The method of obtaining compounds having immunostimulating, antiviral and antibacterial activity, a substance obtained by this method and pharmaceutical composition based on it

 

(57) Abstract:

The invention relates to medicine. The extraction is carried out of the crushed plant material, which is used as plants of the family Dioscoreaceae, plantaginaceae families, Solanaceae, water. Perform centrifugation, aqueous extract, its concentration, the deposition of 96% ethyl alcohol in the presence of sodium chloride. Refresher pereosazhdeniya obtained precipitate salt or acidic agent with subsequent processing of the selected acid peptidoglycan raw alkaline solution or saturated solution of the salt of an alkali metal. Purification of the product is carried out using gel chromatography and drying. The substance obtained in this way, is a water-soluble acid peptidoglycan molecular weight 1200-40000 KD and has a mass ratio between glucose and uronic acids is 1: 2-4. The pharmaceutical composition contains an active ingredient and a carrier. The invention allows to obtain a new substance of plant origin from the available raw materials, with strong immunostimulating, antiviral and antibacterial activity. 3 s and 5 C.p. f-crystals, 12 tab.

The invention oting raw material substances, having immunostimulating, antiviral and antibacterial activity; substances obtained in this way, and pharmaceutical compositions based on it.

A method of obtaining biologically active polysaccharide by grinding plant material, which is used as the sprouts of potato tubers, its extraction boiling water, followed by keeping at 20oC for 16-18 hours, the separation of the extract, extract it for at least 20 days, fractionation its molecular weight and the release of the active substances with a molecular weight of more than 10,000 daltons, followed by concentration and drying (EN 2108800 C1, a 61 K 35/78, 20.04.1998). The substance obtained in this way, it has antiviral and antibacterial activity and is used as a medicinal product as external and internal use. Also known is a method of obtaining herbal polysaccharides having an immunostimulating action, by processing vegetable raw materials with an aqueous solution of formalin, keeping in acidified water, followed by extraction of pectic polysaccharides in an aqueous solution of ammonium oxalate, processing of the extract investmentcode plants, for example, any type of duckweed Lemna spp., pre-chopped fresh aerial part of plants, for example, Campion ordinary Oberna behen(L). (EN 2149642, A 61 K 35/78, 27.05.2000). The closest known solution to the claimed is a method of obtaining compounds having immunostimulating, antiviral and antibacterial activity, the substance obtained in this way is called gamma Plante (-PL), and a pharmaceutical composition based on it (L. A. Czekanowska, A. C. Generals, isolation and characterization of biologically active drug Gamma-Planta from sprouts of the potato Solanum tuberosum", Chem.-Pharm. W-l, T. 34, 3, 2000, S. 51-56). A method of obtaining gamma-Planta includes grinding plant, which is used as the sprouts of potatoes, extraction boiling water, centrifuging an aqueous extract, concentration, precipitation with acetone, cleaned and dried, the target product. Cleaning according to a known method includes dialysis, gelfiltration sorbent Asa, ion exchange chromatography, and at the last stage of the treatment liquid chromatography high pressure. The substance obtained by the method described above, is a glycoprotein with a molecular mass of 70 KD, consisting of Uglevodorody, 3.7% arabinose, 2,08%-xylose, 6,84%-galactose, 0,58-mannose, and 1% of amino sugars, 5,1% of uronic acids. Pharmaceutical composition based gamma-Planta contains the appropriate fillers or carriers, allowing us to obtain various dosage forms. However, these drugs are not high enough immunostimulirutuyu activity that caused, apparently, ballast impurities, including starch, as well as the other structure of the active component.

The task of the invention to provide new substances of plant origin from the available raw materials and product, with a higher immunostimulating, antiviral and antibacterial activity, the development of effective technological method of its production and preparation of pharmaceutical compositions based on it having a high therapeutic effect.

The problem is solved by the proposed method of obtaining compounds having immunostimulating, antiviral and antibacterial activity, by extraction with water crushed plant materials, which use plants of the family Dioscoreaceae, plantaginaceae families, Solanaceae, centrifugation of the aqueous extract, it is s obtained precipitate salt or acidic agent with subsequent processing of the selected acid peptidoglycan raw alkaline solution or saturated solution of the salt of an alkali metal, purification of the target product using gel chromatography and drying.

It has been unexpectedly found that the present method comprising the implementation of the above sequence of operations, allows to obtain a new substance having a strong immunostimulating, antiviral and antibacterial activity, and represents a water-soluble acid peptidoglycan molecular weight 1200-40000 KD, having a mass ratio between glucose and uronic acids is 1: 2-4, and pharmaceutical composition thereof containing a pharmaceutically acceptable carrier or excipient.

