Preparation with polyfunctional medical-biological activity that influences tissue exchange based on fungus strain pleurotus ostreatus vkpm f-819

FIELD: biotechnologies.

SUBSTANCE: preparation with polyfunctional medical-biological activity that influences tissue exchange is produced as a result of liquid-phase depth cultivation of fungus Pleurotus ostreatus All-Russian collection of industrial microorganisms F-819 with further separation of mycelium and cultural liquid and extraction of biologically active substances in the form of a condensed extract from the mycelium. The produced extract, which represents a pharmacological substance of a preparation, is enriched with the agent of the derivative of sterol 4-hydroxy-17P-methylinecisterol with molecular weight of 332.2452 Dalton, polysaccharide β 1-3 glucan and dihydroquercetine with molecular weight of 304.26 Dalton. At the same time the specified components are taken at the following weight ratio: (1):(2):(3):(4) as (79.0-158.0):(1.0-2.0):(10.0-20.0):(10.0-20.0).

EFFECT: invention makes it possible to increase quality of a preparation as a result of increasing its antitumoral activity and reduced toxicity of preparations of antitumoral chemotherapy with simultaneous application with the suggested preparation.

2 cl, 21 dwg, 11 tbl, 2 ex

 

The invention relates to medicine, health and biotech industries, in particular strains-producers of biologically active substances and preparations on their basis, and can be used in the manufacture and application of physiologically active drugs and food additives on the basis of mushrooms - producers.

One of the most promising groups of producers, whose chemical composition of biologically active substances and the properties of these substances currently studied well enough, are higher basidiomycete mushrooms.

The study of therapeutic properties of higher basidiomycetes and their practical application in the field of medicine scientists of several countries over the past 50 years. The results of these studies are particularly widely used in China, Japan, Korea, USA, Canada, Russia and more recently in Norway.

Among the metabolites of higher fungi isolated and identified substances from basidiomycetes, which are immunomodulators and antineoplastic, cardiovascular, antiviral, antibacterial, antiparasitic, hepatoprotective and antidiabetic properties /Materials of IV International conference "Science and practice of mushroom"/ M, the international Association of Mushroom. 1997 - 92 S.; prospects for the use in medicine substances, arr is used basidiomycete mushrooms. Overview /International Journal of Medicinal Mushrooms. Vol.3, 31-62, 1999)/.

About 200 species of mushrooms, which have distinct properties mentioned above, but only about 10 species of fungi are used to obtain drugs. However, most of biologically active substances in the composition of these fungi is not fully specified. For example, in Japan, China, Russia, Ukraine, Belarus, USA, Canada and Norway have developed several drugs-polysaccharides used as medicines. Get them under cultivation in submerged conditions Trametes versicolor (PSK, Krestin; Japan; Glucanova, Norway 2010).

However, the creation of new drugs to date is largely constrained by the complex costly production technology from basidiomycetes lines pure cultures and substances that have the stability required medicinal properties. Primarily this is due to the fact that education is valuable for pharmacology metabolites depends on the cultivation conditions of the producer and, according to most authors, is unstable tamosauskas specific character.

Therefore, at present we do not have modern, safe drugs on the basis of biologically active substances derived from higher basidiomycetes, with polyfunctional specific medical biologists who eskay activity and the ability to enhance the expression of antitumor activity, accompanied hepatoprotective and detoxifying effect with normalization of the lipid spectrum of blood serum.

For the treatment of cancer are widely used anticancer chemotherapy with the use of a variety of cytotoxic agents. To most effectively used are cyclophosphamide, doxorubicin, Vepesid, taxanes, metotrexat, 5-fluorouracil applied in practice, both separately and in combination with each other.

However, a significant limiting factor in achieving maximum efficiency of anticancer therapy is the toxicity of cytotoxic drugs commonly used in chemotherapy. It is toxic manifestations in the course of their application and especially hematologic toxicity, as a rule, limit the provision of adequate treatment. Therapy is often reduced doses of drugs with longer intervals between courses and, as a result, treatment is not always satisfactory.

It is known that long-applicable courses of chemotherapy using known drugs greatly increases the risk of developing liver disease, including steatosis of the liver, the so-called non-alcoholic steatohepatitis (Nash) (fatty infiltration of the liver).

For example, the use of cyclophosphamide, methotrexate and doxorubicin can cause the development of hepatitis the, accompanied by increased activity in blood enzymes of hepatic origin (aminotransferases, gamma-glutamyltranspeptidase, acid and alkaline phosphatase), hyperbilirubinemia, fibrosis, degeneration of the liver cholestasis IVanSaun .N., Lee I.K., Washington, M.K., Matrisian L, Gorden L. High fat diet induced hepatic steatosis establishes a permissive microenvironment for colorectal metastases and promotes primary dysplasia in a murine model // Am. J. Pathol. 2009. V.175(1). P.355-364.; Blokhin H.H., Perevozchikova NI Chemotherapy of neoplastic diseases. M.: Medicine, 1984. 303 S.; Spitzer So, Cirenza E., McAfee, S., Foelber R., Zarzin J., Cahill R., Mazumder A. Phase I-II trial of high-dose cyclophosphamide, carboplatin and autologous bone marrow or peripheral blood steam cell rescue // Bone Marrow Transplant. 1995. V.15, No. 4. P.537-542/.

For this reason, it is difficult to find effective drugs for the treatment of oncological diseases with the development of Nash. Modern approaches to the treatment of this pathology is mainly aimed at the elimination or weakening of the factors leading to the development of steatosis: a low-calorie diet, correction of hyperlipidemia and hyperglycemia, the abolition of potentially hepatotoxic drugs. However, these activities have little therapeutic efficacy and the fact that only a relatively small portion of patients.

More meaningful results could be obtained in patients with cancer, if used in chemotherapy drugs or drugs "support" in the integrated Le is the situation of patients, additionally having antitumor activity, had a hepatoprotective and detoxifying effect, manifested in the lock Nash, and normalize lipid profile in serum.

Morphological term Nash, or in another terminology "fatty infiltration", refers to a pathological process in which the cytoplasm of hepatocytes is progressive accumulation of fat droplets (Yakovenko and others, 2004).

Currently, in the field of prevention and the preservation of human health, the largest and more obvious problem is to stop the destruction of the human body from the chemicals.

That is why in the last 15 years actively developed approaches for the creation and application of new drugs on the basis of biologically active substances that reduce the toxic manifestations of anticancer drugs. To date, however, in practical medicine no drugs, effective enough for widespread use.

Among the well-known recommended by the Ministry of health of the Russian Federation of this kind of herbal medicines, since 1996, we must first of all include: BAA from brown algae "Klemin" /Hygienic certificate of the state Committee of sanitary and the Russian Federation No. 1P - 11/100 from 10.02.1995,/; "Petalon" and "Petalon - M /Hygiene certificate the cat of the state Committee of sanitary and the Russian Federation No. 1P - 11/101 from 10.02.1995,/; the drug from the tissue culture of ginseng "Biosensing" /VFS-42-1890-89/.

Inclusion in the scheme of combined and complex treatment of patients with cancer of the breast, esophagus and rectum above herbal adaptogens allows, on the one hand, reduce the damaging effect of operative trauma, chemotherapy and radiation therapy on the body, to reduce the frequency of specific complications of therapy, and on the other hand, increase protective forces of an organism, to a specific treatment, i.e. to achieve synergistic antitumor activity.

However, the above medicines (herbal adaptogens) do not possess antitumor activity, and their integrated use in combination with chemo - and radiation therapy do not have the ability to modulate the processes of reduction of hematological toxicity, accompanied by detoxifying and hepatoprotective activity and normalization of lipid profile in serum of biological systems, which complicates the complex treatment of cancer patients.

Known inducer of apoptosis polypeptide complex HA-40, which was developed in the early 90-ies in biomedical research and production center Alexis (Tbilisi, Georgia). Raw source of the drug is in the form of peptide lyophilized powder is a mountain plant Polygonatum mountain (Polgonatum varticillatum). After carrying out biochemical studies, quantitative and qualitative composition revealed that the product contains several hundreds of small peptides with molecular weight of 10-50 KD. To determine the composition used high-performance liquid chromatography (HPLC) followed by tandem mass spectrometry using LTQ FT ICR mass spectrometer (Hybrid-2D-Linear Quadrupole Ion Trap, Fourier Transform Ion Cyclotron Resonance Mass Spectrometer) (Thermo Electron Corp). /Great N.N. Report on the determination of the quantitative and qualitative composition of the drug HA-40. Institute of biochemistry. Palladin of NAS of Ukraine, Kyiv, 2007/.

The drug HA-40 passed a series of experiments in vitro and in vivo, which reliably demonstrated some therapeutic effects of its peptide structure in the complex, namely:

the polypeptides of the drug HA-40 interact with the membrane structures of atypical cells and initiate the apoptotic mechanism / Lawrence Loeb, A., "Investigation of molecular mechanisms of the anti-carcinogenic action of the GA-40 preparation" University of Washington, School of Medicine, Department of Pathology; WDC, 2005/;

in in vivo studies found that the drug HA-40 mice at doses of 2, 20 and 200 mg/kg causes a significant increase in the number of antibody productive cells 1.5, 2 and 2.4 times, respectively, compared to animals not treated with the drug;

in vitro experiments established that the joint cultivation of the drug G is -40 at a concentration of 100 µg/ml mononuclear cells of peripheral blood of healthy donors causes a significant increase in the production of interferon-gamma;

- the drug HA-40 possesses strong antioxidant and anti-stress action. It does not cause allergic reactions of immediate type and psevdoallergicakie reactions /Humopehy, Akhmetiev, "Study of immunotropic activity of the drug HA-40", "study of the cellular and molecular mechanisms of drug action HA-40", Institute FHM, Moscow, 2006/.

Analysis of medico-biological activity known drug HA-40 - inducer of apoptosis shows that despite reliably demonstrated some therapeutic effects of the drug HA-40 is not applied in the practice of medicine as a drug "support" in complex therapy of oncological diseases, as it has no ability to remove a manifestation of a General toxicity and hematological toxicity caused by the applied drugs, to have hepatoprotective and detoxifying effect, resulting in blocking the development of Nash and normalize lipid profile in serum of biological systems.

It is for the same reason none of the known methods of treating cancer patients with the use of well-known adaptogenic plants cannot be considered sufficiently effective and widely applicable.

As for modulators that reduce toxic manifestations of the anticancer drug is a biologically active substance, isolated from higher basidiomycetes, with polyfunctional medico-biological activity that is causing or intensifying together with cytostatics cell death of malignant tumors by induction of apoptosis, thus to exert hepatoprotective and detoxifying effect, resulting in blocking the development of steatohepatitis and normalize lipid profile in serum of biological systems, information about similar compounds in the literature are missing.

Known "Drug, influencing tissue metabolism and modeling the processes of immunity in biological systems, and biologically active food Supplement "Mipro - VIT"" /RF patent 2092179, CL AC 35/84, A23L 1/054, 1996/used in medicine for the prevention and treatment of diseases associated with disorders of the metabolism and functioning of the immune system, in particular capable of stimulating the activity of antioxidant systems of the body and, as a consequence, bind and excrete radionuclides, fundamentally violates tissue metabolism in the body due to the inactivation of proteins-enzymes. Famous drug is not used in medicine as a modulator, reduces toxic adverse effects of drugs, and in this direction have not been investigated.

Known "Drug, influencing tissue metabolism and the application of strain of the fungus Fusariu Sambucinum Fuskel var. ossicolum (berk. etcurf) bilai to obtain /patent RF 2040932, CL AC 35/70, 1993/used in the medical industry for the production of food additives and medicine for the prevention and treatment of diseases associated with metabolic disorders, with adaptogenic and immunomodulatory effects. This drug has a positive effect on the disturbed metabolic processes in the body, leading to cardio-vascular diseases, obesity, diabetes, immunodeficiency States, avitaminosis, and combined with traditional therapies, increasing their effect on the body and reducing adverse effects, in particular when carrying out radiotherapy for cancer patients. However, this drug is also not used in medicine as a modulator that reduces the toxic manifestations of anticancer drugs, because in this direction have not been investigated.

