Immunostimulants

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmaceutical industry, specifically to an agent of immunostimulating activity. The immunostimulant which represents a liquid nettle extract is prepared by repercolation under certain conditions.

EFFECT: immunostimulant exhibits effective immunostimulating properties that is expressed in intensified immune response, cell immune response and nonspecific resistance of an organism.

3 tbl, 3 ex

 

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

Recently, much attention has been paid to the study and development of specific tools that stimulate or suppress the immune response of the body. It became apparent that the positive effect of various drugs is due to their ability to increase the overall resistance of the organism, its non-specific resistance, or to stimulate a specific immune response. The increase in total body resistance may be influenced by the activating means (caffeine) and vitamins. The ability to increase the immune reactivity have derived nucleic acids (Derinat), extractive preparations of thymus (timolin, taktivin), lyophilizate cell walls of bacteria (broncho-munal), recombinant analogues of endogenous human interleukins (Roncoleukin, betalain) [6]. However, for these drugs there are some contraindications: individual hypersensitivity, concomitant pathologies, development of tolerance, the occurrence of allergic reactions. So practically important is the search for drugs of natural origin. Many years of experience in the use of drugs grow from inogo raw materials have shown their efficiency, and the lack of toxicity makes possible the use of these drugs in individuals with concomitant pathologies [9].

Herbal drugs that stimulate the immune response, the most famous drugs on the basis of Echinacea purpurea (immunal, immuderm, doctor of Tiss tincture Echinacea) [8, 13]. Drugs immunal and immuderm contain the juice obtained from freshly harvested herbs flowering Echinacea purpurea. The doctor was 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 of the immune system [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 nettle extract obtained by the method of repercolation using 40% ethanol in the ratio 1:1. Standardization of the extract was performed according to the dry residue (GF X1, volume 2, str). The major biologically active substances nettle are vitamins K and C, polysaccharides, carotenoids, organic acids, tannins, mineral salts. In current medical practice nettle leaves are used as a styptic, wound healing, anti-inflammatory and vitamin tool [7].

New in the alleged invention is that the liquid nettle extract first proposed for use as an immunostimulating agent.

The use of liquid nettle extract obtained by the method of repercolation using 40% ethanol as an immunostimulating agent are not described in literature. Application for a new appointment was made possible through the discovery of new properties, namely stimulation of the humoral immune response, cellular immune response and nonspecific resistance. This property is not explicitly derived for the expert from the prior art. Liquid extract of nettle can be used in patients with immune deficiency, in the treatment of chronic, indolent and recurrent diseases.

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

New properties of liquid extract of nettle have been found through experimental research.

To establish the immunostimulatory properties of liquid nettle extract was studied its effect on humoral immune response, cellular immune response and nonspecific resistance of the organism. idci nettle extract was obtained by the method of repercolation using 40% ethanol in the ratio 1:1. Standardization of the extract was performed according to the dry residue (GF X1, 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 nettle extract (EK) 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 received dealcoholizing EC, which 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 introducing EK, EP or distilled water. 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) number antitelomerase the cells (AFC) in the spleen of mice according to the method of Cunningham [12] and antibody titer IgM and IgG in the serum using standard reaction of haemagglutination (DSA) [3].

Experiments to study the effect of nettle extract on cellular immune response were carried out on 18 mice male CBA/CaLac weighing 18-20, Animals were divided into 3 groups (6 mice were administered for 5 days in 0.2 ml of distilled water intragastrically (control); 6 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; 6 mice received dealcoholizing nettle extract (EK), which was administered to the animals in for 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. To assess the impact of drugs on the cellular immune response was used in a reaction of hypersensitivity of the delayed type (GST) [10]. 24 hours after the last injection of extracts of animals was senzibilizirani with sheep red blood cells subcutaneously with 1×107in a volume of 100 μl. Allowing the antigen dose (1×108in volume 20 µl) were injected on day 5 after sensitization under aponeurotic plate of one of the hind legs. In the contralateral paw as a control were injected with saline in equal volume. A record of the intensity of the inflammatory reaction was carried out 24 hours after the introduction of the resolving dose of antigen. The index vospolenie the (IV) was determined as the difference of the masses of the experimental and control legs.

Experiments to study the effect of nettle extract nonspecific resistance of the organism were carried out on 36 mice male CBA/CaLac weighing 18-20, Animals were divided into 3 groups: 12 mice were administered for 5 days in 0.2 ml of distilled water intragastrically (control); 12 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; 12 mice received dealcoholizing nettle extract (EK), which was administered to the animals in for 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 the peripheral blood in the test absorption of latex particles at 4 and 7 days after injection [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 of nettle.