The proposed method involves the use of leaves, stems, roots, tubers and seedlings of plants, i.e. different parts of the plant at any stage of development or maturity.

When presideni as salt agent using cetyltrimethylammonium bromide or calcium chloride or acidic agent is an organic or inorganic acid or an acidic inorganic salt.

The holding of such resultant deposition rates allowed us to identify acid peptidoglycan raw, separating it from numerous co-primii as khromatograficheskogo agent used TSK HW-75F, sepharose 2V or 4V CL.

The substance obtained by this method, characterized by the fact that the peptide portion of the molecule acid peptidoglycan is 133 wt.%.

The amount of peptide was determined by method of Lowry using bovine serum albumin as standard (O. N. Lowry, N. J. Rosenbrough et al., J. Biol. Chem., (1951), 193, 265-275).

The peptide portion of the acid peptidoglycan consists of the following amino acids is presented in table. 1.

From the data table. 1 shows that the quantitatively predominant content of histidine (HIS), alanine (ALA), glutamic acid (GLU), aspartic acid (ASP) and serine (SER).

In the polysaccharide portion of the molecule consists of galacturonic acid, glucose, galactose, mannose, arabinose, rhamnose, when following their mass content: the galacturonic acid 186%, glucose 93%, galactose 5,52%, mannose 0,70,25%, arabinose 3,81,3%, ramnose 1,90,9%.

The amount of acidic sugars determined by color reaction with 3,5-dimethylphenol in concentrated sulfuric acid (A. I. Usov, M. I. Bilan, N. G. Klochkova, Botanica Marina, 1995, 38, 43-51).

Analysis of uronic acids by GLC as their trimethylsilyl derivatives (P. Albersheim, Methods Enzimol., 1987, 118, 3-40) showed mainly the presence of galactron(P. Albersheim, Methods Enzimol., 1987, 118, 3-40).

The following examples illustrate the present invention.

Example 1.

Plant cell root of Dioscorea family Dioscoreaceae (D. cuacasia Lipsky) were grown in culture medium. After incubation, the cell mass is separated by filtration, and the supernatant liquid concentrate, dialist against distilled water and freeze dried.

To 5 g of the dry mixture, obtained from the culture fluid, add 500 ml of distilled water. The extraction was performed at room temperature for 3 hours. Nerastvorim the precipitate was separated by centrifugation. The mother liquor concentrated to a volume of 200 ml, add 200 mg of sodium chloride, dissolve it and poured 600 ml of 96% ethyl alcohol. Sediment allocate by centrifugation at 4000 rpm for 30 minutes the Precipitate washed with 50 ml of 96% ethanol, centrifuged and dried in air. Then re-pereosazhdeniya obtained precipitate acidic agent is an organic acid. For this purpose, 1 g of dry intermediate product is dissolved in 200 ml of distilled water under stirring at room temperature for 1 hour. To the solution add 5 ml of 10% solution of trichloroacetic acid. Dropped the ATEM add 50 ml of distilled water and with stirring was added dropwise a 25% ammonia solution to dissolve the precipitate.

The resulting solution was applied on a column of TSK HW-75F. Column elute with water. Collect the first high molecular weight peak with a molecular weight of from 1200 up to 40,000 KD. The solution is concentrated on a rotary evaporator and freeze-dried. The output of acid peptidoglycan is 39 mg Mass ratio between glucose and uronic acids is 1:3.

The amount of peptide in the sample is 11.2 per cent.

Example 2.

100 g fresh aerial parts (stems) Dioscorea family Dioscoreaceae (D. cuacasia Lipsky) are crushed, add 500 ml of distilled water and extracted by heating 30-40oC for 5 hours.

The mixture is squeezed using a mechanical press. Aqueous extract cialiswhat and concentrate to a volume of 100 ml by evaporation on a rotary evaporator. To the concentrate was added sodium chloride at the rate of 100 mg per 100 ml and 3 volumes of 96% ethanol. Sediment allocate by centrifugation at 4000 rpm for 30 min.

The precipitate was washed with 30 ml of 96% ethanol, centrifuged and dried in air. Then re-pereosazhdeniya obtained precipitate acidic agent is an inorganic acid. For this purpose, 1 g of dry intermediate product is dissolved in 200 ml distilled to techniqu add 5 ml conc. HCl. Dropped the precipitate was separated by centrifugation. Washed with water. Then add 50 ml of distilled water and with stirring was added dropwise a 25% ammonia solution to dissolve the precipitate.

