Inhibitor of influenza a virus reproduction based on extract of basidial fungus laetiporus sulphureus

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

 

The invention relates to antiviral agents, in particular inhibitor reproduction of influenza virus types And subtypes H5N1 and H1N1, and can be used in medicine, Virology and pharmacology.

The influenza virus And is the most famous and common of more than hundreds of viruses that cause infectious diseases of the upper respiratory tract. Annual influenza epidemics in the world lead to the 3.5 million cases of severe illness and to 300-500 thousand deaths [CDC 2005. Centers for Disease Control. Prevention and control of influenza recommendations of the advisory committee on immunization practices (ACIP) // MMWR. - 2005. - V.54 (RR08). - P.1-40. Available from URL: http://www.cdc.gov/mmwr/preview/mmwrhtml/rr5408a1.html].

New epidemique strains of influenza virus And occur every 1-2 years as the result of point mutations in the two surface glycoproteins - hemagglutinin and the neuraminidase (NA). Using the variety and variability of antigenic structure of the virion, the influenza virus is able to bypass the security mechanisms of human immunity, and therefore long-term immunity against the virus no after natural infection or after vaccination unlike smallpox, yellow fever, polio or measles.

Therefore, at present the disease is based on the treatment of influenza using causal, pathogenetic and symptomatic means. The search for new effective from the Oseni influenza drugs is one of the priority health problems.

The fungus Laetiporus sulphureus - sulphur-yellow tinder - belongs to the ecological group of wood-destroying fungi of basidiomycetes. In nature it occurs on the living and the dead trunks of deciduous trees and conifers, which causes brown wood rot. It is known that the fungus has a complex composition of various biologically active substances. In the fruit bodies are the following groups of compounds: triterpenes (0.5%), phenolic compounds (from 0.7 to 4%), amino acids (1-6,5%), carbohydrates: beckons (0,5-2,0%), water-soluble polysaccharides (0,5-2,5%), dilaceration polysaccharides (24-62%), chitin (2-5%), proteins (from 12.8 45,1%), lipids (0,6-1%), organic acids (2-8%), macro - and microelements (3,5-9%). Selected 2 new polysaccharide: latimore and Katipunan 1. For the first time this fungus detected the presence of alkaloids and natural compounds [Agafonova SV abstract. Kida. dis. The study of the chemical composition and characteristics of accumulation of biologically active compounds in the fruit bodies Laetiporus sulphureus (Bull., Fr.), Irkutsk, 2007. - 22 S.].

It was also established that among the secondary metabolites detected in the extract of this fungus were present such as egonol, demethoxyegonol, egonol glucoside [Jordan K. Zjawtony Biologically Active Compounds from Aphyllophorales (Polypore) Fungi. - J. Nat. Prod. - 2004. No. 67(2). - P.300-310].

The nature of the pigment lautaparketin that defines the color of the fruiting bodies was studied in 70-ies of XX is tolete. The pigment in its structure attributed to carotenoids, among which many are characterized by biological activity [Valadon L.R.G., Mummery R.S.A. A new carotenoid from Laetiporus sulphureus I Ann. Bot. - 1969. - Vol.33. - P.879].

On the basis of submerged mycelium Laetiporus sulphureus derived biologically active additives (BAA) - Leipurin. A food additive is non-toxic and recommended in the Republic of Belarus to restore vitamin and mineral deficiencies, improve the immune resistance to colds [Babicka VG, szczerba CENTURIES, gvozdikov FORCE New biologically active additives based on submerged mycelium of basidiomycetes. Advances in medical Mycology. - M.: national Academy of Mycology, 2006. - V.6. - Pp.178-181].

Known antioxidant and antimicrobial activity of the fungus Laetiporus sulphureus [Tikhonova O.V., Ershov EJ, Lurie L.M., Kulaeva CENTURIES, Katrukha G.S., kusakina O.V., Efremenkova O.V., Angelica Y. Antimicrobial properties of individuals Laetiporus sulphureus (Fr.) Bond et sing // Advances in medical Mycology: edited by Y.V. Sergeev. - 2001. 1. - Ch.6. - S-315; Tikhonova O.V., Lurie L. Ershov EJ, Efremenkova O.V., Angelica Y. the Study of deep culture Laetiporus sulphureus // Modern Mycology in Russia. - M., 2002. - S.257; Capic A.N., Gvozdikov T.S., Kuchava Z.B., Nikolaev, S., Shishkin, L.N., Galkin S., Chataka N., Konoplya E.F., Vereshako GG, Khodosy A.M., Rudkovskaya AA antioxidant is danta, radioprotective and anti-virus properties mycelial extract of the fungus Laetiporus sulphureus // Advances in medical Mycology: edited Yevseyeva. - M - 2004. - V.3. - P.146; Turkoglu A. Duru M.E., Mercan N., Kivrak I., Gezer K. Antioxidant and antimicrobial activities of Laetiporus sulphurous (Bull.) Murril // Food Chemistry. - 2007. - 101. - P.267-273]. Mentioned in a number of other basidiomycetes as a producer of compounds that suppress the development of plant virus diseases, such as tobacco mosaic [patent JP 1272508 (A), 1989].

