RU2435599C1 - PREPARATION-4-Hydroxy-17R-Methylincisterol AFFECTING TISSUE EXCHANGE AND APPLICATION OF Pleurotus 1137 FUNGUS STRAIN FOR MAKING IT - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to medicine and biotechnology and concerns a preparation of 4-hydroxy - 17R - methylincisterol affecting tissue exchange and exhibiting anticancer and hypolipidemic activities and a producer of a biologically active substance of sterol 4-hydroxy - 17R - methylincisterol derivative. Substance of the invention involves submerged Pleurotus ostreatus 1137 (Russian National Collection of Industrial Microorganisms, F-819) fungus cultivation followed by mycelium separation from a culture fluid and recovery from the mycelium by extraction in ethanol at acid value pH of a medium of low-molecular 100 to 500 Da biologically active substances and recovery of sterol 4-hydroxy - 17R - methylincisterol derivative with preset molecular weight, gross formula and structural formula by dichloromethane. The invention also concerns application of Pleurotus ostreatus 1137 (Russian National Collection of Industrial Microorganisms, F-819) strain as a producer of the biologically active substance of sterol 4 - hydroxy - 17R - methylincisterol derivative.

EFFECT: advantage of the invention consists in development of such preparation which is able to modulate manifestations of hypolipidemic activity along with anticancer activity.

3 cl, 7 tbl, 15 dwg

Description

The invention relates to medicine, medical and biological industries, in particular to strains of fungi-producers of biologically active substances and preparations based on them, and can be used in the production and use of biologically active preparations and food additives based on mushroom producers.

One of the promising groups of plants, whose chemical composition of biologically active substances and the properties of these substances are currently being studied quite fully, are higher basidiomycetes.

The study of the healing properties of higher basidiomycetes and their practical application in the field of medicine have been carried out by scientists from several countries over the past 50 years. The results of these studies are especially widely used in China, Japan, Korea, the USA, Canada, Russia, and more recently in Norway.

Among the metabolites of higher fungi, substances from cap mushrooms, which are immunomodulators and exhibit significant antitumor, cardiovascular, antiviral, antibacterial, antiparasitic, hepatoprotective and antidiabetic properties, were identified and identified (Materials of the IV International Conference “Mushroom Science and Practice. 1997; Prospects for the use in medicine of substances formed by basidiomycetes. Review (International Journal of Medicinal Mushrooms. Vol.3, 31-62, 1999).

About 200 species of cap mushrooms are known that have the pronounced properties listed above. However, most of the substances that make up the mushrooms are not completely identified and only about 10 types of mushrooms are used to obtain medicines. For example, in Japan, China, Russia, Ukraine, Belarus, the USA, Canada and Norway, several polysaccharide cytostatics have been developed that are used as pharmaceuticals. They are obtained by culturing under submerged conditions Trametes versicolor (PSK, Krestin; Jopan; GLUCANOVA NORWAY 2010).

At the same time, the creation of new drugs to date is largely constrained by the complex and expensive technology for obtaining pure cultures and substances from basidiomycetes that possess the stability of the required therapeutic properties. First of all, this is justified by the fact that the formation of metabolites valuable for pharmacology depends on the culture conditions of the producer and, in the opinion of most authors, is unstable specific strain-specific.

Therefore, at present, we do not have specific antitumor and hyperlipidemic safe, non-toxic agents obtained from higher basidiomycetes.

Regarding the presence of the biologically active substance currently isolated from higher basidiomycetes, one of the ergosterol derivatives 4-hydroxy-17R-methylincisterol, which can prevent the proliferation of cancer cells without toxic effects on the human body or effectively regulate lipid metabolism in the human body, this kind of published information are absent in the literature and unknown to the authors.

Anticancer chemotherapy using a number of cytostatics is widely used to treat cancer. The most effectively used include: cyclophosphamide, doxorubicin, vepezid, taxanes, methotrexate, 5-fluorouracil, which are used in practice both separately and in combination with each other.

In the last decade in pharmacology, the so-called statins have become the No. 1 drugs in terms of volume of use. From a chemical point of view, the group of statins is heterogeneous and is represented by completely non-close compounds. However, the most used statins in pharmacology are sterol derivatives produced by lower fungi from the genus Aspergillus.

