Immunostimulating drug and method thereof

 

(57) Abstract:

The invention relates to medicine, and is concerned medicines immunostimulating action of plant materials and the method of their derivation. The essence of the invention is a drug immunostimulating action, containing as active principle polysaccharides of Pleurotus ostreatus with a molecular mass of 10 - 420 CD. The method of obtaining the drug is in the preliminary drying slices of mushrooms, grinding, removal of lipids by extraction with an alcohol-based solution, the selection of the active principle from the residue by extraction with boiling water, the concentration, the precipitation of polysaccharides by treatment with an alcohol-based solution and dialysis. The technical result is a new drug that has immunostimulating action. 2 S. and 4 C.p. f-crystals, 6 PL.

The invention relates to medicine, namely to medicines immunostimulating action, in particular drugs from plant materials and methods for their preparation.

A known number of biochemical Immunostimulants, in particular, the adjuvant, nukleinat sodium, prodigiozan etc. Drugs produced in powder form, kotono (Zemskov F. M. and other Immunology, 1981, 1, S. 52-55, Morozov Century, and other DAN SSSR, 1977, T. 233, 3, S. 491-494, Mashkovsky M. D. Medicines, M, Medicine, 1993, so 2, S. 169-177).

The disadvantage of these drugs is generally low efficiency in oral application, the presence of negative human impacts arising from adverse biochemical processes that occur in the body during transformations of chemical compounds contained in the drug.

Known use as Immunostimulants solutions and extracts of various plants - ginseng, Siberian ginseng, sea buckthorn, Catnip, mummy, propolis, etc., (C. I. Mashanov, A. A. Pokrovsky. Aromatic plants. M , Agropromizdat, 1991; I. S. Sokolova and other wild and cultivated plants. M, Medicine, 1990).

For all of these drugs have relatively low activity, the presence of a significant number of contraindications.

One of the most promising groups of plants whose properties are studied in the present time is not enough, are higher fungi (Basidiomycetes). There are about 200 species of mushrooms (Auricularia auricula, Tremella fuciformis, Hericium erinaceus, Lentinus edodes and other) components which have immunomodulatory, against the drug has no commercial interest in connection with non-standard raw materials and the lack of sufficiently stable sources (S. R. Wasser, A. L. Weis, Int. J Med. Mushrooms, v.1, 1999, p. 31-62).

The prototype of the claimed group of inventions is a drug on the basis of fungi of the genus Pleurotaceae - Lentinus edodes (S. P. Wasser, A. L. Weis. Int. J Med. Mushrooms, v.1, 1999, p. 31-36).

The drug is the fraction of polysaccharides with immunomodulating and antitumor properties.

The drug is produced by boiling fresh mushrooms Lentinus edodes in water for 8-16 hours, treatment of the extract with alcohol, separation of the precipitated sludge (320 g 200 kg of mushrooms), his presidenial with the subsequent release of impurities by extraction of 20% and 50% aqueous solution of ethanol, dissolving the precipitate of 6% sodium hydroxide and precipitation of the active agent with ethanol and deproteinization method Sevag. Lack of preparation is the lack of sufficient resources of raw materials.

The challenge faced by the authors, was the creation of a new drug that has immunostimulating action on the basis of fungi of the genus Pleurotaceae, having a sufficient resource base and how to obtain it.

The basis of the decision task was the investigation of the potential application as a promising raw material for production of immunomodulators widespread on the territory of Russia mushroom Oyster in which the falsity of cultivation. Now WU is widely used for nutritional purposes. Messages about research medical use of this fungus as immunostimulant in the reviewed literature was not found.

During the research it was found that the immunostimulating properties of a polysaccharide fraction with a molecular mass of from 10 to 420 KD isolated from fruit bodies and/or Strom oyster.

This fraction may be used alone or in a mixture with carbohydrates (glucose, galactose, etc.,) in the ratio (mass.) 5-35:95-65 or a mixture of carbohydrates and proteins WU at a ratio (mass.) 5-9:60-71:20-35.