Well-known drug, influencing tissue metabolism, and the use of strains of the fungus Pleurotus ostreatus 1137 to obtain /patent RF 2192873, CL AC 35/70, 2001/used in the medical and biotechnology industry for the production of physiologically active drugs and food additives on the basis of mushroom producers of biologically active substances used in medicine to reduce the toxic effects of anticancer ven the drugs (prototype).

According to the description and formula of this invention, a known drug has anti-tumor activity and the ability to modulate the process of reducing hematologic toxicity of cytostatics. The preparation is produced by submerged cultivation of the fungus Pleurotus ostreatus 1137 (VKPM F-819) with subsequent separation of the mycelium and the culture fluid and the release of the mycelium extraction with ethanol at acidic or neutral pH environment of biologically active substances with antimicrobial activity, containing amino acids (glycine, alanine, threonine, serine, leucine) and carbohydrates (glucose, glucosamine, arabinose, xylose).

The drug is made in the form of dehydrated granules or tablets, or in the form of gel syrup.

The strain of Pleurotus ostreatus 1137 (VKPM F-819) is used as a producer of physiologically active substances with antitumor activity.

In addition, the drug exhibits antimicrobial activity against both gram-positive and gram-negative bacteria.

On the basis described in the patent strain of the fungus Pleurotus ostreatus 1137 (VKPM F-819) received the drug with a broad spectrum of preventive and curative actions, including antioxidant activity.

Experience in the use of the above product (prototype) shows that the physiological action of the drug is capable of the tee to influence tissue metabolism and modulate processes reduce hematologic toxicity effective and widely used in the clinic anticancer drugs. The use of this drug can improve the effectiveness of cancer chemotherapy on the background of reducing the toxic effects of cytostatics in the complex treatment of oncological diseases.

However in the course of biomedical and clinical trials we found that individual use of the drug, developed in accordance with the patent of the Russian Federation No. 2192873, not effectively induces apoptosis of cancer cells and almost has a low physiological activity to the ability to enhance liver and detoxifying effect and normalize lipid spectrum of blood serum in the complex treatment of cancer patients.

Therefore, in the invention it is the application of strain Pleurotus ostreatus 1137 for creation on its basis of preparation with a polyfunctional medico-biological activity affecting tissue metabolism, which is an inducer of apoptosis, the mechanism of action is based on the ability to induce cell death of malignant tumors and thus to exert hepatoprotective and detoxifying effect, resulting in blocking the development of steatohepatitis with normalization of lipid profile in serum of biological systems and have no contraindications for use.

Apoptosis is a form of cell death, which is manifested in the changing the properties of the cytoplasmic membrane, condensation and fragmentation of chromatin /Imyanitov E.N., Kuligina E.Sh., Belogubova E.V., Togo A.V., Hanson K.P. Mechanisms of lung cancer. Drug Discov. Today: Dis. Mech., 2005a, 2, 213-223/.

Due to the fact that cancer cells lose the ability to differentiation and acquire the ability to uncontrolled reproduction, one of the priorities of cancer therapy is the selection of drugs that induce apoptosis of cancer cells.

The present invention is the development and creation of the drug from the strain of Pleurotus ostreatus 1137 (VKPM F-819) with polyfunctional medico-biological activity affecting tissue metabolism and have anti-tumor activity with the induction of apoptosis of cancer cells, as well as the ability to modulate the process of reducing hematologic toxicity used anticancer drugs, accompanied hepatoprotective and detoxifying effect with normalization of lipid profile in serum of biological systems.

The technical result consists in increasing the quality of the drug by increasing its antitumor activity and lower toxicity of cancer chemotherapy while applying them together with the proposed drug with the ability to inhibit the proliferation of cancer cells, as well as gepatoprotektivnym and detoxifying effect, prolapsus is MSA in blocking the development of steatohepatitis and normalization of the lipid spectrum of blood serum.

The result is achieved that the claimed product with a polyfunctional medico-biological activity affecting tissue metabolism, the resulting liquid submerged culture of the fungus Pleurotus ostreatus 1137 (VKPM F-819) with subsequent separation of the mycelium and the culture fluid and the release of the mycelium of biologically active substances in the form of condensed extracts with antimicrobial activity, which is a pharmacological substance preparation and enriched with active start-derived Sterol 4-hydroxy-17R-metalinsulator with a molecular mass of 332,2452 Dalton, polysaccharide β 1-3 glucan and dihydroquercetin with a molecular mass of 304,26 Dalton, the components are taken in following mass proportions:

(1):(2):(3):(4) as(79,0-158,0):(1,0-2,0):(10,0-20,0):(10,0-20,0).

The drug has the ability to inhibit the proliferation of cancer cells, as well as hepatoprotective and detoxifying effect, resulting in blocking the development of steatohepatitis and normalization of lipid profile in serum of biological systems.

The drug is made in the form of a gel or syrup, or pill, or dehydrated granules.

In the above text the mass ratio of component(1):(2):(3):(4) in the final form of the proposed drug as the 1st component used condensed extracts with antimicrobial AK is ewnetu, which is a pharmacological substance of the drug; as the 2nd component is used, the active principle derived Sterol 4-hydroxy-17R-metalinsulator with a molecular mass of 332,2452 daltons; as the 3rd component used polysaccharide β 1-3 glucan; as the 4th component is used dihydroquercetin with a molecular mass of 304,26 Dalton.

Strain culture of Pleurotus ostreatus 1137 stored in the all-Russian collection of Industrial Microorganisms, state research Institute of genetics under the registration number VKPM F-819 and is characterized by the following features.

Cultural and morphological characteristics

1. Growth on agar media

Agar wort

The mycelium is well developed, vatoobraznye. Growing mycelium creeping, the edge of the colony felt, smooth. The growth rate is high: for 7-9 days after sowing in the centre of Petri dishes 10 cm in diameter, there is a complete fouling agar medium. The color of the mycelium white. Azopigments missing. During prolonged cultivation (up to 30 days) the color of the mycelium is not changed.

Hyphae septate, branching frequent, at an acute or right angle, type of branching monopodial. Formed buckles. The diameter of the majority of hyphae from 2 to 4 microns; also present hyphae with a diameter of from 1 to 8 microns.

Peptone-tripcony agar

The mycelium is well developed and felt. The growth of creeping, Rast is a first edge of the spider, smooth. The growth rate is high. The color of the mycelium white, with long-term cultivation (more than 21 days) possible fawn or light brown areas. Azopigments missing.

Soy agar

Mycelium sparse, spider, through the visible mycelium agar medium. Character growth creeping, growing edge smooth, spider. The growth rate low: after seeding in the centre of Petri dishes 10 cm in diameter, there is a complete fouling agar medium only at 12-14 days of growth. The color of the mycelium white. Azopigments missing.

2. Deep cultivation

Deep cultivation is carried out in flasks with a capacity of 750 ml with medium volume 40...150 ml shaker with 200 rpm. Sowing was performed pieces of agar with mycelium.

The environment wort

The number of colonies depends on the load of seed. 10-14 days of growth forms several dense colonies rounded shape formed around the seed, and/or many minor daughter colonies. The color of the mycelium white to light-beige. The color of the medium during cultivation lightened. The transparency of the environment does not change. The weight of the mycelium is directly dependent on the concentration of the wort in the environment (from 0.6 to 3 ballingal).

Environment with soy flour

The number of points of significant growth, which is associated with the immobilization of mycelium on the particles of soy flour in the environment. Growth gustonaseyolennyh. When resting mycelium practically settles.

Physiological and biochemical characteristics

Strain, as well as other representatives of the genus Pleurotus, grows on various sources of plant raw materials containing cellulose.

The strain utilizes glucose, sucrose, dextrin, starch, cellulose, glycerin.

As a source of nitrogen strain uses peptone, tripton, soy and oat flour.

Grows in the temperature range from 8 to 34°C, maintains viability at temperatures from 0 to 40°C, the optimum temperature for growth of 24°C.

Grows at pH values from 4 to 8, when the optimum from 6 to 7.

Strict aerobe.

Pathogenicity

The strain is not pathogenic for humans and animals, but as the representative of Pleurotus ostreatus, pathogenic for immunocompromised deciduous trees. Basidiospores can cause allergic reactions in people.

Antagonistic properties

During submerged cultivation appears antimicrobial activity against gram-positive, gram-negative bacteria and fungi.

New appointment strain of Pleurotus ostreatus 1137 lies in its use as a producer of biologically active substances to create a product with a polyfunctional medico-biological activity affecting tissue metabolism and has the ability to inhibit the proliferation of cancer cells, and the e gepatoprotektivnym and detoxifying effect, manifested in blocking the development of steatohepatitis and normalization of lipid profile in serum of biological systems.

For surface cultivation of Pleurotus ostreatus 1137 and test strains used tripcony agar and soy agar medium, oatmeal agar medium and wort agar.

Product with a polyfunctional medico-biological activity affecting tissue metabolism, got biotechnical method using fungus Pleurotus ostreatus 1137. This culture was grown deep way in sterile liquid nutrient medium.

The process of obtaining the drug included 6 sequentially executed steps:

STAGE I - growing biomass of mycelium:

- breeding strain;

- preparation of inoculum (on agar medium);

deep cultivation of mycelium (cultivation in liquid medium and separating the mycelium from the culture fluid).

STAGE II - the Production of condensed extract of Pleurotus ostreatus mycelium 1137 in the form of a gel, comprising the pharmacological substance preparation:

extraction;

- filtering;

- evaporation of the extract (in a vacuum).

PHASE III - Selection of the selected part of the condensed extract of Pleurotus ostreatus mycelium 1137 in the form of a gel active principle derived Sterol 4-hydroxy-17R-metalinsulator dichloromethane.

STAGE IV - Vyd is of recycled Pleurotus ostreatus mycelium 1137, after extraction from it condensed extract in the form of a gel, polysaccharide β 1-3 glucan, obtained by enzymatic hydrolysis of the extracted ethanol mycelium.

V STAGE - ready-made forms of dihydroquercetin used in the composition of the drug as additional substances to the condensed extract, isolated from an extract of Pleurotus ostreatus mycelium 1137.

The sixth STAGE is the Preparation of the finished form of the drug in the form of a gel or syrup or tablets or dehydrated granules.

To determine the antimicrobial activity of the extract of Pleurotus ostreatus mycelium 1137 (pharmacologic substance) deep cultivation of mycelium were carried out in flasks with a volume of 750 ml in a medium containing 0.65 balling wort; tap water, pH was not adjusted. Also used the environment Etter, Wednesday, 2663, meat-peptone broth and their modifications. The volume of medium in the flasks was 40...150 ml. For aeration flask was placed on a rotary shaker with 200 rpm Cultivation and incubation was conducted from 7 to 24 days at a temperature of 26... + 28°C in a controlled aseptic conditions in a liquid nutrient medium with vigorous aeration.

Test strains were incubated at a temperature of 28...37°C for 17...24 hours.

Found that among the tested culture media optimal for the growth of mycelium biomass and antimicrobial activity and is 0,65 ballymaloe wort.

To obtain the drug at first, the mycelium was separated from the native solution by centrifugation.

From the mycelium of a biologically active substance was extracted with ethanol at acidic pH values.

The extract was evaporated in vacuo to a gel-like state with the percentage of water up to 30%. For biological tests used one stripped off the concentrate in the form of a gel, isolated from the mycelium at acidic pH values.

Extract of Pleurotus ostreatus mycelium 1137 in the form of a gel is a mixture of low molecular weight biologically active substances with antimicrobial activity.

To determine the antimicrobial activity of the extract of Pleurotus ostreatus mycelium 1137 as the test cultures used gram-positive and gram-negative bacteria.

Gram-positive bacteria:

Bacillus mycoides 537;

Bacillus pumilis NCTC 8241;

Leuconostoc mesenteroides VKPM B-4177;

Micrococcus luteus NCTC 8340;

Staphylococcus aureus FDA 209P (MSSA);

Staphylococcus aureus UHA 00761 (MRSA);

Gram-negative bacteria:

Escherichia coli ATCC 25922;

Pseudomonas aeruginosa ATCC 27853;

Comamonas terrigena VKPM B-7571.