The resulting solution was applied on a column of TSK HW-75F. Column elute with water. Collect the first high molecular weight peak with a molecular weight of from 1200 up to 40,000 KD. The solution is concentrated on a rotary evaporator and freeze-dried. The output of acid peptidoglycan is 16 mg Mass ratio between glucose and uronic acids is 1:2,2.

The amount of peptide in the sample is 14.7%.

Example 3.

100 g freshly harvested leaves of the plantain family, plantaginaceae families (P. major L.) are crushed, add 500 ml of distilled water and extracted by heating 30-40oC for 4 hours.

The mixture is squeezed using a mechanical press. Aqueous extract cialiswhat and concentrate to a volume of 100 ml by evaporation on a rotary evaporator. To the concentrate was added sodium chloride at the rate of 100 mg per 100 ml and 3 volumes of 96% ethanol. Sediment allocate by centrifugation at 4000 rpm for 30 min.

The precipitate was washed with 30 ml of 96% ethanol, centrifuged and what Isletas. For this purpose, 1 g of dry intermediate product is dissolved in 200 ml of distilled water under stirring at room temperature for 1 hour. Nerastvorim sediment cast, and to the liquor add 5 ml conc. HCl. Dropped the precipitate was separated by centrifugation. Washed with water. Then add 50 ml of distilled water and with stirring was added dropwise a 25% ammonia solution to dissolve the precipitate.

The resulting solution was applied on a column of TSK HW-75F. Column elute with water. Collect the first high molecular weight peak with a molecular weight of from 1200 up to 40,000 KD. The solution is concentrated on a rotary evaporator and freeze-dried. The output of acid peptidoglycan is 16 mg Mass ratio between glucose and uronic acids is 1:2,7.

The amount of peptide in the sample is 13.8 per cent.

Example 4.

Similar to example 3, but for planting acid peptidoglycan instead of 5 ml conc. HCl using 200 ml of a saturated solution of inorganic acid ammonium salts MN4NO3or (NH4)2SO4. The yield of the final product is 14 mg Mass ratio between glucose and uronic acids is 1: 2,4.

The amount of peptide in Lescaut, add 10 l of water and extracted at room temperature with stirring for 2 hours. The mixture is squeezed using a mechanical press. Aqueous extract cialiswhat and concentrate to a volume of 1 l by ultrafiltration filter 10 KD.

To the concentrate was added sodium chloride at the rate of 100 mg per 100 ml and 3 volumes of 96% ethanol. Sediment allocate by centrifugation at 4000 rpm for 30 min.

The precipitate was washed with 300 ml of 96% ethanol, centrifuged and dried in air. Then re-pereosazhdeniya obtained precipitate salt agent is calcium chloride. For this purpose, 10 g of dry intermediate product is dissolved in 1 l of distilled water under stirring at room temperature for 1 hour. Nerastvorim sediment cast, and to the liquor add 150 ml of 5% solution of calcium chloride. The precipitation, which represents an acid peptidoglycan raw, separated by centrifugation. Then translate it into soluble form by adding a saturated solution of sodium chloride and incubated the mixture with stirring and heating at 50oWith up to complete dissolution. The resulting solution was applied on a column of TSK HW-75F. Column elute with water, collect the item is barely and freeze-dried. The output of acid peptidoglycan is 170 mg Mass ratio between glucose and uronic acids is 1:4.

Example 6.

Similar to example 4, but as organs of plants use a potato tubers, and as the salt of the agent at re resultant deposition rates of sediment using a 9% solution of cetyltrimethylammonium bromide. The output of acid peptidoglycan is 14 mg Mass ratio between glucose and uronic acids is 1:3,5. The amount of peptide in the sample is 16%.

Example 7. Immunostimulirutuyu activity, manifested in the activation of the synthesis of antibodies.

Introduction acid peptidoglycan (CNG) at the same time with the foreign antigen leads to a significant increase in the intensity of production of antibodies specific to the antigen. In the experiments used mice CBA, C57B1/6 (CBAxC57B1/6)F1 and BALB/c mice. As antigens for immunization of mice used bovine serum albumin (BSA), egg albumin (YA) or sheep red blood cells (BAE). Erythrocytes defibrinating the blood of the lamb thrice washed, centrifuger to 50 times the volume of Hanks solution, then resuspendable in the same solution. The mice were injected intraperitoneally 2 m the cha 4 weeks. J.A. was administered intraperitoneally at a dose of 50 micrograms, repeated immunization J.A. was performed after 2 and 4 weeks after the first immunization.