It is more profitable to get the fraction of the stroma of the fungus, which are by-products of their processing. The product obtained from selected fragments of the fungus fruit bodies and/or stroma), which was pre-dried at a temperature of 60oWith, and crushed, and then it was removed lipids by extraction of the alcohol-containing solution, and the active principle was isolated from the residue by extraction with boiling water, the solution was concentrated, besieged polysaccharides processing of alcohol-containing solution and dialyzed. The resulting product is optionally subjected to additional cleaning, nab missing ingredients to a given composition. As the alcohol-containing solution used, as a rule, the solution containing 85% ethanol.

Technology of production of the product and its properties are described in the following examples.

Example 1. 500 g of fruit bodies and Strom oyster mushrooms were dried at 60oC, and crushed in a ball mill. Received 180 g powder.

100 g of the obtained powder was extracted twice in to conventional Soxhlet extractions 500 ml of 85% ethanol for 4 hours to separate lipids. From the precipitate was isolated polysaccharide fraction two-time processing 350 ml of boiling distilled water for 3 hours.

The extract obtained was filtered and was evaporated under vacuum and was treated with 85% ethanol for 8 hours. The precipitate was subjected to dialysis, gel filtration and lyophilization. The result was obtained 5.8 g of powder of light yellow color, containing biologically active polysaccharides from Strom oyster with a molecular mass of 10-420 KD (1 Drug). The main component of fraction was beta-D-glucan, in particular, beta-(1,6)-D-glyukopiranozil and branched beta-(1,3)-D-glycerin.

Example 2. The study of immunostimulatory properties of the product of example 1 was carried out as follows. At first the JVI was carried out according to a standard method, described by Boyum [Boyum A. Isolation of mononuclear cells and granulocytes from human blood. Isolation of mononuclear cells by centrifugation. // Scanol J. of Clinical and Laboratory Investigation.- 1968. - v. 41, p. 77 - 89] on the gradient ficoll pack (tank density 1078).

The plasma of blood donors was layered on the gradient ficoll pack and centrifuged for 40 minutes at 400 g. To obtain mononuclear cells were collected cells in the interphase on the boundary of the plasma and ficoll pack, washed twice with saline. In the reaction used cells at a concentration of 5 million cells/ml in medium 199 containing 10% serum of fetuses of cows.

Neutrophils were obtained from the sediment after fractionation on the gradient ficoll pack. The admixture of erythrocytes were removed by hypotonic shock with distilled water for 40 seconds. The obtained cell suspension is washed twice in saline.

The influence of the drug 1 on chemotaxis of neutrophils was performed in the test migration under agarose by Nelson [Nelson, K. D. Chemotaxis under agarose // J. Immunol. - 1975. - v. 115.- p. 1650 and 1656]. This was prepared 1% solution of agarose in medium 199 containing 10% serum fruits cows. The agarose gel was poured into 4 ml plastic Petri dishes by "metroliner" diameter 40 mm After solidification of the gel in it punch punched holes with a diameter of 2.2 mm From the ln. cells/ml) in medium 199 supplemented with 10% serum of fetuses of cows. The cell suspension was brought to 0.01 ml to middle hole. In the same quantities in the left well made medium 199, and the right study drug. Petri dishes were placed in CO2incubator (concentration of CO25%, the temperature of the 37oC) for 90 minutes. Upon completion of the reaction in the Cup was poured in 1 ml of 10% formalin solution and left for 12 hours. Then removed the agarose disk, cells were zafiksirovali within 10 minutes 70% ethanol and stained by Romanovsky-Giemsa [Beyer C. A. a Brief guide on Hematology. - L., 1973, S. 43-44.]. The result was taken into account using the projector, measuring with a ruler the length of run of cells for the prototype (to the right of the hole edges) and to control (to the left of the hole edges). The reaction expressed in the indices of migration, which is the ratio of the length of run of cells in the experience of the path length control. Studied as its own chemotactic properties of the drug and the ability of the drug to activate the serum of a person with the formation of strong chemoattractant for neutrophil - Sa. To do this, the serum of the donor were incubated with drug for 30 minutes at 37oWith, then centrifugation was besieged by drug and serum ispolzo peripheral blood was studied using Lomonosovskiy chemiluminescence (CHL), and in NBT-test, based on the ability formed during cell activation superoxide radical to restore salt of tetrazole.