Antimicrobial activity of the extract of Pleurotus ostreatus mycelium 1137 was determined by the method of diffusion in agar.

In Petri dishes 10 cm in diameter was poured into 15 ml tryptanol agar. On the frozen surface of the medium were sown in solid turf culture test.

The concentrate of the extract of Pleurotus ostreatus mycelium 1137 in which Alceste 10 ml were applied to discs of filter paper with a diameter of 6 mm The disks were placed on the surface of agar medium seeded test cultures.

After days of incubation revealed a zone of growth inhibition of the test strain.

It was established that this strain of Pleurotus ostreatus 1137 10-14 days of growth, there is the greatest antimicrobial activity against both gram-positive and gram-negative bacteria (table 1).

The goal of biomedical research claimed the drug was to obtain data to ensure the safety of its use in medical purposes.

Studied and standardized:

- the chemical composition of the drug, comprising: a pharmacological substance, comprising the extract of Pleurotus ostreatus mycelium 1137 in the form of a gel; the active principle derived Sterol 4-hydroxy-17R-methylenetetra; polysaccharide β 1-3 glucan; dihydroquercetin;

- the toxic effect of the drug on mice of both sexes line BAL, kennel breeding RAMS "Pillar", in the acute experiment for two doses of 100 mg/kg 200 mg/kg 100 mg of the finished product comprises: 79,0 mg condensed extract isolated from Pleurotus ostreatus mycelium 1137 constituting a pharmacological substance of the drug; 1.0 mg derived Sterol 4-hydroxy-17R-methylenetetra constituting the active principle of the drug; 10.0 mg of polysaccharide β 1-3 glucan; 10.0 mg of DigiTrak is retina. 200 mg dose of the finished product comprises: 158,0 mg condensed extract isolated from Pleurotus ostreatus mycelium 1137 constituting a pharmacological substance of the drug; 2.0 mg derived Sterol 4-hydroxy-17R-methylenetetra constituting the active principle of the drug; 20.0 mg of the polysaccharide β 1-3 glucan; 20.0 mg of dihydroquercetin;

- the action of the active principle derived Sterol 4-hydroxy-17R-metalinsulator at doses of 1.0 mg/kg and 2.0 mg/kg at the cell level in vitro using a simple system of cultivation of normal cells (fibroblasts) and transformed (Hela);

- antitumor activity of the drug on the model of solid tumor B16 melanoma, transplantable inbred mice BDF1the hybrids of the first generation f1(C57B1/6×DBA2) kennel breeding RAMS "Pole". Antitumor activity of the finished form of the drug and of its constituent substances was studied taking into account the obtained results of toxic action of the drug on the following versions of the applied doses:

- option 1 - pharmacological substance of the drug, including condensed extract of Pleurotus ostreatus mycelium 1137 in the form of a gel at a dose of 100 mg/kg;

- option 2 - the active principle derived Sterol 4-hydroxy-17R-methylenetetra at a dose of 2.0 mg/kg;

- option 3 - the claimed preparation in the form of a gel at a dose of 100 mg/kg;

- option 4 - the claimed preparation in the form of syrup at a dose of 6.0 ml/kg, which corresponds to a dose of 75 mg/kg of the drug in the form of a gel;

- option 5 - the claimed preparation in the form of syrup at a dose of 8.0 ml/kg, which corresponds to a dose of 100 mg/kg of the drug in the form of a gel;

the pharmacological effect of the substance preparation and of cyclophosphamide on hematological parameters of peripheral blood of healthy mice;

- hypolipidemic and hepatoprotective effects of the drug and of its constituent substances in the development of experimental hepatic steatosis on the model of atherogenic diet (AD) nonlinear white lab sexually Mature male rats obtained from the nursery of the FSUE OPH "Manichino". In addition, hypolipidemic and hepatoprotective effects of the drug and of its constituent substances was studied in the following embodiments, the applied doses:

- option 1 - active start (EN PR) derived Sterol 4-hydroxy-17R-methylenetetra at a dose of 2.0 mg/kg;

- option 2 - pharmacological substance (FS PR) extract of Pleurotus ostreatus mycelium 1137 in the form of a gel at a dose of 100 mg/kg;

- option 3 - the claimed preparation in the form of syrup at a dose of 8.0 ml/kg, which corresponds to a dose of 100 mg/kg of the drug in the form of a gel.

On the basis of experimental studies produced the following results.

A. the Study of the chemical composition and standardization of the extract of the mycelium of Pleurotus ostreaus 1137 in the form of a gel, constituting a pharmacological substance of the drug was carried out after evaporation of the ethanol extract in vacuum at t=40-50°C until a homogeneous viscous composition consisting of a mixture of lipophilic and hydrophilic compounds.

As a result of studying the chemical composition of the extract of Pleurotus ostreatus mycelium 1137 in the form of gel was determined as a percentage of the mass included in the extract mass fractions of the following biologically active substances: carbohydrates; amino acids; higher fatty acids; organic acids; vitamins; minerals content in the composition of the pharmacological substance has a significant impact on the effectiveness of medical and biological activity of the finished form of the proposed drug.

To study the chemical composition of the extract of Pleurotus ostreatus mycelium 1137 biologically active substances were sampled condensed extract in the form of a gel obtained within 12 months (since the beginning of January to December inclusive).

Sampling of the extract and their preparation for the analysis was carried out according to GOST 28495, 1511.0-77, GOST 20438-75.

Determination of the mass fraction of carbohydrates was performed by the method of adsorption chromatography based on chromatographic separation of carbohydrates on the analyzer Sugars Boitronic LC 2000 with refractometric detection bicinchoninic and ninhydrin according to the national /GF XI, issue 1, p.95 with changes/.

The determination of the content in the extract of amino acids was performed by the method of ion-exchange chromatography of amino acids on a strong cation-exchanger: sulfonated copolymer of styrene with divinylbenzene by analysis of amino acids on the amino acid analyzer "Hitashi" model 835, Japan /GF XI, issue 1, p.95; S.Moore, D.Sparckman and W.Stein. Chromatography of amino acids on sulfonated polystyrene resins, Analit. Chem., V.30, p.p.1185-1190. 1958/.

The determination of the content in the extract higher fatty acids was performed by the method of quantitative allocation of fatty acids from complex multi-component natural mixtures and subsequent chromatographic analysis of methyl esters /guidelines on quality control methods for P 4.1.1672-03 Ministry of health, 2004, p.24/.

The determination of the content in the extract of organic acids was performed by high performance liquid chromatography on a color chromatograph "Kontron" with UV detector /guidelines on quality control methods for P 4.1.1672-03 Ministry of health, 2004, St/.

The determination of the content in the extract, vitamins E, B1B2B6, C, D was performed by liquid chromatography /guidelines on quality control methods for P 4.1.1672-03 Ministry of health, 2004, 51, 58, 62, 69/.

The determination of the content in the extract vitamins3Inwithand PP were microbiologic the sky method /Official Methods of Analisis, 15th edition, 1990. Association of Official Analytical Chemists. Volume one, p.129/.

The determination of the content in the extract elements of mineral substances was performed by the method of inversion volt-amperometry /guidelines on quality control methods for P 4.1.1672-03 Ministry of health, 2004, St/.

The study found that the condensed extract of Pleurotus ostreatus mycelium 1137 in the form of a gel, comprising the pharmacological substance of the drug, in its composition contains in it the study of biologically-active substances in the following percentage of the masses:

and 47.0-48.0 per cent carbohydrates (glucose, galactose, mannose, arabinose, galactose, xylose, glucosamine - 7 in total). Among them, the glucose content is up to 90%;

- 3.5 to 9.6% of amino acids (aspargine, serine, threonine, glutamine, Proline, glycine, alanine, valine, isoleucine, leucine, lysine, histidine, arginine, cystine, methionine, tyrosine, phenylalanine, ethanolamine, ornithine, oxylipin only 20);

- 0,6-0,9% of higher fatty acids (capric (C10;0), lauric (C12:0), myristic (C14:0), pentadecane (C15:0), pentadecanol (C15:1), palmitic (C16:0), palmitoleic (C16:1), heptadecane (C17:0), heptadecenoic (C17:1), stearic (C18:0), oleic (C18:1), linoleic (C18:2), arachnid (C20:0), erucic (C22:1), 14);

- 1,02-1,26% institutions the institutions acids (oil, lactic, acetic, malic, oxalic - 5);

- 0,6-1,2% vitamin B1B2In3B6BcPP , E, D, C, carotenoids 10);

- 1,4-1,9% minerals (Na), potassium (K), calcium (CA), magnesium (Mg), phosphorus (P), sulfur (S), iron (Fe), zinc (Zn), manganese (Mn), copper (Cu), aluminum (Al), boron (B)barium (BA), silicon (Si), strontium (Sr), lithium (Li) - total 16).

It should be noted that the minimum and maximum values of the ingredients of the chemical composition of biologically active substances in the composition of the extract of Pleurotus ostreatus mycelium 1137 from selected samples of the extract, are characterized by seasonal fluctuations of their content in the composition of the extract obtained within 12 months (since the beginning of January to December inclusive).

The need for additional separation from the structure of condensed extract of Pleurotus ostreatus mycelium 1137 first enriched fractions of the active principle derived Sterol 4-hydroxy-17R-metalinsulator based primarily its very low natural content in the extract of the mycelium, oscillating, depending on the season of the mycelium, for example, from 1.0 mg to 5.0 mg (from 0.001% to 0.005%) 1000,0 mg of extract, which is clearly insufficient for the manifestation of his polyfunctional medico-biological activity in the recommended application dose of the finished form of the proposed drug (100.0 mg/is - 200.0 mg/kg).

Therefore, if you apply the claimed drug without additional enrichment pharmacological active substance starting derived Sterol 4-hydroxy-17R-metalinsulator in the inventive mass ratios ingredient (1,0-2,0 weight of the active principle derived Sterol 4-hydroxy-17R-metalinsulator from the whole mass of the applied dose), the task and the technical result of the claimed invention will not be executed.

As example implementations of obtaining condensed extracts of Pleurotus ostreatus mycelium 1137 derived Sterol 4-hydroxy-17R-metalinsulator below is the technology of its allocation from 1000,0 mg condensed extract in the form of a gel obtained from the mycelium of Pleurotus ostreatus 1137, cultivated in the spring of the year.

For first-enriched fractions as derived Sterol 4-hydroxy-17R-metalinsulator, his selection of the condensed extract of Pleurotus ostreatus mycelium 1137 produced dichloromethane as follows.

Selected a portion of the condensed extract of Pleurotus ostreatus mycelium 1137 in the form of a gel (1000,0 mg) was dissolved in 96% ethanol at a concentration of 100 mg/ml of the resulting solution (aliquots (400 µl) were separated chromatographically on prepreparation HPLC system Gilson, consisting of 2 pumps with heads 25SC, manometric module, mixer, injector with a loop 500 ál, to nochnogo thermostat and flow divider (1:10), UV detector with variable wavelength and the control program. Was used prepreparation column 24*250 mm with the sorbent diasorb ST (10 μm). The composition of the mobile phase: 96% ethanol/0.1% triperoxonane acid in water 60:40, at a flow rate of 10 ml/min and a temperature of 30°C. analysis Time 40 min, detection at a wavelength of 230 nm, interest peak has a retention time of 20-25 minutes Fraction containing the peak of interest, collected, was added 30% ammonium hydroxide solution until a neutral pH of 6-8 and evaporated on a rotary evaporator at a temperature of 45-55°C to 2/3 volume until opalescence of the solution. To the remaining solution was added an equal volume of dichloromethane, was extracted and the organic layer was separated with a separating funnel.