It is known the use of the extract from the culture biomass of the fungus Laetiporus sulphureus as antiviral agents against plant viruses (Japan patent No. 1272508, IPC A01N 63/04, publ. 31.10.1989,).

One of the closest analogue (prototype) is the use of an aqueous extract of the mycelium of the fungus Laetiporus sulphureus as antiviral agents against herpes virus type 1 (HSV-1). Found that extracts of the mycelium of this fungus showed activity against variants of herpes simplex virus type (HSV-1), resistant to inhibitors acyclovir and phosphonooxy acid with high selectivity antiviral effect. Work carried out on the experimental model of herpetic infection in human cultures of glioma - S-6, tumor-derived rat brain and in the kidney cells of the African yellow monkey Vero [Vacheva B, Capic A.N., Votyakov V.I., S. Nikolaev. Antiviral activity of extracts of the mycelium of the basidiomycete Laetiporus sulphureus / Advances in medical Mycology. - 2005. - V.5. - S-273]. To assess the antiviral activity of the extract of the mycelium of the fungus was diluted with water and added a non-toxic concentrations in cell culture simultaneously with infection by the corresponding variant of HSV-1. Before it was determined the maximum tolerated dose of the drug. Antiviral activity of drugs stated reduction of infectious virus activity. When making mycelium extract in different concentrations (0.1 per cent; of 0.2%to 0.4% and 0.8%) in the infected cell culture was observed pronounced virpiniemi effect in respect of all investigated variants of HSV-1 - sensitive and resistant to acyclovir and phosphonooxy acid. Viryinia concentration of mycelium extract (0.2% solution) was 4 times less than its maximum tolerated dose. Reduction of infectious virus activity for different variants of HSV-1 ranged from 2.8 to 3.2 lg50/ml

However, in the prototype was investigated extracts obtained from cultured mycelium of the fungus Laetiporus sulphureus, which have inhibitory activity only against herpes virus type 1 (HSV-1).

The technical result of the claimed technical solution is obespecheniyavozmozhnost receiving means on the basis of an aqueous extract of the basidiomycete Laetiporus sulphureus, possessing inhibitory activity against influenza virus type A.

This technical result is achieved in that the inhibitor reproduction of the influenza a virus is an aqueous extract of the basidiomycete Laetiporus sulphureus, obtained by extraction of biomass chopped mushroom water at a ratio of 1:1 or 1:2 with the subsequent removal of insoluble precipitate.

The biomass of the fungus obtained from fruit bodies and extracted with shredded biomass of the fungus with water at a ratio of 1:2 or by cultivation on liquid nutrient medium on the basis of oatmeal broth and extracted with shredded biomass of the fungus with water at a ratio of 1:1.

In the present invention water extracts and extracted polysaccharides were investigated using a viral model of transplantable culture of MDCK cells infected with influenza virus strains A/chicken/Kurgan/05/2005 (H5N1) and A/Moscow/226/2009 (H1N1)v. Using an inverted microscope registered cytopathic effect (JRC) in a monolayer of cells and determined the presence or absence of virus in the environment of the cultivation by the reaction of haemagglutination (DSA) with 1% chicken erythrocytes. After determining 50%of the energy of infectious doses (EID50) virus (lg50/ml) in control and during the incubation with the drugs was calculated indexes neutralization (JN) virus in vitro (lg):

JN = ID50con the role ID50experience.

On the model of mice infected with influenza virus strains A/chicken/Kurgan/05/2005 (H5N1) and A/Moscow/226/2009 (H1N1)v, the difference between the titres in the lung homogenates of control and the experience was estimated index suppression products (IPP) of the virus under the influence of the drug in vivo.

Below are examples 1-3 receipt of extractives from fruiting bodies, mycelia and polysaccharides fungus Laetiporus sulphureus.

Example 1. Obtaining extracts from fruiting bodies of the fungus. 100 g of chopped frozen fruit bodies suspended in 200 ml of sterile distilled water and kept in a boiling water bath for 30 min, were released from the sediment by centrifugation for 40 min at 10000 rpm, the solids Content was 5 mg/ml (sample No. 08-09). The sample was stored for 6 months at a temperature of 20°C.