The effect of the drug on Lomonosovsky chemiluminescence of whole blood was studied on chemiluminometer (model 1251 LKB, Sweden). The mother liquor lyuminola (0.01 M) was prepared by dissolving 1,77 mg lyuminola in 1 ml of dimethyl sulfoxide. The experiment was carried out as follows: to determine background levels of CHL in polystyrene vials made in 0.7 ml of Hanks solution; 0.1 ml whole blood; 0.2 ml lyuminola at a concentration of 10-4M Level induced chemiluminescence (positive control) was studied in samples that were made: 0.6 ml Hanks solution, 0.1 ml of whole blood; 0.2 ml lyuminola working dilution, 0.1 ml of PMA at a final concentration of 10 ng/ml To study the effects of the studied extracts on chemiluminescence of whole blood in vials made in 0.6 ml of Hanks solution, 0.1 ml of whole blood, 0.2 ml lyuminola and 0.1 ml of the oyster in appropriate dilutions. The chemiluminescence was assessed by the magnitude of the maximum response (maximum response in mV) and the integral intensity of chemiluminescence for 30 minutes (sutasoma in mV /min). Lomonosovskaya PI allows to judge about the whole spectrum of oxygen metabolites and products is a thief narasinga of tetrazole (PCT) in Hanks solution (pH of 7.2). Neutrophils resuspendable at a concentration of 2 million cells/ml in a solution of HCT and contributed 0.1 ml in 96-well card. The solution of the test drug was made 0.01 ml. Board was placed in CO2-incubator. After 1 hour of circuit boards poured the CNT solution, the precipitated cells were dried and fixed with 70% ethanol. Formed in cells in the reduction reaction of NCT of diformate was suirable of 0.12 ml of 2M KOH and 0.14 ml of dimethylsulfoxide (DMSO). The result was considered on multiscale at a wavelength of 640 nm. Positive control reactions were formalistic acetate (PMA) at a final concentration of 10 ng/ml. the results of the reaction expressed in units of extinction.

To study cytokine production in cell suspension of mononuclear cells in 0.1 ml were placed in 96-well card was added 0.01 ml of the study drug, the card was placed in CO2incubator for 18-20 hours. At the end of the incubation were collected conditioned medium and analyzed in the content of cytokines by ELISA. The preparations were studied in the form of a homogeneous suspension in physiological solution. The results of the tests are shown in tables 1-6.

As follows from the data presented in tables 1-4, the drug has the ability visit-tractants for neutrophils human blood, contributes to the accumulation of Sa component of complement induces the synthesis of proinflammatory cytokine - interleukin 1 (IL-1).

I.e. declare the drug belongs to the group of drugs with immunomodulatory, in particular an immunostimulating action.

Example 3. Under the scheme of example 1 were getting polisakharidami fractions with modification of the conditions of receiving the active agent and to create compositions based on them. The results are shown in table 5. The activity of some drugs has been studied on the basis of their impact on Lomonosovsky chemiluminescence of whole human blood (mV for 30 min) according to the method of example 2. The results are shown in table 6.

As shown, when using mixtures becomes possible to reduce the consumption of the most valuable component of polysaccharides, as well as create a variety of drugs with a wider range of applications.

1. Immunostimulating drug on the basis of biologically active substances by fungi of the genus Pleurotaceae, characterized in that the active agent it contains polysaccharides of Pleurotus ostreatus with a molecular mass of 10 - 420 CD.

2. Immunostimulating drug under item 1, the beginning, wt. %:

Polysaccharides - 5 - 35

Carbohydrates - 95 - 65

3. Immunostimulating drug on PP. 1 and 2, characterized in that it additionally contains proteins in the following proportions of active ingredients in the beginning, wt. %:

Polysaccharides - 5 - 9

Carbohydrates - 60 - 71

Proteins - 20 - 35

4. The method of obtaining immunostimulating drug from fungi of the genus Pleurotaceae, which includes the processing of boiling water, precipitation of the active agent with an alcohol-based solution and its treatment, characterized in that the feedstock used stroma Pleurotus ostreatus, which are pre-dried at 60oAnd clear of lipids by treatment with an alcohol-based solution, and the selection of the active principle of the precipitate is carried out by dialysis.

5. The method according to p. 4, characterized in that after dialysis, the drug is subjected to gel filtration with subsequent lyophilization.

6. The method according to p. 4, characterized in that to the drug add carbohydrates and proteins and such.

 

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