The corresponding hinge CNG was dissolved in Hanks solution or physiological NaCl solution for 2-3 hours before using. CNG was administered to mice in doses of 1 to 1000 μg. In the case of immunization of mice EB, BSA or ON simultaneously with the antigen was injected CNG.

The level of immune response in mice immunized with EB, determined by the number of antibody productive cells (AFC), detected in the spleen method EPHE 4-5 days after immunization. The content of antibodies specific to YA or BSA in serum of mice was determined by the method of enzyme-linked immunosorbent assay (ELISA). The isotype of antibodies specific to BSA was determined by ELISA using rabbit antibodies specific for IgM, IgG1, IgG2a, IgG2b or IgG3 mouse.

Joint introduction of CNG with heterologous antigen experimental mice resulted in an immune reaction, considerably more intensive than in the introduction of only one antigen. Thus, the injection CNG with suboptimal immunogenic dose of EB led to 10-fold increased production of antitelomerase specific to EB.

After immunization with BSA in the combination of the higher level of specific antibodies after immunization only BSA (ELISA titer of not more than 1: 500). With the introduction of CNG was significantly increased not only the intensity but the duration of the secondary immune response to BSA. The dominant isotypes of antibodies IgG1, IgG2a and IgG3. The production of IgM and IgG2b specific to BSA was increased under the influence of CNG to a lesser extent.

Adjuvant effect was clearly dose dependent CNG. When the secondary response to BSA optimal immunostimulatory dose was the dose of 1 μg CNG, during the primary immune response to heterologous erythrocytes were optimal dose of 10 µg CNG or more.

The impact of the CSG on the production of antibodies to J.A. presented in table. 2. YA proved to be highly immunogenic antigen for BALB/c mice, titers of antibodies after injection of 50 µg J.A. without any adjuvant reached 1:13000. Joint introduction of CNG with 50 µg J.A. led to a significant increase in the level or antibody-based test response (titer 1:22000). CNG had a stronger adjuvant effect than known immunoadjuvant lipopeptide Pam3Cys-Ser-Lys4(titer 1: 20000), but was inferior adjuvant Freund (titer 1:33000).

Example. 8. Immunostimulirutuyu activity: activation of tissue macrophages.

Fabric mouse macrophages obtained from the peritoneal exudates significantly advance the s and forms, and in changing their metabolic and enzymatic activities.

Cells of peritoneal exudate (KPIs) was obtained by washing the peritoneal cavity of mice (CBAxC57B1/6)F13 ml of medium 199. Received from 10-15 mice KPIs at a concentration of 2-2,51061 ml was collected in siliconized tubes.

Production by macrophages oxidative radicals

Suspension cells of peritoneal exudate was poured into 1 ml tubes of chemiluminometer and incubated at temperature 37oC in humidified atmosphere of 5% CO2in air for 2 hours. After incubation, unattached cells were washed away and adherent to the tube cell 2 times washed with medium 199. Then the tubes were introduced into 1 ml of complete RPMI-1640 medium containing CNG at concentrations from 0.2 to 50 μg in 1 ml and incubated under these conditions for another 24 hours. After incubation were removed adosados, was added 0.5 ml of buffer solution (pH of 7.2), made with Hanks solution (without phenol red) supplemented with 5 mM glucose, 10 mm HEPES-buffer, and 0.62 mM lyuminola (Sigma Chemical Co), and assessed the level of spontaneous and induced simhasanam chemiluminescence. The results of the sum of the three experiments are presented as the average number of counts per minute per 1 million is Tannoy chemiluminescence, but significantly (by 50%) increased the ability of cells to the production of oxidative metabolites in response to zymosan representing components of the cell wall of microorganisms. The increase chemiluminescent activity occurs already at a concentration of CNG 0.2 ág/ml, and the maximum effect is achieved at a concentration of 5.5 μg/ml, which correlates well with the concentration of CNG, leading to maximal activation of macrophages, judging by their morphological characters.

The activity of 5'-nucleotidase (5'-NTD) on the surface of macrophages

Determination of the level of the enzyme 5'-NTD is one of the highly informative ways to assess activation of macrophages. It is established that the decline in activity of this enzyme under the influence of immunomodulators characterizes their immunoadjuvant properties and correlates with high anti-infective activity.