Example 2. Obtaining extracts from fruiting bodies of the fungus. 200 g chopped frozen fruit bodies suspended in 400 ml of sterile distilled water and kept in a boiling water bath for 30 min, were released from the sediment by centrifugation for 40 min at 10000 rpm, the solids Content was 5 mg/ml (sample No. 09-12)Obrazec were stored for 1 year at a temperature of 20°C.

Example 3. Obtaining the extract from the biomass of the fungus obtained by cultivation on a nutrient medium. Used 27-day culture of the fungus, the floor is built by cultivation on liquid nutrient medium on the basis of oatmeal broth in a stationary state. The biomass of the fungus in an amount of 5 g separated from the culture fluid (QOL) through a nylon filter, froze, then crushed, was added 5 ml of distilled water, treated with an ultrasonic disintegrator at an amplitude of 24 μm, brought the volume up to 25 ml, was centrifuged for 20 minutes at 10000 rpm, the solids Content in the sample was 1 mg/ml (sample No. 09-61).

Example 4. Determination of antiviral activity of samples of the extract of the fungus Laetiporus sulphureus on the cell culture.

Cell culture. For testing antiviral drugs used transplantable cell culture MDCK obtained from the collection of cell cultures fsri SRC VB "Vector". In a sterile place, the suspension was diluted with pre-warmed to a temperature of +37°C medium RPMI-1640 containing 5% serum fruits cows, to a concentration of 1.0 to 1.5×105cells/ml 100 ál of cell suspension MDCK were made in 96-well tablets 12-channel automatic pipette. Tablets with cells was placed in a thermostat at a temperature of +37°C, 5% CO2and 100% humidity for 2-3 days before the formation of the cell monolayer.

Determination of the toxicity of the sample. For the detection of toxic doses of drugs extracts were diluted several times and evaluated the available toxicity in culture monolayers of MDCK cells using inverted microsc the PA. This was done by dilution of the initial drug 5 times, 10, 100, 1000, 10000, 100000, 1000000 once the medium RPMI-1640 containing 5% serum fruits cows, made of 150 μl in appropriate wells and placed in a thermostat at a temperature of +37°C, 5% CO2and 100% humidity for 2 days. After 2 days using an inverted microscope to assess the presence of toxic effect in monolayers of MDCK cells, incubated with different concentrations of fungal extracts. As control was used monolayer cultures of MDCK cells without drug. Was determined the minimum toxic concentration (MTC) and the maximum tolerable concentration (MIC)equal to half the concentration of the substance does not affect cell toxicity. In studies of antiviral drugs used IPC or lower concentration.

Determination of antiviral activity of samples in vitro. To assess the antiviral efficacy of aqueous extracts of basidiomycetes on the culture of MDCK cells used a strain of the virus of avian influenza A/chicken/Kurgan/05/2005 (H5N1) and pandemic strain of influenza A/Moscow/226/2009 (H1N1)v from the collection of the fsri SRC VB "Vector", established on 10-day-old chicken embryos (CE). The concentration of virus in the samples was determined by titration on EC or MDCK cells, was calculated and expressed in lg50/ml (is the decimal logarithm of the 50%bear embryo infectious doses per ml) or in lg 50/ml (decimal logarithm of the 50%bear fabric cytopathic doses per ml), respectively, according to the method of Spearman-Cerberus. Developed and used in the series virusbulletin liquid (IMPORTANT) with influenza virus strains were stored at -70°C. the Concentration of the virus in virusbulletin liquid (IMPORTANT) was different experiments from 7.5 to 9.5 lg50/ml.

To determine the antiviral activity of compounds in vitro were used to the maximum tolerated concentration (MIC) of drugs. In the environment Axcevir-MDCK (Stem Alpha, France)containing 2 μg/ml trypsin TRNC (Sigma, USA), prepared 8 dilutions IMPORTANT with a ten-fold increments. To determine the antiviral activity of drugs on the monolayer of MDCK cells contributed 50 ál of the selected dilution and 100 μl 1-8 th dilutions IMPORTANT. Cells were incubated for 2 days at 37°C in an atmosphere of 5% CO2in thermostat TC-1/80 SDA (Russia). 2 days in each well using an inverted microscope registered cytopathic effect in monolayer cells and determined the presence of influenza virus in the environment of the cultivation by the reaction of haemagglutination with 1% chicken erythrocytes. Based on this determined the titers of influenza virus in lg50/ml in the control (EID50in vitro without drug) and experience (EID50in vitro drug), and then calculated the index neutralize him (JN) under the effects of the m preparation: IN = ID 50control - ID50experience (lg).

As control was used:

1. Control MDCK cells cultivated in a nutrient medium Axcevir-MDCK containing 2 μg/ml trypsin trypsin TRNC.

2. Control of reproduction of influenza virus A/chicken/Kurgan/05/2005 (H5N1) or A/Moscow/226/2009 (H1N1)v 1 to 8 with a tenfold dilution step in MDCK cells cultivated in a nutrient medium Axcevir-MDCK containing 2 μg/ml trypsin TRNC.

