Medication possessing immunostimulation action

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to chemical-pharmaceutical industry, namely to creation of medication, stimulating immunostimulating action, which represents liquid extract of burdock greater and woolly burdock roots, taken in ratio 1:1, obtained by method of repercolation with 40% ethanol.

EFFECT: medication extends arsenal of immunostimulating medications, amplifies humoral immune response, phagocytic activity of peripheral blood neutrophilsand peritoneal macrophages.

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

Currently, in the treatment of chronic, indolent and recurrent diseases apply a number of drugs that enhance the immune response [6, 10]. To synthetic Immunostimulants include levamisole, methyluracil, stronger, a number of peptide Immunostimulants (timogen, immunofan and others) [5]. However, the use of preparations obtained by the method of chemical synthesis, it is often not possible due to their precise and weight side effects, in particular the development of allergic, autoimmune and lampropeltis diseases. Herbal drugs that stimulate the immune response, the most famous drugs on the basis of Echinacea purpurea (immunal, immuderm, doctor of Tiss tincture Echinacea) [7, 8, 12]. Drugs immunal and immuderm contain the juice obtained from freshly harvested herbs flowering Echinacea purpurea, Doctor of Tiss - infusion of leaves of Echinacea. The use of medicinal plants is a promising direction for further development, the improvement of immunorehabilitation, as the drugs of natural origin do not have toxic properties characteristic of synthetic drugs, and do not cause imbalance to the system of immunity [1, 2].

The aim of the invention is to expand the Arsenal of immunostimulatory funds.

This goal is achieved by the use as an immunostimulating agent liquid extract of the roots of burdock and felt taken in the ratio 1:1, obtained by the method of repercolation with 40% ethanol.

Standardization of the extract was performed according to the dry residue (GF X1, volume 2, str). The major biologically active substances roots of burdock and felt are polysaccharides (inulin to 45%), containing, fatty acids, salts of potassium, calcium and magnesium. In modern medical practice, the roots of burdock and felt are used as anti-inflammatory, enveloping and extraordinary means [5].

New in the present invention is that the liquid extract of the roots of burdock and felt produced using raw materials in a ratio of 1:1, using 40% ethanol first proposed for use as an immunostimulating agent.

The liquid extract of the roots of burdock and felt obtained by the method of repercolation using 40% ethanol using raw materials in a ratio of 1:1 as an immunostimulating agent are not described in literature. Application for a new appointment was made possible through the detection is its new properties, namely stimulation of the humoral immune response and nonspecific resistance. This property is not explicitly derived for the expert from the prior art. Liquid extract of the roots of burdock and felt can be used in patients with chronic, indolent and recurrent infectious diseases, radiation therapy and treatment with cytostatics.

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

New properties of liquid extract from the roots of burdock and felt were discovered through experimental research.

To establish the immunostimulatory properties of liquid extract from the roots of burdock and felt taken in the ratio 1:1, and obtained by the method of repercolation using 40% ethanol was studied its effect on humoral immune response and nonspecific resistance of the organism. Liquid extract of the roots of burdock and felt were obtained by the method of repercolation using 40% ethanol in the ratio of raw materials used 1:1. To extract: 0,450 kg of air-dry raw burdock shredded in a shredder type traverese to a particle size of 3-4 mm, Then the crushed material using sieves with the side holes of 0.25 mm salt is bidaut from dust (particles less than 0.25 mm) and receive 0,400 kg of raw material. Extraction was carried out by the method of repercolation 4 percolator. For this 0,100 kg powdered and freed from dust raw material is placed in percolator 1 and pour 500 ml of 40% ethanol before the formation of the "mirror" and leave for 12 hours at a temperature of 18-20°C. After 12 hours infusion of percolator 1 is drained 500 ml extract and pour them 0,100 kg of raw material in percolator 2 before the formation of the "mirror" with simultaneous feeding percolator 1 aliquot quantity of fresh extractant. After 12 hours of infusion of percolation 1 and 2 merge successively with 500 ml of extraction. Thus percolator 1 is replenished with fresh extractant, and percolator 2 - extract from percolator 1, percolator 3 - extract from percolator 2. Thus, repeating the operation until the fourth percolator every 12 hours After 12 hours of infusion perform sequential shifting of the extraction in 500 ml of percolator 1 to percolator 4, percolator 4 is poured into a separate flask 100 ml extraction - finished product stage extraction of raw materials and percolator 1 poured the remains of the extraction (by-product) in a separate container, and then the rest in raw ethanol wash twice the amount of water (recuperate). Percolator 1 release from the waste of raw materials. Next, in the same order every 12 hours to produce shifting of the extraction, excluding the tea from raw materials from percolator 2, 3, and produce a discharge of the finished product (liquid extract) from percolator 4. In the stopping period of the last percolator (4) for 12 hours, then poured 100 ml of extract and combine with extracts obtained in the working period, and receive 400 ml of liquid extract, which defend at a temperature of 3-5°C for 3-4 days and filtered through 3 layers of calico. Before the introduction of the extract is subjected to dealcoholization by keeping thermostat at 37°C for 30 minutes.