The organic layer was evaporated on a rotary evaporator without heating and dissolved in 1 ml of 96% ethanol. The resulting solution (aliquot 100 ál) were separated by analytical HPLC system Agilent 1100, consisting of four channel pump with degasser, a column thermostat, autosampler, diode matrix UV detector and a control program ChemStation. Was used analytical column of 4.6*250 mm sorbent Luna 18(2) (5 μm). The composition of the mobile phase: acetonitrile/water 79:21, at a flow rate of 1 ml/min and a temperature of 25°C. analysis Time - 21 min, detection at a wavelength of 230 nm, interesuiusi the peak has a retention time of 15-18 minutes The fraction containing the peak of interest was collected and evaporated on a rotary evaporator at a temperature of 45-55°C to 2/3 volume until opalescence of the solution. To the remaining solution was added an equal volume of dichloromethane, was extracted and the organic layer was separated with a separating funnel. The organic layer was evaporated on a rotary evaporator without heating, was dissolved in 1 ml dichloromethane and dried in a desiccator. In this embodiment, the output of the substance 4-hydroxy-17R-metalinsulator was 5 mg

For producing the required quantity 4-hydroxy-17R-methylenetetra process can recur or be scaled with increasing applied for these purposes, the initial mass of the extract of the mycelium in the form of a gel.

The chromatogram of the starting solution (analysis of the dichloromethane extract on an analytical column (Luna C18(2) (5 μm) of 4.6*150 mm, mobile phase: acetonitrile/water/1% TFU in water=79:16:5, flow rate 1.5 ml/min at 25°C, detection at 230 nm, retention time of interest matter 6-7 min analysis time 13 min) is shown in figure 1.

Example prepreparation separation on an analytical HPLC system: an analysis of the fractions after preparative HPLC on an analytical column (Luna C18(2) (5 μm) of 4.6*250 mm mobile phase: acetonitrile/water 79:21, flow rate 1.0 ml/min at 25°C., UV detection at 230 nm is shown in figure 2 (while holding the air traffic management substance 16 min).

Chromatogram of a purified substance (an aliquot of 10 μl: HPLC on an analytical column (Luna C18(2) (5 μm) of 4.6*250 mm mobile phase: acetonitrile/water 79:21, flow rate 1.0 ml/min at 25°C, detection at 230 nm) is shown in figure 3.

Molecular formula C21H32O3was determined by accurate mass time-of-flight method UPLC/MS/MS mass spectrometry.

The results of determination of molecular weight and gross formula is shown in figure 4, 5, 6.

In figure 4 the difference between the peaks of the 688 and 1021 is 333. 333.16+23(Na+)=356.16, 332.52*2+23=688.04, 332.54*3+23=1020.62.

Figure 5 ion 331.38 corresponds to the mass 332.38. The difference between the peaks 331.38 and 663.97 is 332.59. 332.34*3-1=996.01, 332.28*4-1=1328.12.

Conclusion: molecular weight 332.28-333.16.

Figure 6 shows the determination of the exact mass, which was performed by the method of time-of-flight mass spectrometry (TOF).

355.2295=C21H32O3Na+(15 ppm);

333.2452=C21H33O3+(8 ppm).

Conducted by the program Structure Elucidator/ACDIabs (Canada)" [Journal of Analytical chemistry, 2008, t, No. 1, SS-26] structural studies have revealed the structure selected from the extract substance.

To achieve the formulated objectives and the technical result of the claimed invention, in addition to the enrichment of the first fraction as derived Sterol 4-hydroxy-17R-metalinsulator, drug enrich the second polysaccharide fraction β1-3 glucan and a third fraction dihydro-quercetin.

The necessity of selection of the composition of recycled Pleurotus ostreatus mycelium 1137 macromolecular polysaccharide β 1-3 glucan, after extraction of the extract in the form of a mixture of low molecular weight substances, justified his absence in the condensed extract of Pleurotus ostreatus mycelium 1137 in the form of a gel obtained by the above mentioned technologies.

To obtain the second enriched fraction β 1-3 glucan its secretion from recycled Pleurotus ostreatus mycelium 1137 conducted by the technology of extraction of mycelium, turned into powder by grinding in liquid nitrogen in a high speed blender with 0.5%solution of hot lye (60°C) for 1 hour, followed by deposition of substrate mycelium polysaccharide ethanol and presidenial them in the mass ratio of ethanol: the solution powder mycelium in alkali 1:1, 3:1, 4:1 accordingly /Ohno N, Miura N.N, Narajima M, Yadomae T. Biol Pharm Bull. 2000. Jul; 23(7): 866-872. Antitumor 1,3-betaglucan from cultured fruit body of sparassis crippa/.

The definition of the structure and content of monosaccharides in aqueous-alkaline extract of mycelium was performed according to the developed technique by enzymatic and acid hydrolysis with a further modification of monoglide and chromatographic analysis using reverse-phase HPLC in isocratic mode on column Luna C18(2) 4.6*150 mm 5 um) on the chromatograph Agilent 1100. The collection and processing of chromatograms was carried out using a CR is grams Chem Station, version W.

The assignment of the peaks and the calculation of the concentration of the carbohydrates produced by the internal standard (glucosamine) and external standard containing a mixture of five analyzed carbohydrates at a concentration of 1 g/L.

The results of mass spectrometric analysis of selected polysaccharide is shown in Fig.7, 8.

Analysis of mass spectrometric data shows that formed during acid hydrolysis of the major peak is really a glucose with a molecular weight of 512 daltons. Two peaks with lower intensity have a lot of disaccharide (674 daltons) and degraded in the mass spectrometer to 512 mass (see Fig.7).

Enzymatic hydrolysis of the formed three peaks with similar intensity. The last peak corresponds to the glucose (512 daltons), and the other two have a lot 836 and 674 daltons. Differences in 324 and 162 Dalton correspond to the three - and disaccharide (see Fig).

To obtain third-enriched fractions were applied industrially produced currently dihydroquercetin in the form of amorphous powder (3 beta, 5E, 7E, 10 alpha, 22E) - 9, 10 - Sakerhet - 5,7,22-triene-3-ol molecular weight 304,26 Dalton on THE other 9197-031-02699613-2006 /Certificate of state registration №U from 19.12.2006, permitted for use in food and medical industry (Sanitary-epidemiological conclusion M/.

The definition is the possession of drugs dihydroquercetin (Taxifolin) was performed using reverse-phase HPLC on a column of Luna 18(2)4.6*250 mm (5 um) production Phenomenex USA using chromatograph Agilent 1100. The collection and processing of chromatograms was performed using the program "Chem Station A.09.03" /guidance on the methods of quality control and safety of dietary SUPPLEMENTS R-03. The Ministry Of Health Of Russia. M. 2004. p.134. paragraph 4.11.3. HPLC/.

Chromatogram possible definitions of the indicators of authenticity and quantitative indicators of dihydroquercetin in the product is shown in Fig.9.

In General, the composition of the proposed drug with polyfunctional medico-biological activity affecting tissue metabolism, contains biological substances with the ability to cause cell death of malignant tumors by induction of apoptosis, thus to exert hepatoprotective and detoxifying effect, resulting in blocking the development of steatohepatitis and normalization of the lipid spectrum of blood serum.

These substances can be divided into three main groups. The first group includes substances that cause cancer cell death by running them in the mechanism of apoptosis. The second group contains substances that increase the activity of liver detoxification, linking carcinogens and accelerating their excretion. This group also contains natural antioxidants that activates endogenous antioxidant system. These factors create a reliable protection of the body of patients from the toxic effects of oxygen stress caused by the production of therapy and radiation exposure. The third group consists of compounds that enhance the antitumor immune response, usually suppressed in cancer patients.

All the studied biological substances of plant origin in the development of the chemical composition of the drug from the medical and biological activity may be used in the manufacture of food products and satisfy health indicators San Pin 2.3.2.1078-01 and San Pin 2.3.2.1293-03.

B. Study the toxic effects of the proposed drug with polyfunctional medico-biological activity was carried out using two doses: 100 mg/kg 200 mg/kg

The experiment involved two groups of mice of 10 animals in each group.

Studies were performed on mice of both sexes lines BA LB/C with a mass of ~40 g after a single intragastric route of administration of the drug. Before application of the drug each dose was dissolved in 0.5 ml of distilled water. The observation period was 14 days.

Criteria toxicity of the investigated doses of the drug were as follows: number of dead animals, the time of death of the animals, the clinical picture of intoxication, the change of body weight during the entire observation period, the behavior of animals, autopsy data.

The results of toxic action of the investigated doses of the drug are given in table 2, 3.

The result p is Ogadenia this series of experiments established the following.

After a single intragastric administration of the proposed drug test doses of 100 mg/kg 200 mg/kg in both groups of mice external clinical signs were observed.

The appearance. During the 14 day observation of animals the coat is smooth. On appearance of animals between groups did not differ. Animals were not marked deterioration of appetite. Chair decorated, normal color.

The behavior. During observation of animals depressed General condition of the animals was not observed.

The mortality rate. Mortality occurred from the introduction of the drug was not detected throughout the observation period.

Lots of animals. During the whole observation period produced two measurements. Sudden loss or increase of weight is not known, but in group 2 observed total weight loss at 1 year (see table 4).

Internal organs. At necropsy, mice at the end of the experiment (after 14 days of observation) macroscopic internal organs without pathology. The condition of the internal organs of both groups of mice, they did not differ from each other.

According to a study of the toxic effect of the proposed drug with polyfunctional medical and biological activity revealed that the drug in the dosage used (100 mg/kg and 200 mg/kg) did not show toxic effects and, in accordance with the classification is oxyconti substances, belong to class IV toxicity (toxic substances) /Methodological guidance on experimental (preclinical) study of new pharmacological substances. 2000/.

Given these toxic research, further study of medico-biological properties of the claimed preparation was carried out in two once daily applied doses: 100 mg/kg 200 mg/kg of weight of animal.

Century In the study of the action of the active principle derived Sterol 4-hydroxy-17R-metalinsulator claimed the drug at the cellular level in vitro as a model for testing used cultured cells (normal and transformed).

The objective of these studies was to study the individual effect of the active principle of the drug in the form of 4-hydroxy-17R-metalinsulator at doses of 1.0 mg/kg and 2.0 mg/kg at the cell level using a relatively simple system of cultured cells - normal (human fibroblasts) and transformed (Hela).

Quantitative evaluation of antitumor efficacy of the active principle derived Sterol 4-hydroxy-17R-metalinsulator claimed the drug level in vitro were compared with the antitumor effect of the prototype, i.e. extract of Pleurotus ostreatus mycelium 1137 (VKPM F - 819) and cytostatic cyclophosphamide method/Li J., Karlsson M.O., Brahmer j, Spitz, A., Zhao M.. M. Hidalgo, S.D. Baker CYP3Aphenotyping approach to predict systemic exposure to EGFR tyrosine kinase inhibitors. J. Natl. Cancer Inst, 2006, 98, 1714-1723/.

To quantify the effectiveness of the actions specified active principle derived Sterol 4-hydroxy-17R-metalinsulator claimed the drug was used indicators of the viability of a population of cultured cells, including mitotic and apoptotic indices. Mitotic index reflects the ability of the cell population to reproduce, and the apoptotic index shows the percentage of cells that activates programmed death. It is known that malignant tumors apoptosis is suppressed, which leads to intensive multiplication of cancer cells and the formation of metastasis /Asahina, H., Yamazaki, K., I. Kinoshita, Sukoh N., Harada M., Yokouchi, H., Ishida T., Ogura, S., Kojima T., Okamoto Y., Fujita Y., Dosaka-Akita, H., Isobe, H., Nishimura M. A phase II trial of gefitinib as first-line therapy for advanced non-small cell lung cancer with epidermal growth factor receptor mutations. Br. J. Cancer, 2006, 95, 998-1004/.

The results showed that when an individual action of the prototype, i.e. the extract of Pleurotus ostreatus mycelium 1137 (VKPM F - 819) at doses of 100 mg/kg 200 mg/kg, is a mild inducer of apoptosis and causes abnormal segregation of chromosomes at metaphase and the occurrence of chromosomal aberrations type "bridge" on stage anafazy.

When the combination of the application of the extract of Pleurotus ostreatus mycelium 1137 (VKPM F-819) and cyclophosphamide manifests pronounced synergism, which, depending on their con is entrale (extract 100 mg/kg, cyclophosphamide 50 mg/kg or 100 mg/kg) and how to install them, is expressed in a partial suppression of proliferation and induction of apoptosis.