Statistical data processing. When calculating the 50% infective doses in every single repetition used method of Spearman-Cerberus was determined 95%confidence interval (I95) and were compared by z-test. For several repetitions was determined by the average value (M) and the error of the mean (m), the reliability of differences between mean values was determined using t-student test.

Table 1
Results antiviral activity of the extract of the fruit bodies Laetiporus sulphureus in the culture of MDCK cells infected with A/chicken/Kurgan/05/2005 (H5N1)
No. of drug and its breedingThe dry matter content of the drug (mg/mlThe final concentration of dry substances of the drug in the culture liquid, mg/ml Virus infectivity (titer IMPORTANT) in MDCK cells (EID50in Dtcd50/ml)The neutralization index EID50pin-EID50experience (lg)
08-09 (1:5)5,00,32,57
Control of the virus without medication9,50

Table 1 presents the results of the antiviral activity of the extract of the fruit bodies of the fungus (sample 08-09). It should be noted that the extract had no toxic effect on MDCK cells even in the initial dilutions.

As can be seen from table 1, the extract of the fruit body in comparison with the control showed a high inhibitory effect against influenza virus in birds, the neutralization index was 7 lg acting on the virus dose was 0.3 mg/ml

Despite the fact that during storage of the sample for six months at subzero temperatures (-20°C) neutralization index decreased by 2 lg, its antiviral activity remained high, the index neutralize the virus was 5 lg (table 2).

Table 2
Results antiviral activity of the extract of the fruit bodies Laetiporus sulphureus in the culture of MDCK cells infected with A/chicken/Kurgan/05/2005 (H5N1) after 6 months storage
No. of drug and its breedingThe dry matter content of the drug (mg/mlThe final concentration of dry substances of the drug in the culture liquid, mg/mlVirus infectivity (titer IMPORTANT) in MDCK cells (EID50in TCD50/ml)The neutralization index EID50pin-EID50experience (lg)
08-09 (1:5) storage 6 months5,00,32,55
Control of the virus without medication7,50

Table 3 presents the results of the antiviral activity of the extract of the fruit bodies Laetiporus sulphureus after 1 year of storage.

The results obtained indicate a high antiviral activity of extracts stored in the freezer for a long time. Even when breeding extract 1:100 neutralization index was 2 lg.

Table 3
Results antiviral activity of the extract of the fruit bodies Laetiporus sulphureus in the culture of MDCK cells infected with A/chicken/Kurgan/05/2005 (H5N1) after 1 year storage
No. of drug and its breeding, the period of storage at t -20°CThe dry matter content of the drug (mg/mlThe final concentration of dry substances of the drug in the culture liquid, mg/mlVirus infectivity (titer IMPORTANT) in MDCK cells (EID50in lgL50/ml)The neutralization index EID50pin-EID50experience (lg)
09-12 (Ref.)5,01,672,55
09-12 (Ref.)
1 year storage
5,01,672,55
09-12 1:100
1 year storage
5,00,0675,52
Control of the virus without medication7,50

Comparing the results for all samples, it can be concluded that the antiviral activity of the fungus against influenza virus strain A/chicken/Kurgan/05/2005 (H5N1) depends on the concentration of active components. In the fruit bodies of them, in terms of dry substance contains more than mycelium. Biological components of the fungus can withstand without loss of activity for long-term storage in the frozen state. Minimum dose of extract (as dry matter), exerting inhibitory effects in cell culture, is 0.01 mg/ml, and in laboratory animals (mice) - 1.0 mg per day.

Table 4 presents the results of the antiviral activity of mushroom extracts from the mycelium of the fungus Laetiporus sulphureus, obtained by cultivation on liquid nutrient medium.

As can be seen from table 4, the efficiency of the extract of the mycelium of the fungus (sample 09-61)obtained in culture, was lower than the extracts of the fruit phone But keep in mind that the achievement of a relatively high index of neutralization of 2.5 lg was achieved at the final concentration of dry substances in the preparation of 0.01 mg/ml, while for the extract of the fruit bodies to achieve neutralization index 2 lg the final concentration was 0,067 mg/ml, h is more about 6.7 times. The results show that the mycelium of the fungus obtained in culture, contains, as the fruit body, the active compounds inhibiting infection with influenza virus A/chicken/Kurgan/05/2005 (H5N1) culture of MDCK cells.

In addition, the authors assessed changes in the infectivity of pandemic influenza virus strain A/Moscow/226/2009 (H1N1)v in MDCK cells when introduced into the environment of the cultivation of extract No. 09-12 at a concentration of 5 mg/ml or Tamiflu. The averaged results for several repetitions of these experiments in comparison with the control are presented in table 5.