The most typical of these are lovastatin and simvastine, semi-synthetic modifications of which are available under dozens of names as effective antihyperlipidemic agents. These drugs, in particular Zokor, reduce the risk of strokes and plaque formation by reducing cholesterol and optimizing the ratio of low and high density lipoproteins in human blood.

Active studies of the effect of lovastatin revealed one of, apparently, the main targets of its action. It was shown that lovastatin (and, possibly, a number of other statins) is an inhibitor of one of the enzymes for the synthesis of cholesterol from mevalonate, namely 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase). At the same time, it is clear that there may be more targets for the action of statins, in accordance with the stages of cholesterol synthesis and metabolism.

Recently, extremely intriguing data has been published that statins have oncoprotective properties, while the mechanisms of this effect remain unknown. Interestingly, a combination of the same multidirectional effects is attributed by eastern medicine to the so-called “medical” mushrooms, in particular oyster mushrooms from the genus Pleurotus. Lyophilized dried powders of the fruiting bodies of these mushrooms are widely used in oriental medicine, although there are no chemically identified preparations from these mushrooms.

Meanwhile, the toxicity of cytostatics and statins is a significant limitation in achieving the maximum effectiveness of antitumor or antihyperlipidemic therapy. It is toxic manifestations during their use, as a rule, that limit the conduct of adequate treatment. Therapy is often carried out with reduced doses of drugs with increasing intervals between courses, and, as a result, treatment is not always satisfactory.

Therefore, approaches to reducing the toxic manifestations of antitumor and lipid-lowering drugs are actively being developed. However, to date, in practical medicine there is no effective enough for widespread use of such drugs.

As for the modulators of toxicity of higher basidiomycetes isolated from biologically active substances, there is no information on such compounds in the literature, except for the biologically active food supplement “Oyster mushroom extract“ developed in accordance with RF patent No. 2192873 and currently commercially available in Russia OVODORIN ”in the form of a gel (Certificate of state registration No. 77.99.23.3.U.11397.12.09 of 12/14/2009) and in the form of syrup (Certificate of state registration No. 77.99.23.3.U.11398.12.09 of 14.12.2009 g.)

A known drug that affects tissue metabolism, and the use of the strain of the fungus Pleurotus ostreatus 1137 to obtain it (RF patent 2192873, class A61K 35/70, 2001), used in biotechnology and medicine in the production and use of biologically active food additives on basis of mushroom producers.

According to the description and claims of this invention, the known preparation has antitumor activity and the ability to modulate the manifestation of hematological toxicity of cytostatics. The drug is produced by deep cultivation of the fungus Pleurotus ostreatus 1137 (VKPM, F-819), followed by separation of the mycelium and the culture fluid and isolation from the mycelium by extraction with ethanol at acidic or neutral pH values of biologically active substances with antimicrobial activity containing amino acids (glycine, alanine, threonine, series, leucine) and carbohydrates (glucose, glucosamine, arabinose, xylose).

The drug is made in the form of dehydrated granules or tablets or in the form of a gel-like syrup.

The strain 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 of the strain of the fungus Pleurotus ostreatus 1137 described in the patent (VKPM, F-819), a preparation was obtained that possesses a wide range of therapeutic and prophylactic effects, including and antioxidant activity.

However, to date, studies on the isolation of a sterol derivative (4-hydroxy-17R-methylincisterol) from the developed oyster mushroom mycelium extract (RF patent No. 2192873 dated 05/08/2001) and its identification have not been carried out.

The objective of the present invention is to develop a biologically active drug based on a strain of the fungus Pleurotus ostreatus 1137 with effective multifunctional biomedical activity that affects tissue metabolism without any adverse side effects, including toxicity to the human body.

The technical result consists in expanding the arsenal of action of a drug released from the biologically active substances of the mycelium extract Pleurotus ostreatus 1137, and reducing toxicity when used as a drug with antitumor and lipid-lowering activities.

The result is achieved by the fact that the drug affecting tissue metabolism was obtained by deep cultivation of the fungus Pleurotus ostreatus 1137 (VKPM, F-819), followed by separation of the mycelium from the culture fluid and isolation from the mycelium by extraction with ethanol at an acidic pH of low molecular weight from 100 to 500 daltons of biologically active substances, followed by isolation from them with dichloromethane of the sterol derivative 4-hydroxy-17R-methylincisterol with an established molecular weight of 332.2452 by UPLC / MS / MS and TOF mass spectrometry, gross formula C ₂₁ H ₃₂ O ₃ meto House of TOF mass spectrometry and structural formula by NMR: 13C, APT, HMQC, NMVC and COZY data

and has the ability to inhibit the growth of cancer cells and regulate the lipid metabolism of the body.