To determine the impact of CNG on level 5'-NTD peritoneal macrophages mice (CBAxC57B1/6)F1was administered intraperitoneally solution of 30 µg of CNG volume of 0.5 ml of the Control animals were injected with 0.5 ml of saline. 24 hours after the injection was given cells of peritoneal exudate by washing the peritoneal cavity of mice is Stolovich Petri dishes with a diameter of 100 mm, 10 ml of the suspension of the ECP on the Cup. After removal of not adhering cells remaining adherent cells were removed from the surface of the Cup using a rubber spatula, and drove concentration KPIs to 2 million cells in a volume of 50 μl. This volume of the suspension was introduced into the wells of 96-well plates, were added 5'-adenosinemonophosphate, which is the substrate 5'-NTD, and incubated for 60 minutes in the above conditions. The enzyme activity was determined photometrically (wavelength 620 nm) on the intensity of staining molybdenum reagent, included in the sample of cells after incubation. The results were expressed in units of optical density in terms of 1 million KPIs and relative activity of 5'-NTD in percent of the reference level.

The activity of CNG compared with the activity of immunomodulator "Polyoxidonium", which is well known as an activator of phagocytes (table. 3). The results obtained testify to the efficient activation of macrophages in vivo as CNG and polyoxidonium. When CNG was superior to the comparator drug in its activation effect on macrophages. So, under the influence of polyoxidonium activity of 5'-NTD decreased in 2 times, and under the influence of CNG - 6 times, reaching 16% of the control level. These data show the high bactericidal potential, they prove high together with immunomodulating activity CNG.

Example 9. Immunomodulating activity: activation of cells of the human immune system (NK cells, monocytes, granulocytes).

In vitro investigated the impact of CNG on immune cells circulating in the peripheral blood of man.

The activation of NK-cells

Method three-color laser flow cytometry was found, the rapid emergence of the activation marker on NK cells after exposure to CNG.

Blood from the cubital vein were obtained from healthy donors in the morning on an empty stomach in tubes with anticoagulant heparin sodium using system Vacutainer" (firm "Becton Dickinson"). The drug CNG was dissolved in a concentration of 1 mg/ml in medium RPMI-1640 and filtered through a 0.22 μm filter. Prepared the necessary dilution in complete medium RPMI-1640 in the range from 0,016 to 100 μg/ml and added to 0.2 ml in wells of a 48-hole culture plate (firm Nunc"). As a negative control was made 0.2 ml of complete RPMI-1640 medium. All wells were added to 0.2 ml of heparinised whole blood. Samples were incubated at 37oC and 5% CO2for 3-48 hours. After incubation, 0.05 ml of blood sample was placed in 1,ICRIN ("Pharmingen"), anti-D3-rR (firm "Becton Dickinson"). The tubes were shaken for 5 s and then incubated at room temperature in the dark for 20 minutes. To each tube was added 1 ml of lyse-fixing solution (Becton Dickinson) and kept for 15 minutes at room temperature. The tubes were centrifuged for 10 min at 300 g, the supernatant was removed, and the cell sediment was reasponsible in 0.5 ml isotonic. Analysis of fluorescence was performed on flow-laser cytometer "FAX Calibur" Becton Dickinson" using the software "Cell Quest".

Without adding CNG only 7% of NK-cells expressed the activation marker CD69, and under the action of CNG at a concentration of 10 μg/ml, almost all (96%) of NK-cells were activated.

The increased cytolytic activity of NK-cells under the influence of CNG

From whole heparinized blood viselli mononuclear cell fraction by centrifugation (30 min 300g at room temperature) in a stepwise density gradient ficoll ("pharmacy"). The selected cells were washed in medium 199 and diluted to a concentration of 2 million/ml medium RPMI-1640 containing 10% fetal calf serum, 20 mm HEPES buffer (pH 7.4), 10 μg/ml of Ghent is nsii and added 1 ml PS (control) or 1 ml CNG 20 μg/ml, or 1 ml of solution of standard activator of NK-cell interleukin-2 (IL-2, 20 IU/ml). Samples were incubated for 3 hours at 37oC in an atmosphere of 5% CO2. After incubation, the cells were collected into centrifuge tubes, besieged by centrifugation (10 min 300g) and brought a concentration of up to 5 million cells in 1 ml of PS. The obtained cells were used as effectors of cytolysis. A series of 2-fold dilutions in TS cells effector was poured into 0.1 ml in triplets in wells of 96-well round-bottom of the tablet. All wells were added to 10 thousand labelled3H-uridine target cells K-562 in a volume of 0.1 ml PS. The mixture of cells effector and target cells were incubated for 4 hours at 37oC in an atmosphere of 5% CO2. After incubation, the cells were transferred to paper filters with collector cells Titertek Cell Harvester 550 and counting the radioactivity on the counter Wallac 1409.