Table 5
The infectivity of the influenza virus strain A/Moscow/226/2009 (H1N1)v in MDCK cells during incubation with the extract of basidiomycetes or Tamiflu and Control (M±m)
The strain of influenza virusThe infectivity of the virus in MDCK cells (EID50in lg50/ml) after 3 days after infection during the incubation with drugs:
09-12 (5 mg/ml)TamifluControl
A/Moscow/226/20092,3±0,1*2,7±0,1*5,4±0,1
(H1N1)vn=1n=4n=4
Note: * - significant difference from control group (p=0.05); n is the number of repetitions.

Presented in table 5, the data indicate significant loss of infectivity of the influenza virus in the culture of MDCK cells during incubation with the extract 09-12 3.1 lg. The infectivity of influenza virus in MDCK cells using Tamiflu was also lower than in the control 2.7 lg.

Next was studied antiviral activity of this mushroom extract in their ability to suppress the production of influenza virus strains A/chicken/Kurgan/05/2005 (H5N1) and A/Moscow/226/2009 (H1N1)v in the evaluation of the suppression index reproduction (IPR) of virus in the lungs from mice 4 days after infection (see table 6 below).

Example 5. Determination of the antiviral activity of the fungus Laetiporus sulphureus on animals. The study was performed on the linear Balb/c mice, obtained from the nursery of laboratory animals fsri SRC VB "Vector", weight 14-17, research and content of the animals was carried out in accordance with the "Rules of work with the use of experimental animals" [the Annex to the order of the Ministry of health of the USSR from 12.08.1977, No. 755].

Infection of mice with influenza virus is produced intranasally under light ether anesthesia when injected into both nostrils total of 40 μl of each dilution of virus. Mice infected with influenza virus strains A/chicken/Kurgan/05/2005 (H5N1) in a dose of 10 LD50(50%bear lethal doses)equal to 1,6±0,4 lg50/goal., and A/Moscow/226/2009 (H1N1)v in a dose of 10 EID50(50%of cases of infectious doses), equal to 1.3±0,3 lg50/goal. The concentration of BR in the lungs was performed at 4 days after infection with influenza virus during the titration of the combined lung homogenates of mice of each group at the 9-daily EC was calculated by the method of Spearman-Cerberus and expressed in lg50/ml.

As the comparison drug in vivo used Tamiflu (oseltamivir), which was administered to mice once a day orally for 30 ug/g mass in a volume of 0.2 ml immediately after infection and continuing for 4 days after infection with influenza virus.

Statistical data processing. When calculating titers of influenza virus in biological samples in every single repetition used method of Spearman-Cerberus, was determined by the 95%confidence interval (I95) and were compared by z-test. For several repetitions was determined by the average value (M) and the error of the mean (m), the reliability of differences between mean values was determined using t-student test.

Table 6 presents the results of determining the titers of influenza virus in the lungs of mice 4 days after infection with strains A/chicken/Kurgan/05/2005 (H5N1) or A/Moscow/226/2009 (H11)v.

As can be seen from table 6, with the drug 09-12 at a concentration of 5 mg/ml titres in lungs from infected mice were significantly lower than in the corresponding control group (table 6). It was shown that the introduction of the drug 09-12 mice infected with influenza virus strain A/chicken/Kurgan/05/2005 (H5N1), the suppression index of production (IIP) of virus in the lungs was 1,16 lg, and the introduction of Tamiflu he was equal to 1.33 lg. However, 4 days later, the concentration of influenza virus in the lung homogenates of mice infected with influenza virus strain A/Moscow/226/2009 (H1N1)v, treated with drugs 09-12 and Tamiflu, was significantly lower than control 0.75 lg.

Table 6
The titers of influenza virus strains A/chicken/Kurgan/05/2005 (H5N1) or A/Moscow/226/2009 (H1N1)v in the lungs of mice 4 days after infection by oral administration of mushroom extract from Laetiporus sulphureus or Tamiflu in comparison with control
The strain of influenza virusThe titers of influenza virus (lg50/ml±I95in homogenates of lungs from infected mice treated with drugs:
09-12 (5 mg/ml)TamifluControl
A/chicken/Kurgan/05/2005 (H5N1)4,67±0,6*4,5±0,62*of 5.83±0,55
A/Moscow/226/2009 (H1N1)v6,0±0,57*6,0±0,69*6,75±0,49
Note: * - significant difference from control group at p<0,05.

As can be seen from tables 3, 5 and 6, the extract 09-12 in the initial concentration after one year of storage showed a significant reduction in virus titre of avian influenza A(H5N1) and pandemic influenza A(H1N1/09)v not only in cell culture but also in laboratory animals.