Example 1

Control mice were administered for 5 days in 0.2 ml of distilled water intragastrically. Mice 1-the experimental group was 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 nettle extract for 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 intraperitoneally in a volume of 0.2 ml). On the 4th and 7th days after immunization were determined by the total number of splenocytes (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 at 4 and 7 days after immunization and the absolute number of antitelomerase on 4 days of the experiment compared with the control (table 1). In addition, there was an increase in the titer of immunoglobulin G in 4 days. Analysis of the results presented in table 1, shows that exchange rate 5-day administration of the extract of nettle led to the increase in the total cellularity of lymphoid organ on the 7th day of the study, the relative number of antibody productive cells and IgG titer relative to the animals of the control group during all periods of the experiment. Compared with mice treated with tincture of Echinacea was significantly increased relative and absolute number of cells-antitelomerase is in 7 days.

Table 1
The effect of nettle extract (EK) and tinctures of Echinacea purpurea (EP) on humoral immune response of mice CBA/CA Lac, (X±m)
A group of miceIndicatorsTime 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, log2T2±0,321,2±0,49
IgG, log2T10,4±0,25*10±0,32*
Experience 2 (EC+EB)ACS, 106/authority151,80±8,68171,60±16,07*
AOK, %9,12±0,44*#8,48±0,32*#
AOK, 106/authority13,87±1,29#14,30±1,70#
IgM, log2T1,2±0,20,6±0,25
IgG, log2T10,4±0,4*9,6±0,4*
Note: here and in tables 2, 3
* - 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 of the 1st experimental group 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; mouse 2nd experimental group received dealcoholizing nettle extract (EK), 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. 24 hours after the last injection of extracts of animals was senzibilizirani with sheep red blood cells subcutaneously with 1×107in a volume of 100 μl. Allowing the antigen dose (1×108in volume 20 µl) were injected on day 5 after sensitization under aponeurotic plate of one of the hind legs. In the contralateral paw as a control were injected with saline in equal volume. A record of the intensity of the inflammatory reaction was carried out 24 hours after the introduction of the resolving dose of antigen. Index of inflammation (IV) was determined as the difference of the masses of the experimental and control legs.

Table 2
The effect of nettle extract and tinctures of Echinacea purpurea (EP) on the cellular immune response michelinie CBA/CA Lac, (X±m)
Groups of miceControlExperience 1 (EP)Experience 2 (EK)
Index of inflammation18,05±1,0418,53±1,9627,33±1,45*#

In table 2 the results of the experiment indicate a significant stimulating effect of nettle extract on the formation of a clone of antigen-specific T lymphocytes.

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 nettle extract (EK) 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 the last injection were 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 (ferociter the e number PC).

Table 3
The effect of nettle extract (EK) 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 (EK)41,8±3,81*#7,46±0,74*#26±3,45*2,32±0,15

In table 3 the results of these experiments show that nettle extract significantly stimulates the absorption activity of neutrophils in peripheral blood (compared with the control, so I1 experimental group) as due to the increased proportion of neutrophils, capable of absorbing latex (PHI), and by increasing the amount absorbed by each cell particles (FC) on 4 days of the experiment. To 7 days phagocytic activity of the investigated cells in animals of the 2nd experimental group was also significantly higher than in mice of the control group.

Thus, liquid nettle extract exerts immunostimulatory properties, which is reflected in the humoral immune response (an increase in antibody productive cells and the titer of specific hemagglutinin), cellular immune response (stimulation of the formation of a clone of antigen-specific T-lymphocytes and phagocytic activity of neutrophils peripheral blood, exceeding in severity tincture of Echinacea purpurea.

Sources of information

1. Amosov B.N., Zuev, H.E., Razin YEAR, Krylov YEAR Medicinal plants as a means of adjunctive therapy for treatment of tumors, Byull. the experts. Biol. - 2003. - Mgr. 2. - Pp.24-34.

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

6. Mashkovsky M. D. Medicines. Moscow: Publishing house "New wave", 2006. - 1205 S.

7. Polozhii AV, Goldberg ED, Mrs. I.I. Medicinal plants of Siberia. Tomsk: Izd. University, 1995. - 325 C.

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. Bruises A.F. Herbal medicine against cancer. - M., 1997. - 198 S.

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

11. 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.

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

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

Means, having an immunostimulating action, characterized by the fact that is a liquid extract of nettle, obtained by the method of repercolation using 40%ethanol in the ratio 1:1.



 

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