Found that the combined use of the extract of Pleurotus ostreatus mycelium 1137 (VKPM F-819) and cyclophosphamide determines the ability of cancer cells to enable their apoptotic death. Thus the antitumor effect of extracts of Pleurotus ostreatus mycelium 1137 (VKPM F-819) is not directly related to its cytotoxic effect. However, this effect is not evident in normal human fibroblasts.

According to a study of the active principle 4-hydroxy-17R-metalinsulator sausage of the drug is established that it is used in doses of 1.0 mg/kg or 2.0 mg/kg in cultured transformed Hela cells after 24 hours causes a pronounced apoptotic effect.

Included in the claimed composition of the drug the active principle 4-hydroxy-17R-metalinsulator in the dosage used is a potent inducer of apoptosis in cultured transformed cells, increasing 20 to 30 times, compared to the prototype (an extract of Pleurotus ostreatus mycelium 1137 in the form of a gel), the number of apoptotic cells, which is 10-15 times more efficient than the application of well-known drug HA - 40, which has no apoptotic and experimental mitostatic effect in normal fibres the asty /Lawrence Loeb,A.,"Investigation of molecular mechanisms of the anti-carcinogenic action of the GA-40 preparation" University of Washington, School of Medicine, Department of Pathology; WDC, 2005/.

On the basis of the results of the cytological studies of cultured cells, it was found that the main mechanism of the antitumor effect of active principle 4-hydroxy-17R-metalinsulator the proposed drug is to stimulate apoptosis in cancer cells. Therefore, entered in the developed preparation of the active principle in the form of 4-hydroxy-17R-metalinsulator in the amount of 1.0 to 2.0 weight from the total weight of the proposed drug is an inducer of apoptosis of cancer cells, increasing at the same time, as mentioned above, 20-30 times, compared with the prototype, the number of apoptotic cells in cultured transformed Hela cells.

, In the study of antitumor activity of the claimed product with a polyfunctional medico-biological activity in animals on the model of solid tumor melanoma b-16 were conducted four series of experiments.

a) In the first series of experiments, we studied the antitumor activity of the claimed preparation in the form of gel and syrup and its component parts on the model of solid tumor melanoma b-16.

The experiments were carried out on inbred mice BDF1the hybrids of the first generation f1(C57Bl6×DBA2) kennel breeding RAMS "Pole". In the experiments used 100 mice, males weighing 18-20 g

as tumor test systems was transplantable solid tumor animal melanoma b-16. The tumor was perepevalas in accordance with standard regulations under the skin of the right flank of mice crushed fragments of tumor tissue contained 0.3 ml of physiological sodium chloride solution /Tresalia E.M., Zhukov O.S, Gerasimov G., Andronova, NV, Garin A.M. "guidelines for the study of antitumor activity of pharmacological substances" // Book "Manual on experimental (preclinical) study of new pharmacological substances" (as amended Ruhanie), edition 2, M, "Medicine", 2005, s-651/.

In the first series of experiment involved 5 groups of mice (8 mice in each group):

1st group - intact control transplanted mice solid tumor melanoma b-16;

2nd group - transplantirovannam mice solid tumor melanoma b-16 with the introduction of the condensed extract of Pleurotus ostreatus mycelium 1137 in the form of a gel, comprising the pharmacological substance of the drug in a dose of 100 mg/kg, oral, ten times daily, during the first 10 days. On the first day the drug was administered within 1 hour after transplantation mice tumor and then in the next 9 hours daily;

Group 3 - transplantirovannam mice solid tumor melanoma b-16 with the introduction of the active agent 4-hydroxy-17R-metalinsulator drug in the dose of 2 mg/kg, oral, ten times daily, for 1...10 days. IU the year of introduction of the active principle of the drug is similar to the introduction of the second group;

4-I group - transplantirovannam mice solid tumor melanoma b-16 with the introduction of the proposed drug in the form of a gel at a dose of 100 mg/kg, oral, ten times daily, for 1...10 days.

5-I group - transplantirovannam mice solid tumor melanoma b-16 with the introduction of the claimed preparation in the form of syrup at a dose of 6.0 ml/kg, oral, ten times daily, for 1...10 days.

b) In the second series of experiment involved 3 groups of mice (8 mice in each group):

1st group - intact control transplanted mice solid tumor melanoma b-16;

2nd group - transplantirovannam mice solid tumor melanoma b-16 with the introduction of cyclophosphamide (CP) at a dose of 100 mg/kg, intraperitoneally, once, on the first day;

Group 3 - transplantirovannam mice solid tumor melanoma b-16 with the introduction of combined use ZF in a dose of 100 mg/kg intraperitoneally once daily, in the first day and pharmacological substance of the drug in the form of a gel at a dose of 100 mg/kg, oral, ten times daily, for 1...10 days. On the first day the drug was administered within 1 hour after transplantation mice tumor and then in the next 9 days daily.

in the third series of the experiment involved 3 groups of mice (8 mice in each group):

1st group - intact control with transplanted mashamaite tumor melanoma b-16;

2nd group - transplantirovannam mice solid tumor melanoma b-16 with the introduction of CP at a dose of 50 mg/kg, intraperitoneally, once, on the first day;

Group 3 - transplantirovannam mice solid tumor melanoma b-16 with the introduction of combined use CP at a dose of 50 mg/kg, intraperitoneally, once, on the first day, and the active agent 4-hydroxy-17R-methylincisterol drug in the dose of 2 mg/kg, oral, ten times daily, for 1...10 days.

g) In the fourth series of the experiment involved 3 groups of mice (8 mice in each group):

1st group - intact control transplanted mice solid tumor melanoma b-16;

2nd group - transplantirovannam mice solid tumor melanoma b-16 with the introduction of CP at a dose of 50 mg/kg, intraperitoneally, once, on the first day;

Group 3 - transplantirovannam mice solid tumor melanoma b-16 with the introduction of combined use CP at a dose of 50 mg/kg intraperitoneally once daily, in the first day and the claimed preparation in the form of syrup at a dose of 8.0 ml/kg, oral, ten times daily, for 1...10 days.

Daily in all groups of four series of experiment conducted inspection of the animal to assess the presence and severity of the tumor (visually and by palpation), and signs of possible intoxication. Recorded: average tumor weight (g); growth inhibition is the tumor (SRW) (in %); the average life expectancy of animals (day); increase the life expectancy of the animals of the experimental groups relative to the life span of the animals of the control group (in %).

The research results of four series of experiments are given in table 4-7 and figure 10-18.

In particular, figure 10 shows the results of antitumor activity of condensed extract of Pleurotus ostreatus mycelium 1137 in the form of a gel, comprising the pharmacological substance of the drug, on the model of solid tumor melanoma b-16 where:

1 - a group of intact control mice with transplanted solid tumor melanoma b-16;

2 - experimental group of mice with transplanted solid tumor melanoma b-16 with the introduction of pharmacological substances of the drug in a dose of 100 mg/kg, oral, ten times daily, for 1...10 days.

Figure 11 shows the results of antitumor activity of the active principle derived Sterol 4-hydroxy-17R-metalinsulator the proposed drug on the model of solid tumor melanoma b-16 where:

1 - a group of intact control mice with transplanted solid tumor melanoma b-16;

2 - experimental group of mice with transplanted solid tumor melanoma b-16 and with the introduction of the active principle derived Sterol 4-hydroxy-17R-metalinsulator in to the e 2 mg/kg, oral, ten times daily, for 1...10 days.

On Fig shows the results of antitumor activity of the claimed preparation in the form of gel on the model of solid tumor melanoma b-16 where:

1 - a group of intact control mice with transplanted solid tumor melanoma b-16;

2 - experimental group of mice with transplanted solid tumor melanoma b-16 and with the introduction of the proposed drug in the form of a gel at a dose of 100 mg/kg, oral, ten times daily, for 1...10 days.

On Fig shows the results of inhibition of growth of a solid tumor melanoma b-16 in mice under the influence entered the claimed preparation in the form of a gel, where:

1 - a group of intact control mice with transplanted solid tumor melanoma b-16;

2 - experimental group of mice with transplanted solid tumor melanoma b-16 and with the introduction of the proposed drug in the form of a gel at a dose of 100 mg/kg, oral, ten times daily, within 1...10 days.

On Fig chart shows the survival of mice with established tumor melanoma b-16 in mice that received the claimed preparation in the form of a gel, where:

1 - a group of intact control mice with transplanted solid tumor melanoma b-16;

2 - experimental group of mice with transplanted solid tumor melanoma b-16 and entered the eating of the claimed preparation in the form of a gel at a dose of 100 mg/kg, oral, ten times daily, for 1...10 days.

On Fig shows the results of antitumor activity of the claimed preparation in the form of syrup on the model of solid tumor melanoma b-16 where:

1 - a group of intact control mice with transplanted solid tumor melanoma b-16;

2 - experimental group of mice with transplanted solid tumor melanoma b-16 and with the introduction of the claimed preparation in the form of syrup at a dose of 6.0 ml/kg, oral, ten times daily, for 1...10 days.

On Fig shows the results of inhibition of growth of a solid tumor melanoma b-16 in mice under the influence entered the claimed preparation in the form of syrup, where:

1 - a group of intact control mice with transplanted solid tumor melanoma b-16;

2 - experimental group of mice with transplanted solid tumor melanoma b-16 and with the introduction of the claimed preparation in the form of syrup at a dose of 6.0 ml/kg, oral, ten times daily, for 1...10 days.

On Fig chart shows the survival of mice with established tumor melanoma b-16 in mice that received the claimed preparation in the form of syrup, where:

1 - a group of intact control mice with transplanted solid tumor melanoma b-16;

2 - experimental group of mice with transplanted solid tumor melanoma b-6 and with the introduction of the claimed preparation in the form of syrup at a dose of 6.0 ml/kg, oral, ten times daily, for 1...10 days.

On Fig shows the results of antitumor activity of combined use of the proposed drug in the form of a syrup of cyclophosphamide on the model of solid tumor melanoma b-16 (4th series of experiments), where:

1 - a group of intact control mice with transplanted solid tumor melanoma b-16;

2 - experimental group of mice with transplanted solid tumor melanoma b-16 with the introduction of CP at a dose of 50 mg/kg, intraperitoneally, once, on the first day;

3 - experimental group of mice with transplanted solid tumor melanoma b-16 with the introduction of combined use CP at a dose of 50 mg/kg, intraperitoneally, once, on the first day and the claimed preparation in the form of syrup at a dose of 8.0 ml/kg, oral, ten times daily, for 1...10 days.

In the analysis presented in table 4 and shown in figure 10-17 data change dynamics of the controlled characteristics of antitumor activity of substances, the dependence of their activity on the type of the applied composition. Studied four samples of individual application of drugs, the greatest inhibition of tumor growth caused the claimed drugs with polyfunctional medico-biological activity in the form of a gel (4) and syrup (5th group), which accordingly is about 21 days after inoculation the mice tumor was 61% and 55%. The average tumor volume in mice in these groups, compared with the intact control (group 1), 21 days decreased, respectively, 2.9 and 2.5 times.

The average life expectancy of animals treated with the claimed preparation, increased compared with the intact control group, respectively, at 117% 109,5% and exceeded the average life expectancy of animals treated with pharmacological substance of the proposed drug (2nd group) or its active principle (group 3).

To estimate the antitumor effect of the claimed preparation in the form of gel and syrup all over rustinhibiting effect of tumor melanoma b-16 presents the data obtained in the form of dependencies that characterize the dynamics of change in tumor size in treated animals in percent relative to the control (indicator T/C%, see Fig, 16).

Shown in these figures according confirm the efficiency of inhibition of tumor growth during the observed period of its development, which on the severity and nature of the antitumor activity have their individuality.

The claimed preparation in the form of a gel or syrup after 11 days after completing therapy provides sustained antitumor activity, remaining at the level of 55-60%.

After anticancer research the activity of the proposed drug in animals after their death (43 day of tumor inoculation) were extracted from the remaining part of the tumor, which were carried out histological studies specialists of cell biology and Cytology. As a result of the research it was found that at high magnification (CA.×40) fragments of cross-sections of treated tumors no clear zoning. Under striated muscles are multiple foci of necrotic cell death. Under a layer of muscle fibers are vessels with red blood cells and numerous apoptotic cells.