In the process of suppressing the reproduction of influenza virus is involved with a wide range of chemical compounds included in the composition of the fungus, providing anti-viral action of drugs on the basis of fungus Laetiporus sulphureus.

1. Inhibitor reproduction of the influenza a virus, which is an aqueous extract of a basidiomycete Laetiporus sulphureus, obtained by extraction of biomass chopped mushroom water at a ratio of 1:1 or 1:2 with the subsequent removal of insoluble precipitate.

2. Inhibitor reproduction of the influenza a virus according to claim 1, wherein receiving the biomass of the fungus from fruit bodies and extracted with shredded biomass of the fungus with water at a ratio of 1:2.

3. Inhibitor reproduction Viru is and flu And according to claim 1, wherein the receive biomass of the fungus by cultivation on liquid nutrient medium on the basis of oatmeal broth and extracted with shredded biomass of the fungus with water at a ratio of 1:1.



 

Same patents:

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: selenium-containing complex is an autolysate of fungal strain Cephaliophora tropica D3, obtained by growing fungus in a liquid culture medium with subsequent autolysis of the obtained biomass for 12 hours at temperature of 53-55°C and for 5-6 hours at temperature of 63-65°C. The culture medium contains the following trace elements in form of water-soluble salts: selenium 0.003-0.04 g/l, iodine 0.001-0.035 g/l, cobalt 0.001-0.017 g/l, copper 0.001-0.02 g/l, zinc 0.01-0.07 g/l, manganese 0.02-0.42 g/l, magnesium 0.02-0.8 g/l, iron 0.1-0.95 g/l, boron 0.0005-0.108 g/l, calcium 0.4-3.5 g/l.

EFFECT: obtained complex is capable of optimising metabolism of selenium and iodine in the body and can be used as a biologically active additive.

7 ex

FIELD: chemistry.

SUBSTANCE: method involves culturing a strain of Laetiporus sulphureus BKM-F-4276D on a cabbage medium which contains glucose in amount of 2%, milk whey in amount of 1%, with addition of sodium selenite in concentration of 15-25 mg/l.

EFFECT: invention increases content of selenium in the mycellium of the strain.

8 dwg, 4 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: method of obtaining proteinase-activator of protein C in blood plasma includes cultivation of strains of Aspergillus ochraceus VKM F-4104D or Aspergillus ochraceus VKM F-4105D or Aspergillus ochraceus VKM F-4106D or Aspergillus ochraceus VKM F-4107D on nutritional medium at initial value pH 6.5. Nutritional medium contains (%): glucose 3.2-3.8, starch 0.1-1.0, fish flour hydrolysate 0.5-1.0, peptone 0.1-0.5, NaCl 0.1-0.2, KH2PO4 0.04-0.06, MgSO4-7H2O 0.04-0.06, water to 100%. Anticoagulant activity of solution of proteins of culture liquid of strains on elongation of activated partial thromboplastin time (CPTT) constitutes 1020-1032%. Activity of protein C activator in culture liquid of strains constitutes 79.8-89.9 units/ml.

EFFECT: increased activator activity.

2 tbl, 8 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology. Spores of filamentous fungi Cunninghamella are grown on wort agar at temperature 27-28°C for 5-6 days. Trehalose in amount of 0.2% is added to the wort agar layer. The spores are washed and held for 20-30 minutes in sterile tap water while slightly shaking. The washed off spores are fermented as inoculum for 90-96 hours at temperature 29±0.5°C on a culture medium containing glucose, ammonium nitrate, magnesium sulphate heptahydrate, potassium dihydrophosphate, a yeast extract and 0.1-0.2% D-glucosamine or 0.11-0.15% N-acetyl-B-glucosamine. The obtained biomass is separated and lyophilised. Lipids are extracted by shaking the lyophilised biomass with a mixture of chloroform and ethyl alcohol in ratio 2:1 on a shaker.

EFFECT: method increases output of lipids.

8 ex

FIELD: chemistry.

SUBSTANCE: powder of coarsely ground lichen fronds undergoes solid-phase mechanochemical treatment in a bead mill at 1500 rpm for 3 minutes with addition of 0.5 wt % sodium bicarbonate.

EFFECT: invention improves quality, antibacterial action of the preparation and simplifies the process of producing said preparation.

3 tbl, 5 dwg

FIELD: medicine.

SUBSTANCE: what is offered is the Trichoderma harzianum rifai M 99/51 strain deposited in the Russian National Collection of Industrial Microorganisms, No. F-1027. The strain is characterised by synthesis of active anticancer non-peptide compounds having no amide bonds. Besides, the strain is marked by producing the anticancer compounds. The strain has high cytotoxic activity with respect to the anticancer cell lines (human HaCaT-keratinocytes; THP-1 macrophage-like human cell line; A431 - human epidermoid carcinoma line; JurKat - human T-cells; K562 - chronic myeloid leukemia cell line; HEK293T - human embryonic kidney cells) and may be used for producing anticancer preparations.