The drug can be made in the form of dehydrated granules or tablets or in the form of a water-alcohol solution or in the form of a syrup.

The strain Pleurotus ostreatus 1137 (VKPM, F-819) is used as a producer of the biologically active substance of the sterol derivative 4-hydroxy-17R-methylincisterol.

The strain of culture Pleurotus ostreatus 1137 is stored in the All-Russian collection of Industrial Microorganisms, State Research Institute of Genetics under registration number VKPM, F-819 and is characterized by the following features.

Cultural and morphological characteristics

1. Growth on agar media

Agarized Wort

The mycelium is well developed, cotton-like. The growing mycelium is creeping, the edge of the colony is felt, even. The growth rate is high: for 7-9 days after seeding in the center of a Petri dish with a diameter of 10 cm, complete agar medium fouling is observed. The color of the mycelium is white. There is no exopigment. With prolonged cultivation (up to 30 days), the color of the mycelium does not change.

Hyphae are septate, branching is frequent, at an acute or right angle, the branching type is monopodial. Buckles form. The diameter of most hyphae is from 2 to 4 microns; hyphae with diameters from 1 to 8 microns are also present.

Peptone tryptone agar

Mycelium is well developed, felt. The growth is creeping, the growing edge is spider, even. The growth rate is high. The color of the mycelium is white, with prolonged cultivation (more than 21 days) pale yellow or light brown areas are possible. There is no exopigment.

Soya Agar

The mycelium is sparse, arachnoid, an agar medium is visible through the mycelium. The nature of growth is creeping, the growing edge is flat, spider. The growth rate is low: after plating in the center of a Petri dish with a diameter of 10 cm, complete agar medium fouling is observed only on the 12-14th day of growth. The color of the mycelium is white. There is no exopigment.

2. Deep cultivation

Depth cultivation is carried out in 750 ml flasks with a medium volume of 40 ... 150 ml on a shaker with 200 rpm. Inoculation was carried out with pieces of agar with mycelium.

Wednesday with a must

The number of colonies depends on the load of the seed. Over 10-14 days of growth, several dense round-shaped colonies are formed that have formed around the seed, and / or many small daughter colonies. The color of the mycelium is from white to light beige. The color of the medium during cultivation is clarified. The transparency of the environment does not change. The weight of the mycelium is in direct proportion to the concentration of the wort in the medium (from 0.6 to 3 ballings).

Soya flour medium

The number of growth points is significant, which is associated with the immobilization of mycelium on soy flour particles in the medium. Thick, mushy growth. When defending the mycelium practically does not settle.

Physiological and biochemical characteristics

The strain, like other representatives of the Pleurotus genus, grows on various sources of plant materials containing cellulose.

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

The strain uses peptone, tryptone, soy and oat flour as a source of nitrogen.

It grows in the temperature range from 8 to 34 ° C, maintains viability at a temperature of 0 to 40 ° C, the optimum temperature for growth is 24 ° C.

It grows at pH values from 4 to 8, with an optimum of 6 to 7.

Strict aerob.

Pathogenicity

The strain is not pathogenic for humans and animals, but, as a representative of the Pleurotus ostreatus species, it is phytopathogenic for weakened deciduous trees. Basidiospores can cause allergic reactions in humans.

Antagonistic properties

Under conditions of deep cultivation, antimicrobial activity is manifested against gram-positive, gram-negative bacteria and fungi.

A new purpose of the strain Pleurotus ostreatus 1137 is its use as a producer of the biologically active substance of the sterol derivative 4-hydroxy-17R-methylincisterol.

The basis of the invention is the use of the strain Pleurotus ostreatus 1137 to create on its basis the preparation 4-hydroxy-17R-methylincisterol, which has the ability to inhibit the proliferation of cancer cells and regulate the lipid metabolism of the body.

Tryptone agar was used for surface cultivation of Pleurotus ostreatus 1137 and test strains, as well as soy agar medium, oat agar medium and wort agar.