Numerous small vessels penetrate the tumor cell which is characterized by basophilia, kernel heterogeny in size and shape. In tumors there are numerous melanocytes with granules. Extensive foci of necrosis penetrate deep into the tumor. In the composition of the tumor on the border of leukocyte shaft, often in some of the gaps identified apoptotic cells with distinctive appendages membranes and heteropterinae cores. The zone of necrosis surrounded by leukocyte shaft (inflammation) of lymphocytes, macrophages and neutrophils.

Thus, the individual application of the proposed product in the first 1-10 days after inoculation mice solid tumor melanoma b-16 concluded that remaining after treatment of the tumor penetrated by numerous foci of necrotic and apoptotic cell death. Tumor AK is actively destroyed.

In the analysis presented in tables 5, 6 and 7 and shown in figure 18 data change dynamics of the controlled characteristics of antitumor activity of the investigational pharmacological active substance and the beginning of 4-hydroxy-17R-metalinsulator, the proposed drug (see table 5, 6)and directly claimed preparation in the form of syrup (see table 7, Fig) when combined with cyclophosphamide (CP), the dependence of activity from the applied composition.

As a result of a series of experimenal shown that the joint use of the investigational compounds substances and CP causes a profound inhibition of tumor growth of melanoma b-16, which varies from 70% to 96%, compared with untreated intact control mice.

The average tumor volume in mice in these groups compared with the intact control (group 1) decreased almost throughout when tested pharmacological substance with the fit of 14.3 times (see table 5), when tested active principle 4-hydroxy-17R-metalinsulator drug with ZF 3.75 times (see table 6) when testing of the proposed drug in syrup with ZF 23 times (see table 7, Fig).

Summarizing the results of a study of the effectiveness of combined combined application of the proposed drug with polyfunctional medico-biological activity in the form of siropas dose of 8.0 ml/kg during the first 1...10 days and fit in a dose of 50 mg/kg once, in the first days after pereprivivki tumors almost leads to a complete inhibition of the tumor compared with the intact untreated control, without adverse effect on the antitumor effect of the cytostatic agent.

Moreover, it must be emphasized that having a detoxifying effect, the claimed drug significantly improves the clinical picture of "quality of life" animals (activity, appearance, hair condition, behaviour, body weight, no diarrhea) compared to animals receiving only cyclophora and compared with untreated mice intact control.

D. the study of the effects of pharmacological substance of the proposed drug

and fits on hematological indices of peripheral blood of healthy animals experiments were conducted on mice C57B L/6, age 8 weeks, weight 23-30,

Cells of a solid tumor In-16 were grown in RPMI medium with 10% fetal serum, collected by trypsinization, washed three times with medium, resuspension cooled to 4°C phosphate-buffered saline PBS and kept on ice (time of storage did not exceed 30 min). Perebivka of the tumor was carried out by subcutaneous injection in the back of the animal 30 μl of the suspension containing 10 cells. On the fifth day after inoculation of the tumor animal among animals, shows the observed steady growth of the tumor, were formed 4 groups of mice (4 mice in each group).

1st group - intact control transplanted mice solid tumor melanoma b-16;

2nd group - transplantirovannam mice solid tumor melanoma b-16 with the introduction of CP at a dose of 300 mg/kg, intraperitoneally, once, on the 5th day after inoculation of the tumor;

Group 3 - transplantirovannam mice solid tumor melanoma b-16 with the introduction of a pharmacological substance of the proposed drug in the form of a gel at a dose of 100 mg/kg, intraperitoneally, ten times daily, for 1...10 days after administration of CP. On the first day the drug substance in the form of a gel was administered 2 hours after injection ZF;

4-I group - transplantirovannam mice solid tumor melanoma b-16 with the introduction of combined use CP at a dose of 300 mg/kg, intraperitoneally, once daily, 5 days after inoculation of the tumor and pharmacological substance of the proposed drug in the form of a gel at a dose of 100 mg/kg, intraperitoneally, ten times daily, for 1...10 days after administration of CP.

During the experiment we measured the number of leukocytes in peripheral blood (before the introduction of the ZF and the first, fourth and seventh days after the introduction of the fit).

Measuring the number of cells was carried out according to standard methods /Eaast. Handbook of clinical laboratory methods the research is. M, Medicine, 1975/.

The results of this series of experiments are given in table 8 and shown in Fig where:

- group 1 - intact control (IR);

- group 2 - introduction to the mice ZF;

- group 3 - introduction to the mice pharmacological substance of the proposed drug (FS PR);

- group 4 - introduction to the mice combined use ZF and pharmacological substance of the proposed drug (FS PR+CPH).

Counting the number of leukocytes in peripheral blood showed that pharmacological substance improves the recovery of white blood cells of animals after treatment ZF (see Fig, table 8).

In the 4th group treated with CP and pharmacological substance, the number of cells on the next day after administration of CP was higher than the number of cells than in the 2nd group received only ZF (respectively, 43% and 29% of the original). On the third day, the number of leukocytes in the 4th group had grown to 51%, while the 2nd group fell slightly, to 27%. On the seventh day, the number of leukocytes was normal in all mice.

Thus, the use of pharmacological substance of the proposed drug causes earlier and more intensive recovery in the number of leukocytes in the peripheral blood of immunized mice after the application of the fit and reduces hematologic toxicity, in particular, the severity and duration of the radiation caused by the introduction of animal is m CP at a dose of 300 mg/kg body weight.

That is, If the study gipolipidemiceski and hepatoprotective activity of the drug and of its constituent substances in animal model of atherogenic diet (AD) in the experiment involved 5 groups of outbred laboratory of Mature male rats with an initial body weight of 300-350 g (8 rats in each group):

1st group - intact control (IR);

2nd group - atherogenic diet (AD);

Group 3 - the HELL with the active beginning of the 4-hydroxy-17R-metalinsulator drug in the dose of 2 mg/kg, oral, ten times daily, after the first 4 days of application of HELL (EN PR);

4 - AD with the use of pharmacological substance of the proposed drug in a dose of 100 mg/kg, oral, ten times daily, after the first 4 days of applying AD(AD+FS PR);

5-I group - the HELL with the drug in the form of syrup at a dose of 8 ml/kg, oral, ten times daily, after the first 4 days of applying AD (AD+PR syrup).

All test substances were administered to rats orally at intervals of 3 hours after the atherogenic burden in the last 10 days of HELL.

During the whole experiment conducted observation sheet: survival, General appearance, condition and behavior of animals, change of body weight (rats weighing was performed once a week).

At the end of the experiment the animals after 18 hours of fasting took the blood from the tail vein into centrifuge the wading is I. The blood was centrifuged and selected serum for biochemical analyses, namely: the content of total cholesterol (total cholesterol); cholesterol high density lipoprotein (HDL cholesterol); cholesterol low-density lipoprotein (LDL)cholesterol very low density lipoprotein (VLDL cholesterol), triglycerides (TG), atherogenic index (IA); alanine aminotransferase (Alt); aspartate aminotransferase (AST); Alp (alkaline phosphatase).

Analyses were performed standardized methods for biochemical photometer "Stat Fax 1904+" made in the USA using standard kits.

Currently for studying the early stages of the development of Nash and testing the various tools necessary for its treatment, the proposed experimental model, in which the development of fatty liver induced by the artificial increase in the number of lipids in the blood plasma of laboratory animals /Fan et al., 2003; VanSaun et al., 2009; Bivalkevich, Pocket, 2010/.

A similar model is used to study the validity of the proposed drug and its constituent substances in the early stages of the development of Nash.

For histological and ultrastructural analysis of the liver of the studied groups of rats the material was taken from just slaughtered animals. A small piece of the liver was separated by scissors, was transferred to a 4% solution of glucaro the CSOs aldehyde in 0.1 M phosphate buffer of Serensen. The fragment was cut with a razor blade into pieces of about 1 mm3obtained samples was transferred into fresh fixative mixture of the same composition. Fixation was carried out at room temperature for 4 hours. Then the samples were washed in the distillated water and postvaccinial 1% aqueous solution of osmium tetroxide for 12 hours at +4°C. the Samples were obezvozhivani in increasing ethanol concentrations (70% ethanol contained 2% uranyl acetate, acetone and concluded in epoxy resin according to standard procedures.

For histological analysis were made "half-slices about 1 µm thick, were stained sections dye methylene blue and examined in a light microscope using a lens ×10 and ×100.

For electron microscopy, ultrathin sections with a thickness of about 80 nm were contrasted with 1%aqueous solution of uranyl acetate for 20 minutes and lead citrate and photographed in the electron microscope (Hitachi H-700 (Japan).

The research results of this series of experiments is shown in Fig, 21 and table, 10, 11.

In particular, Fig shows the biochemical parameters of lipid-lowering activity of the proposed drug and its constituent substances.

On Fig shown biochemical parameters hepatoprotective activity of the drug and of its constituent substances.

When analyzing the results, the light is howling and electron microscopy sections of the group of intact rats of control was established, that "half" sections of the liver is clearly seen the orderly arrangement of the liver cells (hepatocytes) in the form of strands (beams). Between the beams pass vnutritrekovye blood capillaries, which are blood cells, primarily red blood cells. Hepatocytes are one and binucleate cells. The cores have 1 or 2 nucleoli, chromatin structure typical for cells with normal metabolic activity. In the uniform density of the cytoplasm of hepatocytes are rendered numerous dark globule corresponding detectable histological methods basophilic granules. On electronmicroscopy drugs these zones are identified as separate areas of granular EPR. Lipid inclusions in the form of separate drops are not detectable. Hepatocytes with signs of necrotic death or apoptosis are absent.

When analyzing the results of light and electron microscopy group with AD found to be in breach of the location of the liver cells (hepatocytes). Typical of normal liver location of liver cells (hepatocytes) in the form of strands (beams) is violated due to the strong increase of hepatocytes. Such changes affect the entire area of the hepatic lobules. Accordingly, loss of ordered orientation vnutrizonovyh capillaries, along which are formed gaps of different size and shape, containing increased compared with the control, the number of erythrocytes and leukocytes. In General, the structure of vnutrizonovyh capillaries and riches in the gaps of the elements of the blood pattern corresponds to venous hyperemia.

At the cellular level of HELL leads to the formation in hepatocytes numerous lipid droplets. Significant changes are observed in the structure of nuclei: they acquire an irregular shape (apparently due to the obstruction of lipid inclusions), chromatin and nucleoli appear more compact in comparison with the control. This structure of nuclei occurs in case of partial damage of cell membranes and can be identified as "parametros", i.e. reversible functional state of the cell, leading to necrosis. Hepatocytes with signs of necrotic death or apoptosis are absent.

In the 2nd group of rats compared to the 1st group of rats IR after HELL increasing the level of cholesterol by 34% and triglycerides by 50%, reducing the fraction of HDL cholesterol by 17%, a sharp increase of 78% of LDL cholesterol and 40% increase in VLDL cholesterol (see Fig, table).

When analyzing the results of light and electron microscopy 3-th and 4-th and 5-th groups of experimental rats with hepatoprotective effects of the proposed drug and its constituent substances, it was found that the introduction simultaneously with cholesterol of different forms of the drug blokiruy the development of fatty liver, called HELL, but their activity is aimed at the normalization of pathology, varies greatly.

The greatest efficiency, as detected by histological indicators, has the active principle 4-hydroxy-17R-metalinsulator of the proposed drug (group 3).

It is established that the introduction simultaneously with cholesterol this medication blocks the development of fatty degeneration, activated HELL. In the liver remains the orderly arrangement of hepatocytes in the form of beams and the normal structure vnutrizonovyh capillaries. The structure and location of basophilic granules remain close to normal, the signs of fatty degeneration in hepatocytes is poorly expressed. The nucleus of hepatocytes have the correct form and the General organization of chromatin correspond to the control. Thus, on the basis of histological studies it can be argued that the active principle of the drug is pronounced hepatoprotective properties. However, it is clear that the histological analysis, with all its credibility cannot be the only criterion for a finding of preventing the development of steatosis. For a more precise description of the used drugs as possible hepatoprotectors, were mapped data of histological analysis with biochemical blood indices obtained for all five studied groups of animals.