EFFECT: using this strain as a producer for making the based anticancer preparations.

8 dwg, 6 ex

FIELD: medicine.

SUBSTANCE: Aspergillus ochraceus strain is recovered from carbonate chernozem samples and deposited in Russian National Collection of Microorganisms, No. F-4106D. The strain produces proteinase being a human blood plasma protein C activator into a culture fluid while grown on a medium containing carbohydrate and protein compounds, and mineral salts. Anticoagulant activity of the strain to prolong activated partial thromboplastin time by 2 cultivation days makes 960%. Proteinase activity of the strain Aspergillus ochraceus in the culture fluid determined with the use of chromogenic peptide substrate pGlu-Pro-Arg-pNA makes 77.9 units/ml.

EFFECT: higher proteinase activity of the strain.

1 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: Aspergillus ochraceus strain is recovered from plant residue samples on wort agar and deposited in Russian National Collection of Microorganisms, No. F-4105D. The strain produces proteinase being a human blood plasma protein C activator into a culture fluid while grown on a medium containing carbohydrate and protein compounds, and mineral salts. Anticoagulant activity of the strain to prolong activated partial thromboplastin time by 2 cultivation days makes 920%. Proteinase activity of the strain Aspergillus ochraceus in the culture fluid determined with the use of chromogenic peptide substrate pGlu-Pro-Arg-pNA makes 72.5 units/ml.

EFFECT: higher proteinase activity of the strain.

1 tbl, 2 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: medicine, pharmaceutics.

SUBSTANCE: claimed invention relates to radiopharmaceutical composition for determination of presence, localisation and/or quantity of one or more amyloid deposits on an organ or area of subject's body. Claimed composition contains compound of formula

:

Z represents S, NR', O or C(R')2, where each R' independently represents H or C1-6alkyl, Y represents hydrogen, halogeno, OR' or SR', (R1 = H or C1-6alkyl), or Y represents -NR1R2; and each R1-10 is independently selected from group, consisting of hydrogen, C1-6alkyl, C2-6alkenyl, C2-6alkinyl, C1-6alcoxy, C4-6cycloalkyl, hydroxyl, C1-6hydroxyalkyl, C2-6hydroxyalkenyl, C2-6hydroxyalkinyl, thiol, C1-6thioalkyl, C2-6thioalkenyl, C2-6thioalkinyl, C1-6thioalkoxy, halogeno, C1-6halogenoalkyl, C2-6halogenoalkenyl, C2-6halogenoalkinyl, C1-6halogenoalkoxy, amino, C1-6aminoalkyl, C2-6aminoalkenyl, C2-6aminoalkinyl, C1-6aminoalkoxy, cyano, C1-6cyanoalkyl, C2-6cyanoalkenyl, C2-6cyanoalkinyl and C1-6cyanoalkoxy; nitro, C1-6nitroalkyl, C2-6nitroalkenyl, C2-6nitroalkinyl and C1-6nitroalkoxy. Composition also contains biocompatible carrier-medium and 0.05-5.0% wt/vol of polysorbate at pH from 4.0 to 10.5. One atom of said formula I compound represents radioactive isotope, suitable for visualisation in vivo. Also claimed is method of obtaining said radiopharmaceutical composition.

EFFECT: invention ensures reduced loss of derivatives of thioflavin derivatives and high radioactivity.

22 cl, 5 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to medicine, namely to medication for promotion of survival of obtained from bone marrow mesenchymal stem cells in vivo and differentiation in cadiomyocytes. Application of traditional Chinese medical composition for obtaining medication for promotion of survival of obtained from bone marrow mesenchymal stem cells in vivo and differentiation in cadiomyocytes. Traditional Chinese medication composition (versions). Method of treatment and prevention of cardiovascular disease, including introduction to patients who need it of efficient quantity of traditional Chinese medical composition, and mesenchymal stem cells, obtained from bone marrow.

EFFECT: composition is efficient for promotion of in vivo survival of obtained from bone marrow mesenchymal stem cells and their differentiation in cardiomyocytes.

15 cl, 8 dwg, 1 tbl, 1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to medicine, namely to medication for promotion of survival of obtained from bone marrow mesenchymal stem cells in vivo and differentiation in cadiomyocytes. Application of traditional Chinese medical composition for obtaining medication for promotion of survival of obtained from bone marrow mesenchymal stem cells in vivo and differentiation in cadiomyocytes. Traditional Chinese medication composition (versions). Method of treatment and prevention of cardiovascular disease, including introduction to patients who need it of efficient quantity of traditional Chinese medical composition, and mesenchymal stem cells, obtained from bone marrow.