After 3-hour incubation of cells effectors in the presence of CNG 29% NK-cells expressed CD69, has been activated. Testing the cytolytic activity of cells-effectors, pre-activated CNG, against C target cells showed a dramatic increase in the ability of NK cells to lyse target. It is noticeable at low relationship effector : target and. If we compare the action of CNG with IL-2, a known stimulator of NK-cells shows that CNG is several times more effective than IL2, as by expression of CD69 and lysis of target cells C.

Activation of granulocytes

In examining the impact of CNG on the expression of activation markers on different types of cells, it was found that, in addition to monocytes and NK-cells, the activation marker CD69 is expressed on granulocytes. After 24 hours incubation in the presence of CNG activation of granulocytes was become considerable, to this date, about 60% of the cells expressed CD69.

Example 10. Immunostimulatory effects: activation of synthesis and secretion of cytokines.

CNG activates the production of cytokines by human blood cells. In particular, CNG starts the production of interleukin-1 (IL-1), tumor necrosis factor (TNF), interleukin-8 (IL-8). Blood from the cubital vein were obtained from healthy donors in the morning on an empty stomach in tubes with anticoagulant heparin sodium using system Vacutainer" (firm "Becton Dickinson"). The drug CNG was dissolved in a concentration of 1 mg/ml in medium RPMI-1640 and filtered through a 0.22 μm filter. Prepared the necessary dilution in complete medium RPMI-1640 in the range from 0,016 to 100 μg/ml and who wore 0.2 ml of complete RPMI-1640 medium. All wells were added to 0.2 ml of heparinised whole blood. Samples were incubated at 37oC and 5% CO2for 3-48 hours. After incubation were collected on 0.2 ml of culture medium in 0.5 ml centrifuge tubes and centrifuged at 10,000 rpm for 15 minutes. The supernatant was frozen at -70oWith and used for determination of secreted cytokines. Cells remaining in the hole, carefully pietravalle and transferred into 1.2 ml plastic tubes for the detection of surface markers of activation.

IL-1 was determined in the culture medium enzyme-linked immunosorbent assay. To measure the concentration of IL-1 used set of firm "Immunotec" (France) in accordance with the instructions for its use. A (TNF-a were determined using kits "Innogenetics" (Belgium). Interleukin-8 (IL-8) were determined using kits "Innogenetics" (Belgium). The colour intensity was measured at a wavelength of 450 nm on an automatic reader "Dynatec".

Determination of the intracellular content of cytokines in monocytes human blood was performed by the method of three-color laser flow cytofluorometry.

Whole blood was diluted 1:1 in medium RPMI-1640 containing various concentric who has bated at 37oC and 5% CO2within 5 hours. After the end of the incubation were selected to 0.1 ml of the suspension was added 1 ml of lyse-fixing solution (Becton Dickinson"). Kept for 15 minutes at room temperature. The tubes were centrifuged for 10 min at 300 g, the supernatant was aspirated and added to cell draught 0.5 ml solution for increasing the permeability of the cell membrane (Becton Dickinson) and kept for 10 minutes at room temperature. This procedure allows you to paint intracellular contents. After adding 5 ml of isotonic solution containing 0.5% BSA and 0.1% sodium azide, cells were besieged by centrifugation (10 minutes at 300 g), the supernatant was aspirated. To the cell precipitate was added to 5 μl of the antibody. For determination of IL-1 in monocytes used the following antibodies: anti-IL-1-FITC ("Caltech"), anti-D14-RE ("Caltech") and anti-CD45-PerCP (Becton Dickinson"). Likewise determined the intracellular content-TNF in monocytes.

From the data obtained it follows that CNG stimulates the production of TNF in monocytes and that monocytes are a source of TNF in the environment. Indeed, if in the incubation medium was added brefeldin, inhibitor of secretion of b is environment - at the level of the background. If the inhibitor of the transport protein is absent, the accumulation of TNF in monocytes does not occur, and the concentration of TNF in the environment increases directly proportional to the concentration of the PDA. From the dose dependence of the accumulation of TNF in monocytes can be seen that already at a concentration of 40 ng/ml CNG monocytes significantly increase the synthesis of TNF. At the dose of 0.4 µg/ml is quite a high production of IL-1, TNF and IL-8. If we compare the concentration of cytokines in the extracellular environment, it is clear that IL-8 is produced several times more than that of IL-1 and TNF.