The mouth is attached, the greatest effect of preventing dyslipidemia in hepatocytes with normalization of biochemical parameters of blood Alt, AST and alkaline phosphatase has claimed the drug in the form of syrup (5th group, see Fig, table 10).

In the blood of animals treated with this drug compared with AD have lower levels of cholesterol by 23%. The level of triglycerides decreased by 53% and falls below control values by 30%, increases the amount of HDL cholesterol by 16%, reduced LDL cholesterol levels by 19%, reduced the level of VLDL cholesterol by 58%. Convincing evidence of the hepatoprotective activity of the drug is a significant reduction IA 38% in animals with high-calorie diet received the claimed preparation in the form of syrup (see Fig, table).

According to the results of pathomorphological study of the status of the internal organs of experimental animals established that the studied organs of the thoracic and abdominal cavities of rats, lungs, thyroid gland, thymus, heart, mucous of esophagus, stomach, small and large intestine, pancreas, spleen, kidneys, bladder, liver) in laboratory animals, 3rd, 4th and 5th groups did not differ from the 1-St group IR rats.

The average body mass of animals are presented in tabl.

Thus, the application of the proposed drug with polyfunctional medico-biological activity, the effect of the future on tissue metabolism, it was found that after use of the drug development of destructive changes in the liver is blocked at the level of the overall organization of the liver, and at the cellular level. A slight amount of fat inclusions formed only in single cells, with nuclei, mitochondria and other cytoplasmic organelles retain structural native characteristics of bacterial. That the drug possesses cytoprotective properties evidenced by biochemical tests. On the background of the action of the drug close to the norm stored indicators of destructive processes in the liver (XC), and dyslipidemia (triglycerides and lipoproteins of different density).

The claimed product with a polyfunctional medico-biological activity get biotechnical method using:

culture of the fungus Pleurotus ostreatus 1137 (VKPM F-819) for growing biomass of mycelium;

- microbiological agar according to GOST 17206-84;

- wort beer food according to GOST 51-174-98;

mycelia of the fungus Pleurotus (strain 1137, PMBC, F-819), grown in submerged conditions in a sterile liquid nutrient medium on THE 9317-01-87552538-08;

- condensed extract in the form of a gel consisting of a mixture of low molecular weight substances isolated from Pleurotus ostreatus mycelium 1137 on THE 9317-01-87552538-08;

- the active agent in the form of biologically active substances derived Sterol 4-HYDR the XI-17R-metalinsulator, additionally allocated from the condensed extract of Pleurotus ostreatus mycelium 1137 (gel) according to the developed technology;

- polysaccharide β 1-3 glucan, first isolated from recycled Pleurotus ostreatus mycelium 1137 after extraction of the extract in the form of a mixture of low molecular weight biologically active substances;

- dihydroquercetin, manufactured in the form of amorphous powder with the molecular weight 304,26 Dalton on THE other 9197-031-02699613-2006;

- ethyl alcohol rectified according to GOST R-2000;

- acid salt according to GOST 3118-77;

- drinking water according to GOST 2874, GOST R 51232-98;

- fructose crystalline, CF4118136AG, Pharmacopoeia Germany DAB;

- citric acid food according to GOST 908-79, highest grade;

- sodium benzoate, 3382SE023494C4, Pharmacopoeia Germany DAB;

- reagent-grade dichloromethane, THE 2631-019-44493179-98, Russia.

The process of obtaining a product with a polyfunctional medico-biological activity industrial method using a culture of the fungus Pleurotus ostreatus includes 6 stages and consists of the following operations.

At the first stage of growing biomass of mycelium breeding strain (maintaining strain) is conducted by subculture onto fresh agar medium. For selection work carried out sieving dispute, the selection monoporosa options and evaluation of their properties, which allows to maintain the target in the Naki culture.

Seed grown in test tubes with beveled agar medium, which contains beer wort, agar and water. After planting tubes put in 10-12 days in a thermostat with a temperature of 28°C.

Deep cultivation of mycelium is carried out in a liquid medium consisting of an aqueous solution wort (10%). Seed mycelium with beveled agar introduced into the environment at a rate of 1 vial of seed per 1 liter of medium. Duration of cultivation 12-14 days with constant aeration. The cultivation is carried out in a thermostatted room, equipped with sensors monitoring recorders at temperature t=28°C.

Department of grown biomass of mycelium from the culture fluid is produced by filtration through, for example, gauze until the full expiration of liquids from biomass. The obtained wet mycelium is subjected to extraction.

At the second stage, the production of condensed extract in the form of a gel consisting of a mixture of low molecular weight substances, formed by pharmacological substance of the proposed drug.

At this stage of preparation of the extract of the mycelium is separated from the culture liquid of the wet mycelial mass mechanically ground to a minimum of fines and pour ethyl alcohol rectified with added hydrochloric acid in a concentration of 0.1%. E is straccia is carried out for 20 hours at 4°C in the refrigeration chamber.

After the end of extraction, the mycelium is separated by filtering through cheesecloth, and the resulting alcohol extract evaporated in a vacuum unit to the state of a viscous homogeneous concentrate in the form of a condensed extract in the form of a gel. Thus to control the compliance of the performed pharmacological substance take a portion of the extract and determine the content of the chemical composition (carbohydrates, amino acids, higher fatty acids, organic acids, vitamins, minerals and water).

The third stage produces an additional allocation of active principle derived Sterol 4-hydroxy-17R-metalinsulator of the selected part of the condensed extract of the mycelium in the form of a gel by fractionation of dichloromethane followed by filtration and lyophilization for further use in the preparation as enriching substances introduced into pharmacological substance from the calculation: 79,0 or 156 mass parts of pharmacological substances to 1.0 or 2.0 mass parts active gachala as derived Sterol 4-hydroxy-17R-metalinsulator.

The allocation derived Sterol from condensed extract of Pleurotus ostreatus mycelium 1137 as produced above is described in the invention method.

In the fourth stage from recycled Pleurotus ostreatus mycelium 1137 after extraction of the extract in VI is e mixture of low molecular weight substances produce a selection of high-molecular polysaccharide β 1-3 glucan by enzymatic hydrolysis of extracted ethanol mycelium to further the use of β 1-3 glucan in the drug as enriching substances introduced into pharmacological substance from the calculation: 79,0 or 156 mass parts pharmacological substance - 10,0 20,0 or mass parts β1-3 glucan.

The selection of β 1-3 glucans from recycled mycelium produced above in the invention method. Experiments have shown that, for example, 1000,0 mg dry recycled Pleurotus ostreatus mycelium 1137 allocate to 700,0-750,0 mg β1-3 glucan.

In the fifth stage produce the commodity form of dihydroquercetin used in the composition of the drug as an added antioxidant in pharmacological substance of the extract of Pleurotus ostreatus mycelium 1137, to further its use in the preparation as enriching substances introduced into pharmacological substance from the calculation:

on 79,0 or 156 mass parts pharmacological substance - 10,0 20,0 or mass parts of dihydroquercetin.

For the application of dihydroquercetin in the present preparation is performed according to claim 2 in the form of a gel prior to enrichment pharmacological substance of the extract of Pleurotus ostreatus mycelium 1137 this substance before its mixing with the composition of the extract in the form of a gel, amorphous powder dihydroquercetin crushed to fine powder (powder).

For the application of dihydroquercetin in the present is the drug executed according to claim 2 in the form of syrup, amorphous powder dihydroquercetin is dissolved in the prepared hot syrup fructose at a temperature of 60°C.

The use of amorphous powder of dihydroquercetin in the inventive preparation according to claim 2 in the form of tablets or dehydrated granules produced by dosing in pharmacological substance extract of Pleurotus ostreatus 1137 prepared in the form of ground crystalline powder.

At the sixth stage produces the preparation of the finished form of the drug with polyfunctional medico-biological activity in the form of a gel or syrup, or pill, or dehydrated granules.

During the preparation of the finished form of the drug in one or another of the claimed according to claim 2 dosage forms of a pharmaceutical substance-containing preparation the condensed extract low molecular weight biologically active substances (carbohydrates, amino acids, higher fatty acids, organic acids, vitamins, minerals and enriched with active start-derived Sterol 4-hydroxy-17R-metalinsulator with a molecular mass of 332,2452 Dalton, polysaccharide β 1-3 glucan and dihydroquercetin with a molecular mass of 304,26 Dalton, produced in the following mass proportions:

(1):(2) (3):(4) as(79,0-158,0):(1,0-2,0):(10,0-20,0):(10,0-20,0),

the minimum and maximum values ingredient is adopted in which the composition of the drug based on the requirements of Toxicological safety and medico-biological activity of the proposed drug, proven results toxic and medico-biological testing of the finished form of the drug and its components the component.

Examples of the preparation form of the claimed preparation:

Example 1

Preparation of the final form of the proposed drug in the form of a gel is carried out by ordinary careful consistent hashing prepared in advance in the desired weight ratio of part of the preparation of the 1-St, 2-nd, 3-th and 4-th components of biologically active substances.

In the first stage, based on a 100.0 mass parts of the finished form of the proposed drug, weighed 79,0 mass parts of condensed extract of Pleurotus ostreatus mycelium 1137 (1-th component of the drug) and heated to kabulasoke fluid state in a water bath at t=40-50°C.

At the second stage in 79,0 mass parts warmed up to t=40-50°C condensed extract is administered to 1.0 mass part derived Sterol 4-hydroxy-17R-METI-lanzisera (2-th component of the drug) from a 100.0 mass parts of the finished form of the proposed drug with subsequent thorough mixing of these components at a fixed temperature (t=40-50°C) until a smooth homogeneous state.

At the third stage in the prepared 2-component mixture of condensed extract of Pleurotus ostreatus mycelium 1137 and derived Sterol 4-hydroxy-17R-metalinsulator introducing the mass of 10.0 parts of polysaccharide β 1-3 glucan (3rd component of the drug) from a 100.0 mass parts of the finished form of the proposed drug with subsequent thorough mixing of these components at the specified temperature (t=40-50°C) until a smooth homogeneous state.

At the fourth stage in the prepared 3-component mixture of condensed extract of Pleurotus ostreatus mycelium 1137 derived Sterol 4-hydroxy-17R-metalinsulator, polysaccharide β 1-3 glucan is administered to 10.0 mass parts of dihydroquercetin (4-th component of the drug) from a 100.0 mass parts of the finished form of the proposed drug with subsequent thorough mixing of these components at a fixed temperature (t=40-50°C) until a smooth homogeneous state.

In the fifth stage analyze the quality of the cooked finished product in the form of gel and produce packaging 100.0 mg or 200.0 mg, for example, in a tightly closed glass vials and their product packaging.

Example 2

Preparation of the final form of the proposed drug in syrup carry out simple thorough consistent hashing prepared in advance in the desired weight ratio of part of the preparation of a syrup consisting of a mixture 64,0 x mass parts of crystalline fructose, 0,85-t mass parts citric acid, and 0.15 g-mass part of sodium benzoate, 34,0 x mass parts of water and 1.0-second mass part of the finished form of the drug in the form of a gel using 1-St, 2-nd, 3-th and 4-th components of biologically active substances of the proposed drug made with regard to the availa able scientific C with the above-described example 1.

In the first stage, prepare the syrup distilled water. In designed to prepare syrup tank fill 34,0 x mass parts of water by weight of the total syrup and heat it to a temperature of t=45-50°C.

After heating the water in it enter 64,0 mass parts of crystalline fructose by weight of the total syrup and thoroughly dissolve it in water at t=45-50°C.

After receiving a 2-component mixture of syrup in its composition is administered 0,85 mass parts of citric acid by weight of the total syrup and thoroughly dissolve it in syrup at t=45-50°C.

After receiving a 3-component mixture of syrup in its composition is administered to 0.15 mass part of sodium benzoate by weight of the total syrup and thoroughly dissolve the syrup at t=45-50°C.

In the second stage, prepare the finished form of the claimed preparation in the form of a gel using 1-St, 2-nd, 3-th and 4-th components set of biologically active dietary substances claimed preparation, prepared as described above in example 1.

At the third stage in the prepared 99,0 mass parts of the composition of the syrup is administered to 1.0 mass part of the finished form of the claimed preparation in the form of a gel using 1-St, 2-nd, 3-th and 4-th components of biologically active substances of the proposed drug at t=45-50°C and carefully peremeshivayte until a smooth homogeneous state.