Standardization of the extract was performed according to the dry residue (GF XI, volume 2, str). As the comparison drug used tincture of Echinacea purpurea (Galenofarm", Saint-Petersburg, approved for use in Russia, reg. No. 000167.01-2000).

Experiments to study the effect of the extract of the roots of burdock and felt (E), taken in the ratio 1:1, and obtained by the method of repercolation using 40% ethanol, on the humoral immune response were carried out on 60 mice male CBA/CaLac weighing 18-20, Animals were divided into 3 groups: 20 mice were administered for 5 days in 0.2 ml of distilled water intragastrically (control); 20 mice received dealcoholizing 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 (experiment 1) prototype; 20 mice in which Uchali dealcoholizing E, introduced animals within 5 days at a dose of 50 mg/kg in a volume of 0.2 ml of distilled water intragastrically (experiment 2) - the proposed remedy. In the study of humoral immune response of mice of all groups were immunized with sheep erythrocytes (DL) - 6×108/mouse (once intraperitoneally in a volume of 0.2 ml) at the end of the 5-day course of introduction of E, EP or distilled water [9]. On the 4th and 7th days after immunization were determined by standard methods [4] the total cellularity of the spleen (ACS)and relative (%) and absolute (×106) the number of antibody productive cells (AFC) in the spleen of mice according to the method of Cunningham [11] and antibody titer IgM and IgG in the serum using standard reaction of haemagglutination (DSA) [3].

Experiments to study the effect of the extract of the roots of burdock and felt taken in the ratio 1:1, and obtained by the method of repercolation using 40% ethanol, nonspecific resistance of the organism were carried out on 30 mice male CBA/CaLac weighing 18-20, Animals were divided into 3 groups: 10 mice were administered for 5 days in 0.2 ml of distilled water intragastrically (control); 10 mice received dealcoholizing 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 intragastric (experiment 1) prototype; 10 mice policeoccasionally extract of the roots of burdock and felt (E), introduced animals within 5 days at a dose of 50 mg/kg in a volume of 0.2 ml of distilled water intragastrically (experiment 2) - the proposed remedy. Evaluation of nonspecific resistance was conducted by investigating the phagocytic activity of neutrophils in peripheral blood and peritoneal macrophages in test uptake of latex particles [4].

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

Results confirming immunostimulatory properties of liquid extract from the roots of burdock and felt taken in the ratio 1:1, and obtained by the method of repercolation with 40% ethanol.

Example 1. Control mice were administered for 5 days in 0.2 ml of distilled water intragastrically. Mice of the 1st experimental group were injected dealcoholizing tincture of Echinacea purpurea (EP) 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 were injected dealcoholizing extract of the roots of burdock and felt (E) within 5 days at a dose of 50 mg/kg in a volume of 0.2 ml of distilled water intragastrically. Then the mice of all groups were immunized with sheep erythrocytes - 6×108/mouse (once inside rushino in a volume of 0.2 ml). On the 4th and 7th days after immunization were determined by the total cellularity of the spleen (ACS)and relative (%) and absolute (×106) the number of antibody productive cells (AFC) in the spleen of mice and antibody titer IgM and IgG in serum. Course introduction tinctures of Echinacea purpurea resulted in a significant increase in the relative amount of antibody productive cells on the 4th and 7th day after immunization and the absolute number of antitelomerase on the 4th day of the experiment compared with the control (table 1). In addition, there was an increase in the titer of immunoglobulin G on the 4th day. Analysis of the results presented in table 1, shows that exchange rate 5-day administration of the extract of the roots of burdock and felt led to increase in the total cellularity of lymphoid organ, the relative and absolute number of antibody productive cells and IgM titer relative to animals from the control and 1 experimental groups in all study periods.

Table 1
The effects of an extract of the roots of burdock and felt (E) and tinctures of Echinacea purpurea (EP) on humoral immune response of mice CBA/CA Lac, (X±m)
A group of miceWhile ately Time studies
4 days7 days
Control (EB)ACS, 106/authority159,20±16,50149,8±7,71
AOK, %of 7.36±0,107±0,21
AOK, 106/authority11,72±1,23of 10.47±0,57
IgM, log2T2±0,322,2±0,32
IgG, log2T8,4±0,757,30±0,50
Experience 1 (EP+EB)ACS, 106/authority190,00±one-22.67163,00±10,85
AOK, %11,44±0,49*5,64±0,19*
AOK, 106/authority21,89±3,20*which 9.22±0,77
IgM, log2T 2±0,321,2±0,49
IgG, log2T10,4±0,25*10±0,32
Experience 2 (E+EB)ACS, 106/authority208,6±a 10.74*224,6±10,04*#
AOK, %of 14.76±0,67*#of 12.76±0,31*#
AOK, 106/authority30,67±1,55*#28,76±1,91*#
IgM, log2T4,8±0,26*#3,9±0,22*#
IgG, log2T8,2±1,129,2±0,50
Note: here and in tables 2
* - the differences with the corresponding values of the control are significant;
# - differences with the corresponding values of the group "experiment 1" valid.