Example 11. Induction of the synthesis of interferons.

The study of the interferon-inducing activity CNG conducted in vitro in human cultured human cells infected with virus encephalomyelitis (VEM). The intensity of the induction of interferon was evaluated for antiviral activity CNG. In the experiments used a line of human cells L-41 (fibroblast-like cells used for titration of IFN - and IFN-) and J-96 (line monocytopenia cells). Cells at a concentration of 200,000 cells/ml were inoculated in 96-well flat-bottomed culture plates in medium 199 (J-96) or Needle (L-41) with addition of 10% serum of cattle embryos, glutamine (300 μg/ml) and Dustin (NGOs "Vector", Novosibirsk, Russia). Antiviral (interferonogenna) effect was evaluated by the minimum effective concentration (highest dilution of the drug, effectively the vast growth of the virus) drug that protects 50% of the cells from cytopathogenic actions VEM (JRC50). Interferonogenna considered to be a substance that reduces the concentration of virus in cell cultures of 1.7 to 2.0 lg. To determine the JRS virus used inverted microscope (Leitz magnification 200x). Each study drug in cultural tablets with cells L-41 or J-96 cells was titrated in increments of 1:2 24 hole 3 times each dilution. The experiments were repeated 3 times.

In table. 4 and 5 presents data showing the minimum effective concentration and the most effective cultivation, in which investigational drugs are interferonogenic activity in cell culture L-41 and J-96. The data presented prove that CNG has a pronounced antiviral activity in vitro at concentrations above 6-50 μg/ml Comparison of CNG with Radostina known antiviral drug interferon inducer, testifies interferonogenna action CNG.

Example 12. Activation of antibacterial tamborini mice. The experiments were performed on mice of both sexes weighing 12-14, the Drug was administered subcutaneously at different doses (3.3 µg, 10 µg or 30 µg) for 24 hours prior to infection with Salmonella. Infection of mice were made intraperitoneally with doses of 102, 103, 104or 105microbial cells in the mouse. The observation of the death of animals were carried out over 20 days. Efficacy was assessed by its impact on the survival of infected animals, LD50infection (EID50and life expectancy.

The results obtained are presented in table. 6. It is seen that a single subcutaneous injection of CNG in doses of 10-30 μg resulted in a significant increase in the resistance of mice to infection. Increased the percentage of surviving animals, the average life expectancy of infected and, particularly, the dose of infection, leading to 50% mortality (EID50).

Antimicrobial protection, reinforced CNG is not limited to biological species of microbe or owner. For example, CNG effectively strengthens the protection of chickens from staphylococcal infections.

In the work were used chicken egg directions. CNG was administered per os at a dose of 10 μg (chicken) twice with an interval of 96 hours between doses. For comparison, in the control group e of the second injection chickens were infected by intramuscular introduction Staphylococcus aureus 209R dose 0,75109, 109or 2109microbial phone After infection of chickens developed acute septicaemia, they died within 10 days. So, 10 days after infection dose 2109microbial cells were killed 95% of infected chickens (table. 7). The death of chickens in groups that received CNG or polyoxidonium was significantly less, it amounted to 45% and 55%, respectively. When using sub-lethal infective doses CNG and polyoxidonium defended 100% of infected chickens, while in the control of infection has killed 40% when infection 0,75109S. aureus and 60% when infection 109S. aureus.

In chemotherapeutic experiments used antibacterial drug nebulin, which was administered in the dose of 10 mg/kg per os 20 minutes after infection of chickens 1,5109S. aureus. The results obtained are presented in table. 8. Treatment Nevolina allowed to increase the survival rate of Chicks up to 70% compared with 40% in the control infection. The combination of CNG with Nevolina defended all infected chickens.

The results obtained indicate high activity of the drug CNG. CNG significantly increased the resistance of chickens to infection with different doses of Staphylococcus aureus 209R. To activate anti-infective immunity CNG was the

Antiviral effect CNG investigated in vivo in outbred (b/n) mice model of herpes simplex virus type 1 (HSV-1) strain P2 (Shubladze A. K. , Russian S. J., "a Brief course in practical Virology", 1954; Pshenichnov C. A., Semenov, B. F., Teterow E., "Standardization of methods for Virology research, 1974, pages 123-128). Previously, the virus had 3 passage on the white b/n mice weighing 7-8 g with intracerebral infection. The titer of the virus was 7 lg LD50/0,03 ml In the primary experience mice weighing 10-12 g were infected intraperitoneally with a virus in the form of a 10% suspension of mouse brain at a dose of 1000 LD50. The titer of the virus intraperitoneal infection was equal to 3.5 lg/0,2 ml of the Drug CNG animals were injected intraperitoneally at various doses: 1.0 microgram/mouse, 3,0 µg/mouse, 10 μg/mouse, 30 μg/mouse on one of the following schemes:

1) for 48 hours before infection,

2) for 48 and 24 hours before infection,

3) 24 hours before infection,

4) 24 hours after infection,

5) after 24 and 48 hours after infection,

6) at 48 hours after infection.