The drug 4-hydroxy-17R-methylincisterol, which affects tissue metabolism, was obtained biotechnologically using the fungus Pleurotus ostreatus 1137. In accordance with the patent of the Russian Federation No. 2192873 dated 05/08/2001, this culture was grown by the deep method in a sterile liquid nutrient medium.

The process of obtaining the drug 4-hydroxy-17R-methylincisterol included 4 sequentially performed steps:

I STAGE - growing mycelium biomass:

- breeding work with the strain;

- preparation of seed (on an agar medium);

- deep cultivation of mycelium (cultivation in liquid media and separation of mycelium from the culture fluid).

II STAGE - Preparation of the condensed extract from the mycelium Pleurotus ostreatus 1137 in the form of a gel:

- extraction;

- filtering;

- evaporation of the extract (in vacuum).

III STAGE - Isolation of the concentrated mycelium extract Pleurotus ostreatus 1137 in the form of a gel of the biologically active substance of the sterol derivative 4-hydroxy-17R-methylincisterol with dichloromethane.

IV STAGE - Preparation of the dosage form of 4-hydroxy-17R-methylincisterol in the form of dehydrated granules or tablets or in the form of an aqueous-alcoholic solution or in the form of a syrup.

The deep cultivation of mycelium was carried out in 750 ml flasks in a medium containing 0.65 wort ballings; tap water, pH was not adjusted. Etter medium, medium 2663, meat and peptone broth and their modifications were also used. The volume of medium in the flasks was 40 ... 150 ml. For aeration, flasks were placed on a rotary shaker at 200 rpm. Incubation was carried out from 7 to 24 days at a temperature of 26 ... 28 ° C.

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

It was established that of the tested nutrient media, the most optimal for the mycelium growth in biomass and antimicrobial activity is 0.65 balling wort.

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

Biologically active substances were isolated from mycelium by extraction with ethanol at an acidic pH.

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

The mycelium extract of Pleurotus ostreatus 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 the mycelium Pleurotus ostreatus 1137, gram-positive and gram-negative bacteria were used as test cultures.

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.

The antimicrobial activity of Pleurotus ostreatus 1137 mycelium extract was determined by agar diffusion.

15 ml of tryptone agar was poured into Petri dishes with a diameter of 10 cm. The test culture was sown on a solid surface of the medium with a continuous lawn.

A concentrate of Pleurotus ostreatus 1137 mycelium extract in an amount of 10 ml was applied to 6 mm diameter filter paper disks. Disks were placed on the surface of an agar medium seeded with test cultures.

After a day of incubation, zones of growth inhibition of the test strain were detected.

As a result, it was found that the studied strain of Pleurotus ostreatus 1137 exhibits antimicrobial activity against both gram-positive and gram-negative bacteria (Table 1).

Based on the above strain, a preparation of 4-hydroxy-17R-methylincisterol was obtained, which has the ability to inhibit the proliferation of cancer cells and regulate the lipid metabolism of the body.

Isolation of the condensed extract of the mycelium Pleurotus ostreatus 1137 as a gel of the biologically active substance of the sterol derivative 4-hydroxy-17R-methylincisterol with dichloromethane was carried out as follows.

The gel extract of Pleurotus ostreatus 1137 mycelium (1 g) was dissolved in 96% ethanol at a concentration of 100 mg / ml. The resulting solution (aliquots of 400 μl) was separated chromatographically on a Gilson semi-preparative HPLC system, consisting of 2 pumps with 25SC heads, a pressure gauge module, a mixer, an injector with a 500 μl loop, a column thermostat and a flow divider (1:10), Variable wavelength UV detector and control program. A semi-preparative 24 × 250 mm column with a Diasorb C16T sorbent (10 μm) was used. The composition of the mobile phase: 96% ethanol / 0.1% trifluoroacetic acid in water 60:40, at a flow rate of 10 ml / min and a temperature of 30 ° C. It is possible to use a similar chromatographic system and a similar reversed-phase column. The analysis time is 40 minutes, detection at a wavelength of 230 nm, the peak of interest has a retention time of 20-25 minutes. The fraction containing the peak of interest was collected, 30% ammonia solution was added to a neutral pH of 6-8 and evaporated on a rotary evaporator at a temperature of 45-55 ° C for 2/3 by volume until the solution became opalescent. An equal volume of dichloromethane was added to the remaining solution, extracted and the organic layer was separated on a separatory funnel.