Example 2. Control mice were administered for 5 days in 0.2 ml of distilled water intragastrically (control)animals 1-the second experimental group received dealcoholizing tincture of Echinacea purpurea (EP), which animals were injected for 5 days at a dose of 50 mg/kg in a volume of 0.2 ml of distilled water intragastrically; mouse 2nd experimental group received dealcoholizing extract of the roots of burdock and felt (E), 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. At 4 and 7 days after injection was evaluated phagocytic activity of neutrophils in the peripheral blood of mice of all groups on the ability of these cells to ingest latex particles (concentration of suspended particles - 60 million/ml); take into account the percentage of neutrophils, absorbing particles (phagocytic index - PHI) and the average number of particles trapped in one cell (phagocytic number FC).

In table 2 the results of the experiments show that the extract of the roots of burdock and felt taken in the ratio 1:1, and obtained by the method of repercolation using 40% ethanol, significantly stimulates the absorption activity of neutrophils in peripheral blood (compared with the control group) as due to the increased proportion of neutrophils that can absorb latex (PHI), and by increasing the amount absorbed by each cell particles (FC) on 4 days of the experiment.

Table 2
The effects of an extract of the roots of burdock and felt (E) and tinctures of Echinacea purpurea (EP) on the phagocytic activity of neutrophils in the peripheral blood of mice CBA/CA Lac (X±m)
A group of mice4 days7 days
PHI (%)FCPHI (%)FC
Control22±1,22of 2.06±0,23to 17.25±1,803,79±0,62
Experience 1 (EP)32±3,91*4,72±0,45*23,8±2,13to 4.52±0,23
Experience 2 (E)28,5±3,12*6,64±0,08*#21,6±0,984,93±0,31

Example 3. Control mice were administered for 5 days in 0.2 ml of distilled water intragastrically. Mice of the 1st experimental group were injected dealcoholizing tincture of Echinacea purpurea (EP) 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 were injected dealcoholizing the hydrated extract of the roots of burdock and felt (E) within 5 days at a dose of 50 mg/kg in a volume of 0.2 ml of distilled water intragastrically. At 4 and 7 days after injection was evaluated phagocytic activity of peritoneal macrophages of mice of all groups on the ability of these cells to ingest latex particles (concentration of suspended particles - 60 million/ml); take into account the percentage of macrophages that engulfed particles (phagocytic index - PI), and the average number of particles trapped in one cell (phagocytic number FC). Introduction animal extract of the roots of burdock and felt led to a pronounced enhancement of phagocytic activity of macrophages in both the studied parameters during all periods of the experiment as compared to the control and 1 experimental group. The use of tinctures of Echinacea caused the increase in phagocytic index compared to the control mice only 4 days.

Table 3
The effects of an extract of the roots of burdock and felt (E) and tinctures of Echinacea purpurea (EP) on the phagocytic activity of peritoneal macrophages of mice CBA/CA Lac (X±m)
A group of mice4 days7 days
PHI (%)FCPHI (%) FC
Control24,6±3,592,18±0,1824,4±1,632,64±0,29
Experience 1 (EP)32,8±2,482,02±0,1632±2,30*2,20±0,21
Experience 2 (E)41,4±2,05*#3,44±0,07*#48,8±1,32*#3,82±0,20*#

Thus, the liquid extract from the roots of burdock and felt taken in the ratio 1:1, and obtained by the method of repercolation using 40% ethanol, exhibits immunostimulatory properties, which is reflected in the humoral immune response (increase in the total cellularity of lymphoid organ, the number of antibody productive cells and the titer of specific hemagglutinin) and phagocytic activity of neutrophils peripheral blood, comparable in intensity with the tincture of Echinacea purpurea. According to the degree of stimulation of phagocytic activity of peritoneal macrophages liquid extract from the roots of burdock and felt significantly exceeds the tincture of Echinacea purpurea.

References

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2. Vershinin SF, Patavina E.V. the Use of natural bioregulators in Oncology /Oncology Issues. - 2003. - V.49. No. 2. - C-151.

3. Immunological methods / edited Grimes. - M.: Medicine, 1987. - S-218.

4. Immunology: Workshop / Eweber, VoD, Our, Naiviety. - K.: visa SHK. Publishing house in Kyiv. University, 1989. - S-204.

5. Closev P. A. Complete Handbook of medicinal plants. - M.: Publishing house Eksmo, 2003. - 922 S.

6. Leskov's VP Immunostimulants // Allergy, asthma and clinical immunology. - 1999. No. 4. - P.12-25.

7. Linova BM, Barnaul OD, Krylov V.A. and other New drugs from plants of Siberia and the Far East. - Tomsk, 1986. - S.89-90.

8. Samorodov, VN, Pospelov SV, Moses GF and other Phytochemical composition of the genus Echinacea (Echinacea Moench.) and its pharmacological properties (review) // pharmaceutical journal. - 1996. No. 4. - P.32-37.

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

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

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

12. Percival SS Use of echinacea in medicine // Biochem. Pharmcol. - 2000. - Vol.60. No. 2. - R-158.

Means, having an immunostimulating action, characterized by the fact that it is a liquid extract of the roots of burdock and felt taken in the ratio 1:1, obtained by the method of repercolation 40% ethanol.



 

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