As a positive control were used Radostin (known anti-herpes drug, interferon inducer), at a dose of 100 μg/mouse. The reference product was administered to mice by razrabotaete,

4) at 48 hours after infection.

The control group consisted of mice infected with HSV-1 and treated with placebo. Each group consisted of 20 animals. The follow - up period was 15 days.

Antiviral effect of the preparations was assessed by survival of mice (% survival, % protection, life expectancy ALE), as well as in the experiments of biological titration of the brain of infected animals (5 mice per point) on VERO cells. The presence of HSV-1 (titer) in the brain tissue experimental and control mice, taken on day 7 after infection, was determined in 3 replays on VERO cells. The titer was determined in the processing of cell culture 10-multiple dilutions of a suspension of brain. Monitoring and accounting JRC carried out in 3-4 days after contact with infectious materials.

Assessment of the activity of the drugs was performed, taking into account:

1) harmonic average value of the life time () for each group of mice, which includes information on all individuals, calculated by the formula: 1/= (X1/t1+X2/t2+...+Xn/tn):n, where t is the day of death, X is the number of dead animals in day t, n is the number of animals in the group (20 mice);

2) rate protection (SC) - multiplicity reduce the number of KAB the animals in the control group /% dead animals in the experimental group;

3) the performance index of the drug (IE), calculated by the formula:

IE=[(KS-1)/KZ]100%.

The experimental results showed that the drug CNG has a preventive and therapeutic effect in experimental models of HSV-1 infection in mice. The drug was highly effective (percent protection 40-60%) during routine introduction once for 48 hours in a dose of 3 μg/mouse or twice for 48 and 24 hours in a dose of 3-30 ág/mouse (PL.9). This observed high values of mean life span (p<0.001) and compared with control. Data are also in good agreement with the revealed suppression of viral replication in the brain of mice at 2.5 lg (PL. 10). SC and IE also have high values of 7.0 and 85.7%, respectively.

Obtained evidence of antiviral activity of the CNG in the experiments of biological titration on VERO cells. It is established that the maximum suppression of the reproduction of HSV-1 was observed with the drug for prevention scheme, the drug reduced the infectious titer of the virus in 100-1000 times (i.e. 2-3 lg). With the introduction of drug treatment scheme, the titer of HSV-1 was decreased by 1.5-2.0 lg.

1. The method of obtaining compounds having immunostimulating, antiviral and antibacterial activestrategy, precipitation, purification by chromatography and drying of the target product, characterized in that as the plant materials used plants of the family Dioscoreaceae or plantaginaceae families, or Solanaceae, sedimentation spend 96% ethyl alcohol in the presence of sodium chloride, followed by repeated pereosazhdeniya obtained precipitate salt or acidic agent with subsequent processing of the selected acid peptidoglycan raw alkaline solution or saturated solution of the salt of an alkali metal.

2. The method according to p. 1, characterized in that as the plant materials used leaves, stems, roots, tubers or seed plants.

3. The method according to any of paragraphs. 1 and 2, characterized in that for deposition using 96% ethanol and sodium chloride in the ratio of the volume/weight of 3000: 1.

4. The method according to any of paragraphs. 1-3, characterized in that the second presideni as salt agent use cetavlon or calcium chloride, and as the acidic agent is an organic or inorganic acid or an acidic inorganic salt.

5. A substance having immunostimulating, antiviral and antibacterial activity, obtained by extraction of the crushed grow the reaceae or plantaginaceae families, or Solanaceae, and represents a water-soluble acid peptidoglycan molecular weight 1200-40000 KD, having a mass ratio between glucose and uronic acids is 1: 2-4.

6. Substance under item 5, characterized in that the peptide portion of the molecule acid peptidoglycan is (133) wt. %.

7. Pharmaceutical composition having immunostimulating, antiviral and antibacterial activity, characterized in that it contains the active substance under item 6 in an effective amount and a pharmaceutically acceptable carrier or excipient.

8. The pharmaceutical composition according to p. 7, characterized in that it is made in the form of a solution.

 

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