The organic layer was evaporated on a rotary evaporator without heating and dissolved in 1 ml of 96% ethanol. The resulting solution (aliquots of 100 μl) was separated using analytical HPLC on an Agilent 1100 system consisting of a four-channel pump with a degasser, a column thermostat, an autosampler, a diode array UV detector, and a ChemStation control program. An analytical column of 4.6 × 250 mm with a Luna C 18 (2) sorbent (5 μm) was used. It is possible to use a similar chromatographic system and a similar reversed-phase column. The composition of the mobile phase: acetonitrile / water 79:21, at a flow rate of 1 ml / min and a temperature of 25 ° C. The analysis time is 21 minutes, detection at a wavelength of 230 nm, the peak of interest 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 for 2/3 by volume until the solution becomes opalescent. An equal volume of dichloromethane was added to the remaining solution, extracted and the organic layer was separated on a separatory funnel. The organic layer was evaporated on a rotary evaporator without heating, dissolved in 1 ml of dichloromethane and dried in a desiccator. The yield of 4-hydroxy-17R-methylincisterol was 8 mg.

Chromatogram of the initial solution (analysis of the extract with dichloromethane on a Luna C18 (2) analytical column (5 μm) 4.6 × 150 mm, mobile phase: acetonitrile / water / 1% TFA in water = 79: 16: 5, flow rate 1.5 ml / min at 25 ° C, detection at 230 nm, retention time of the substance of interest 6-7 min, analysis time 13 min) is shown in figure 1.

Example of semi-preparative separation on an analytical HPLC system: fraction analysis after preparative HPLC on an Luna C18 (2) analytical column (5 μm) 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 FIG. 2 (substance retention time 16 min).

Chromatogram of the purified substance (aliquot of 10 μl: HPLC on a Luna C18 (2) analytical column (5 μm) 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 Fig.3.

The molecular formula C ₂₁ H ₃₂ O ₃ was determined by the exact mass by time-of-flight method UPLC / MS / MS mass spectrometry. Spectrum 13C contains 21 signals of carbon atoms. The number of protons attached to each atom was determined by combining the 13C, 1H, APT, and HMQC spectra. Chemical shifts and multiplicativity of carbon signals are shown in table 2.

The results of determining the molecular weight and gross formula are shown in figure 4, 5, 6.

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

In figure 5, ion 331.38 corresponds to a mass of 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 it.

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

355.2295 = C ₂₁ H ₃₂ O ₃ Na + (15ppm);

333.2452 = C ₂₁ H ₃₃ O ₃ + (8ppm).

Structural studies carried out under the program “Structure Elucidator / ACDlabs (Canada)” [Journal of Analytical Chemistry, 2008, vol. 63, No.1, cc. 18-26] established the structure of the drug.

The structure was determined by combining 13C, APT, HMQC, NMVC, and COSY data. To determine the relative configuration of optically active centers, the ROESY spectrum was used.

The program generates a set of structures that correspond to the initial data on chemical shifts and 2D correlations.

The COZY spectrum contains 27 unambiguous correlations, which are shown on the proposed structure as a dashed line (Fig. 7). Almost all theoretically possible correlations of COSY are present in the spectrum. Only correlations between two pairs of atoms were not found in the spectrum, mainly due to masking by other correlations with a close chemical shift. For example, the correlation between atoms 8 and 12 (1.49-1.88 ppm) is very close in chemical shift to the correlation between two protons attached to atom 7 (1.48-1.92). The correlation between atoms 7 and 15 (1.48-1.50 ppm) is too close to the intense diagonal peaks. Additionally, the spectrum contains one correlation between atoms 14 and 17, shown in Fig. 7 as a broken line with dots. The information obtained from the COZY spectrum made it possible to unambiguously establish the skeleton of the molecule.

The cyclic part of the molecule contains several Quaternary carbon atoms, which cannot be established using the COZY spectrum. Therefore, to determine this part of the molecule, the NMVS spectrum was used. The NMVS spectrum contains 85 correlations (Fig. 8 and Table 2). All theoretically possible correlations (2J and 3J) were found in the NMVS spectrum. An additional 4 long-range correlations (4J) are present in the spectrum. These correlations are highlighted in Fig. 8 as a broken line with dots.

The established structure has the best agreement with the carbon spectrum (the average deviation between the calculated and experimental values is 2.6 ppm).

All of the above suggests that the structure of the drug is uniquely established.

