption activity of peripheral blood neutrophils (compared with the control group) on the 4th day of the experiment. By day 7, the phagocytic activity of the studied cells in animals of the 2nd experimental group was significantly higher than in mice of the control and 1st experimental groups, both by increasing the proportion of neutrophils capable of absorbing latex (PI) and by increasing the number of absorbed each cell particles (PS).

Table 3 The effect of marshmallow root extract (EA) and Echinacea purpurea (EP) tincture on the phagocytic activity of peripheral blood neutrophils from CBA / Ca Lac mice (X ± m) Group of mice 4 days 7 days FI (%) Ff FI (%) Ff The control 22 ± 1.22 2.06 ± 0.23 17.25 ± 1.80 3.79 ± 0.62 Experience 1 (EP) 32 ± 3.91 * 4.72 ± 0.45 * 23.8 ± 2.13 4.52 ± 0.23 Experience 2 (EA) 18.4 ± 1.99 # 5.81 ± 0.68 * 32.2 ± 1.93 * # 6.76 ± 0.35 * #

Thus, the liquid extract from the roots of the medicinal marshmallow exhibits immunostimulating properties, which is manifested in an increase in the humoral immune response (increase in the total cellularity of the lymphoid organ, the number of antibody-forming cells and the titer of specific hemagglutinins), the cellular immune response (stimulation of the formation of a clone of antigen-specific T-lymphocytes ) and phagocytic activity of peripheral blood neutrophils, comparable in severity with tincture of Echinacea purpurea.

Sources of literature

1. Amosova E.N., Zueva E.P., Razina T.G., Krylova S.G. Medicinal plants as a means of additional therapy for the treatment of tumors // Bull. expert. biol. - 2003. - App. 2. - S. 24-34.

2. Vershinina S.F., Potyavina E.V. The use of natural bioregulators in oncology // Questions of oncology. - 2003. - T. 49. - No. 2. - S.145-151.

3. Immunological methods. / Ed. G. Fremel. - M .: Medicine, 1987. - S.211-218.

4. Immunology: Workshop. / E.U. Pasteur, V.V. Ovod, V.K. Pozur, N.E. Vikhot. - K .: High school. Publishing House at Kiev. Un-te, 1989 .-- S.202-204.

5. Leskov V.P. Immunostimulants // Allergy, asthma and clinical immunology. - 1999. - No. 4. - S.12-25.

6. Linov B.M., Barnaulov O.D., Krylov V.A. and other new drugs from plants in Siberia and the Far East. - Tomsk, 1986. - S.89-90.

7. Polozhy A.V., Goldberg E.D., Gureeva I.I. Medicinal plants of Siberia. Tomsk: Publishing House Tom. University, 1995 .-- 325 p.

8. Samorodov V.N., Pospelov S.V., Moiseeva G.F. et al. Phytochemical composition of representatives of the genus Echinacea (Echinacea Moench.) and its pharmacological properties (review) // Chemical and Pharmaceutical Journal. - 1996. - No. 4. - S. 32-37.

9. Khaitov P.M., Gushchin I.S., Pinegin B.V. et al. An experimental study of the immunotropic activity of pharmacological preparations // Vedomosti Pharm. Committee. - 1999. - No. 1. - S. 31-36.

10. Khaitov P.M., Pinegin B.V. Immunomodulators and some aspects of their clinical use // Clinical medicine. - 1996. - T.74. - No. 8. - S.7-12.

11. Curnningham A.J. A method of increased sensitivity for detecting single anti-bodyforming cells // Nature. - 1965. - V.207, No. 5001. - P.1106-1107.

Claims (1)

A tool with an immunostimulating effect, characterized in that it is a liquid extract of the roots of the marshmallow obtained by repercolation with 40% ethanol.