EFFECT: composition is efficient for promotion of in vivo survival of obtained from bone marrow mesenchymal stem cells and their differentiation in cardiomyocytes.

15 cl, 8 dwg, 1 tbl, 1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to medicine, namely to medication for promotion of survival of obtained from bone marrow mesenchymal stem cells in vivo and differentiation in cadiomyocytes. Application of traditional Chinese medical composition for obtaining medication for promotion of survival of obtained from bone marrow mesenchymal stem cells in vivo and differentiation in cadiomyocytes. Traditional Chinese medication composition (versions). Method of treatment and prevention of cardiovascular disease, including introduction to patients who need it of efficient quantity of traditional Chinese medical composition, and mesenchymal stem cells, obtained from bone marrow.

EFFECT: composition is efficient for promotion of in vivo survival of obtained from bone marrow mesenchymal stem cells and their differentiation in cardiomyocytes.

15 cl, 8 dwg, 1 tbl, 1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to medicine, namely to medication for promotion of survival of obtained from bone marrow mesenchymal stem cells in vivo and differentiation in cadiomyocytes. Application of traditional Chinese medical composition for obtaining medication for promotion of survival of obtained from bone marrow mesenchymal stem cells in vivo and differentiation in cadiomyocytes. Traditional Chinese medication composition (versions). Method of treatment and prevention of cardiovascular disease, including introduction to patients who need it of efficient quantity of traditional Chinese medical composition, and mesenchymal stem cells, obtained from bone marrow.

EFFECT: composition is efficient for promotion of in vivo survival of obtained from bone marrow mesenchymal stem cells and their differentiation in cardiomyocytes.

15 cl, 8 dwg, 1 tbl, 1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to medicine, namely to medication for promotion of survival of obtained from bone marrow mesenchymal stem cells in vivo and differentiation in cadiomyocytes. Application of traditional Chinese medical composition for obtaining medication for promotion of survival of obtained from bone marrow mesenchymal stem cells in vivo and differentiation in cadiomyocytes. Traditional Chinese medication composition (versions). Method of treatment and prevention of cardiovascular disease, including introduction to patients who need it of efficient quantity of traditional Chinese medical composition, and mesenchymal stem cells, obtained from bone marrow.

EFFECT: composition is efficient for promotion of in vivo survival of obtained from bone marrow mesenchymal stem cells and their differentiation in cardiomyocytes.

15 cl, 8 dwg, 1 tbl, 1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to medicine, namely to medication for promotion of survival of obtained from bone marrow mesenchymal stem cells in vivo and differentiation in cadiomyocytes. Application of traditional Chinese medical composition for obtaining medication for promotion of survival of obtained from bone marrow mesenchymal stem cells in vivo and differentiation in cadiomyocytes. Traditional Chinese medication composition (versions). Method of treatment and prevention of cardiovascular disease, including introduction to patients who need it of efficient quantity of traditional Chinese medical composition, and mesenchymal stem cells, obtained from bone marrow.

EFFECT: composition is efficient for promotion of in vivo survival of obtained from bone marrow mesenchymal stem cells and their differentiation in cardiomyocytes.

15 cl, 8 dwg, 1 tbl, 1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to medicine, namely to medication for promotion of survival of obtained from bone marrow mesenchymal stem cells in vivo and differentiation in cadiomyocytes. Application of traditional Chinese medical composition for obtaining medication for promotion of survival of obtained from bone marrow mesenchymal stem cells in vivo and differentiation in cadiomyocytes. Traditional Chinese medication composition (versions). Method of treatment and prevention of cardiovascular disease, including introduction to patients who need it of efficient quantity of traditional Chinese medical composition, and mesenchymal stem cells, obtained from bone marrow.

EFFECT: composition is efficient for promotion of in vivo survival of obtained from bone marrow mesenchymal stem cells and their differentiation in cardiomyocytes.

15 cl, 8 dwg, 1 tbl, 1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to medicine, namely to medication for promotion of survival of obtained from bone marrow mesenchymal stem cells in vivo and differentiation in cadiomyocytes. Application of traditional Chinese medical composition for obtaining medication for promotion of survival of obtained from bone marrow mesenchymal stem cells in vivo and differentiation in cadiomyocytes. Traditional Chinese medication composition (versions). Method of treatment and prevention of cardiovascular disease, including introduction to patients who need it of efficient quantity of traditional Chinese medical composition, and mesenchymal stem cells, obtained from bone marrow.

EFFECT: composition is efficient for promotion of in vivo survival of obtained from bone marrow mesenchymal stem cells and their differentiation in cardiomyocytes.

15 cl, 8 dwg, 1 tbl, 1 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

Up!