In the fourth stage prepared p is apart in the form of syrup to cool to room temperature, analyze quality and produce packaging, for example, 80,0 ml or 160,0 ml sealed glass vials and their product packaging.

Thus, research of the proposed drug, influencing tissue metabolism, confirmed its high physiological effect from the effective multifunctional biomedical activity.

The physiological action of the drug, as an inducer of apoptosis, with polyfunctional medico-biological activity lies in its ability to influence tissue metabolism and inhibit the reproduction of cancer cells, and cause liver and detoxifying effect, resulting in blocking the development of steatohepatitis and normalization of lipid profile in serum of biological systems.

The use of the proposed drug from the strain of Pleurotus ostreatus 1137 (VKPM F-819) with polyfunctional medico-biological activity affecting tissue metabolism, will increase the antitumor activity when used in combination with drugs cancer chemotherapy, and reduce toxic manifestations in the body in their application due to its hepatoprotective and detoxifying action that will significantly improve the quality of life of cancer patients in the prevention and Le is the situation of cancer.

1. Product with a polyfunctional medico-biological activity affecting tissue metabolism, the resulting liquid submerged culture of the fungus Pleurotus ostreatus 1137 (VKPM F-819) with subsequent separation of the mycelium and the culture fluid and the release of the mycelium of biologically active substances in the form of condensed extracts with antimicrobial activity, which is a pharmacological substance preparation and enriched with active start-derived Sterol 4-hydroxy-17R-metalinsulator with a molecular mass of 332,2452 Yes, the polysaccharide β 1-3 glucan and dihydroquercetin with a molecular mass of 304,26 Yes, at the following weight ratio:
(1):(2):(3):(4)as(79,0-158,0):(1,0-2,0):(10,0-20,0):(10,0-20,0)
and has the ability to inhibit the proliferation of cancer cells, as well as hepatoprotective and detoxifying effect, resulting in blocking the development of steatohepatitis and normalization of lipid profile in serum of biological systems.

2. The preparation according to claim 1, made in the form of GE is I or syrup, or tablets, or dehydrated granules.



 

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4 ex

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2 cl, 6 tbl, 5 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: inhibitor of influenza A virus reproduction represents water extract of basidial Laetiporus sulphureus, obtained by extraction of crushed fungus biomass with water at ratio 1:1 or 1:2 with further removal of insoluble deposit from extract. Claimed inhibitor demonstrates high inhibiting effect with respect to influenza A virus. Index of virus neutralisation in culture of MDCK cells constitutes 2.5-7 lg. Titres of influenza A virus in homogenates of light infected mice constitute (4.67±0.6)-(6.0±0.57) lgEID50/ml±I95.

EFFECT: high inhibitor activity.

3 cl, 6 tbl, 4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: inhibitor of influenza A virus reproduction represents water extract of basidial fungus Phallus impudicus, obtained by extraction of crushed fungus with water at ratio 1:5 with further removal of insoluble deposit from extract.

EFFECT: inhibitor possesses high activity against human and avian influenza of A type.

3 cl, 1 tbl, 3 ex

FIELD: biotechnologies.

SUBSTANCE: strain Trichoderma harzianum Rifai, having L-lysine-alpha-oxidase activity is deposited in the Russian National Collection of Industrial Microorganisms (RNCIM) under the registration number RNCIM F-180 and may be used in agricultural biotechnology and plant growing.

EFFECT: invention makes it possible to reduce losses of decorative and vegetable crops.

2 ex

FIELD: chemistry.

SUBSTANCE: method involves: (1) obtaining a prepolymer of formula: where R1 is a divalent polycarbarbonate polyol radical, R2 is an aliphatic or cycloaliphatic polyisocyanate radical, R3 is a low-molecular weight diol radical, optionally substituted with ionic groups, n ranges from 1 to 5 and m ranges from 1 to 5; by reacting: (1a) at least one polycarbonate polyol, (1b) one or more aliphatic or cycloaliphatic polyisocyanates, and (1c) at least one low-molecular weight diol, optionally substituted with ionic groups; (2) reacting said prepolymer with at least one polyalkylene oxide with monohydroxyl functional groups with number-average molecular weight equal to less than about 3000; (3) elongating the chain of said prepolymer by reacting with (3a) at least one chain-elongating agent of formula: H2N-R4-NH2, where R4 is a an alkylene or alkylene oxide radical which is not substituted with ionic or potentially ionic groups, and in the presence of an organic solvent in order to form polyurethane; (4) dispersing the polyurethane in water; (5) removing the organic solvent to obtain an aqueous dispersion of polyurethane; where the polyalkylene oxide radical with monohydroxyl functional groups makes up between about 0.1 wt % to about 5 wt % per weight of polyurethane. Disclosed is a suntan lotion which contains said dispersion.

EFFECT: obtaining hair fixing compositions using said polyurethane dispersion, which demonstrate excellent adhesion to hair, reduce separation, provide improved moisture retention, higher lustre and a natural feel, as well as a suntan lotion having perfect water-repellence, is uniformly applied, gives a good feel and no clumping.

11 cl, 6 ex

FIELD: chemistry.

SUBSTANCE: method involves: (1) obtaining a prepolymer of formula: where R1 is a divalent polycarbarbonate polyol radical, R2 is an aliphatic or cycloaliphatic polyisocyanate radical, R3 is a low-molecular weight diol radical, optionally substituted with ionic groups, n ranges from 1 to 5 and m ranges from 1 to 5; by reacting: (1a) at least one polycarbonate polyol, (1b) one or more aliphatic or cycloaliphatic polyisocyanates, and (1c) at least one low-molecular weight diol, optionally substituted with ionic groups; (2) reacting said prepolymer with at least one polyalkylene oxide with monohydroxyl functional groups with number-average molecular weight equal to less than about 3000; (3) elongating the chain of said prepolymer by reacting with (3a) at least one chain-elongating agent of formula: H2N-R4-NH2, where R4 is a an alkylene or alkylene oxide radical which is not substituted with ionic or potentially ionic groups, and in the presence of an organic solvent in order to form polyurethane; (4) dispersing the polyurethane in water; (5) removing the organic solvent to obtain an aqueous dispersion of polyurethane; where the polyalkylene oxide radical with monohydroxyl functional groups makes up between about 0.1 wt % to about 5 wt % per weight of polyurethane. Disclosed is a suntan lotion which contains said dispersion.

EFFECT: obtaining hair fixing compositions using said polyurethane dispersion, which demonstrate excellent adhesion to hair, reduce separation, provide improved moisture retention, higher lustre and a natural feel, as well as a suntan lotion having perfect water-repellence, is uniformly applied, gives a good feel and no clumping.

11 cl, 6 ex

FIELD: medicine.

SUBSTANCE: invention refers to veterinary science, and aims at animal growth stimulation. The method implies using a herbal tea collected in the period of intensive sap flow including stems and bark of Acer Platanoides. The raw material is grinded to 5-35 mm, heated for 1.5-3 hours at 160-280°C. Simultaneously, the volatile particles produced with material decomposition when heated are cooled and condensed. A light fraction is recovered and added to prepared gastric juices in ratio 1:10 respectively. The prepared solution is kept for 18-20 days at temperature 38-39°C and periodically agitated, filtered, reduced by concentrated sodium or potassium hydrate to pH 4.5-5.0, kept for another day at temperature 38-39°C, filtered and bottled. Then it is sterilised for 15-20 min at temperature 110-120°C and pressure 1 atm; the bottles are tightly sealed.

EFFECT: preparation produced by the declared method is safe, provides increased body weight, and average daily growth in the animals.

9 tbl, 2 cl

FIELD: medicine.

SUBSTANCE: invention refers to cosmetic industry, particularly it represents a composition for dry damaged hair, containing at least one lactone combined with trehalose.

EFFECT: invention provides improving the manageability of dry damaged hair to be styled.

5 cl, 1 ex

FIELD: medicine.

SUBSTANCE: invention refers to cosmetic industry, particularly it represents a composition for dry damaged hair, containing at least one lactone combined with trehalose.

EFFECT: invention provides improving the manageability of dry damaged hair to be styled.

5 cl, 1 ex

FIELD: medicine.

SUBSTANCE: group of inventions concerns oral care compositions. The presented compositions contain an amorphous silica abrasive having a particle size characterised by the fact that 90% of the particles have a particle size less than 50 mcm, and a fluoride source. One version of the composition also contains stannous ions and peroxide, and pH of the composition is less than 5. The use of amorphous silica with these characteristics provides improved fluoride stability and other active agents in the oral care composition. The compositions with pH lower than usual, e.g. falling within the range of 3.5 to 5 is allowed to be used.

EFFECT: using the presented compositions enables the effective and safe oral cleansing.

11 cl, 2 ex, 30 dwg

FIELD: medicine.

SUBSTANCE: group of inventions concerns oral care compositions. The presented compositions contain an amorphous silica abrasive having a particle size characterised by the fact that 90% of the particles have a particle size less than 50 mcm, and a fluoride source. One version of the composition also contains stannous ions and peroxide, and pH of the composition is less than 5. The use of amorphous silica with these characteristics provides improved fluoride stability and other active agents in the oral care composition. The compositions with pH lower than usual, e.g. falling within the range of 3.5 to 5 is allowed to be used.

EFFECT: using the presented compositions enables the effective and safe oral cleansing.

11 cl, 2 ex, 30 dwg

FIELD: medicine.

SUBSTANCE: group of inventions concerns oral care compositions. The presented compositions contain an amorphous silica abrasive having a particle size characterised by the fact that 90% of the particles have a particle size less than 50 mcm, and a fluoride source. One version of the composition also contains stannous ions and peroxide, and pH of the composition is less than 5. The use of amorphous silica with these characteristics provides improved fluoride stability and other active agents in the oral care composition. The compositions with pH lower than usual, e.g. falling within the range of 3.5 to 5 is allowed to be used.

EFFECT: using the presented compositions enables the effective and safe oral cleansing.

11 cl, 2 ex, 30 dwg

FIELD: medicine.

SUBSTANCE: present invention refers to dentistry, namely to oral care products. The presented nonabrasive toothpaste contains the enzyme papain, harpagophytum extract, D,L-pyrrolidone carboxylate N-cocoyl ethylarginate and sodium fluoride, as well as a carrier - sodium carboxymethyl cellulose, an emulsifier, a preserving agent, a flavouring agent, sorbitol 70% and dimeralised water in certain proportions. Using the toothpaste for oral care cleansing enables reducing plaque formation on the dental surface with no abrasive materials to be used by enzymatic plaque destruction.

EFFECT: toothpaste possesses manifested cleansing, anti-inflammatory and haemostatic action.

9 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: present invention refers to dentistry, namely to oral care products. The presented nonabrasive toothpaste contains the enzyme papain, harpagophytum extract, D,L-pyrrolidone carboxylate N-cocoyl ethylarginate and sodium fluoride, as well as a carrier - sodium carboxymethyl cellulose, an emulsifier, a preserving agent, a flavouring agent, sorbitol 70% and dimeralised water in certain proportions. Using the toothpaste for oral care cleansing enables reducing plaque formation on the dental surface with no abrasive materials to be used by enzymatic plaque destruction.

EFFECT: toothpaste possesses manifested cleansing, anti-inflammatory and haemostatic action.

9 tbl, 2 ex

FIELD: medicine, oncology, amino acids.

SUBSTANCE: invention relates, in particular, to the development of an antitumor preparation based on natural substances. Invention relates to an amino acid preparation comprising at least one modified essential amino acid obtained by treatment of amino acid by ultraviolet radiation (UV) at wavelength 250-350 nm for 12-80 h at temperature 15-30oC or with ozone at temperature 15-25oC. The modified amino acid has no toxicity for health cells. Also, invention relates to a method for preparing such preparation. Invention provides the development of an antitumor preparation based on modified amino acids and expanded assortment of antitumor preparations being without cytotoxicity for normal cells.

EFFECT: valuable medicinal antitumor properties of preparation.

8 cl, 4 tbl, 2 dwg, 4 ex

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