Definition of stereo configuration

The E configuration of the double bond was determined on the basis of the spin – spin interaction constant (15.1 Hz). To determine the relative stereo configuration of the four stereo centers of the cyclic system, the ROESY spectrum was used. Two stereo centers in the side chain were not considered (due to the presence of free rotation). For all possible 16 (24) stereoisomers, the distances between the atoms were calculated and compared with the corresponding distances determined from the peak volumes of the ROESY spectrum. The most probable stereoisomer configuration is shown in FIG. 9 and FIG. 10.

9, stereo centers with an undefined configuration are marked with an asterisk.

Figure 10 shows a 3D model of the structure with key correlations of NOE.

It should be noted that the described method is suitable only for determining the relative stereo configuration and the connection may have a mirror stereo configuration. However, this configuration is preferred since in common parts coincides with the configuration of known natural compounds.

Chemical shifts ¹ H shown in Fig.11.

Chemical shifts ¹³ C are shown in FIG.

The task of biomedical research of the drug 4-hydroxy-17R-methylincisterol was to obtain data guaranteeing the safety of its use for medicinal purposes.

Studied:

- the toxic effect of the drug on mice of both sexes of the BAL B / C line, breeding at the Stolbovaya RAMS nursery, in an acute experiment;

- the effect of the drug at the cellular level in vitro when using a relatively simple system of cultured cells - normal (human fibroblasts) and transformed (Hela);

- antitumor activity of the drug on a model of a solid tumor of melanoma B-16 inoculated to inbred mice BDF ₁ - first-generation hybrids f1 (C ₅₇ B 1/6 × DBA ₂ ) breeding RAMS "Pillar";

- lipid-lowering activity of the drug on the model of an atherogenic cholesterol diet with white non-linear laboratory sexually mature male rats obtained from the nursery of FSUE OPH Manikhino.

Studies of the toxic effects of 4-hydroxy-17R-methylincisterol were performed using two doses: 100 mg / kg, 200 mg / kg.

In the experiment, two groups of mice were involved, 10 animals in each group.

Studies were performed on mice weighing ~ 40 g with a single intragastric route of administration of the drug. Before using the drug, each dose was dissolved in 0.5 ml of distilled water. The observation period was 14 days.

The criteria for the toxic effect of the studied doses of the drug were: the number of dead animals, the time of death of the animals, the clinical picture of intoxication, changes in body weight during the entire observation period, animal behavior, autopsy data.

The results of the toxic effects of the studied doses of the drug are given in tables 3, 4.

As a result of this series of experiments, the following was established.

With a single intragastric administration of the drug in test doses of 100 mg / kg, 200 mg / kg in both groups of mice, external clinical manifestations were not observed.

Appearance. Within 14 days of observation of animals, the coat is smooth. In appearance, the animals did not differ between the groups. Animals showed no deterioration in appetite. The chair is decorated in the usual color.

Behavior. During the observation of animals, suppression of the general condition of the animals was not observed.

Mortality. Mortality resulting from drug administration was not detected throughout the observation period. In the administration group of the drug at a dose of 100 mg / kg on day 13, one animal fell due to aggression of other individuals (see table 3).

The mass of animals. During the entire observation period, two control weighings were made. A sharp loss or increase in mass was not detected (see table 4).

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

Given the toxicity data, further study of the biomedical properties of 4-hydroxy-17R-methylincisterol was carried out in two once-daily doses: 100 mg / kg, 200 mg / kg of animal weight.

When studying the effect of the drug at the cellular level in vitro, cultured cells (normal and transformed) were used as a model for testing. The objective of these studies was to study the individual action of the drug at the cellular level using a relatively simple system of cultured cells - normal (human fibroblasts) and transformed (Hela).

A quantitative assessment of the antitumor efficacy of the 4-hydroxy-17R-methylincisterol preparation at the in vitro level was carried out in comparison with the antitumor effect of the Pleurotus ostreatus 1137 mycelium extract (VKPM, F-819) and the cyclophosphamide cytostatic.

To quantify the effectiveness of these drugs, we used indicators characterizing the viability of the population of cultured cells, including mitotic and apoptotic indices. The mitotic index reflects the ability of the cell population to reproduce, and the apoptotic index shows the proportion of cells that activate programmed death. It is known that in malignant tumors apoptosis is suppressed, which leads to intensive multiplication of cancer cells and the formation of metastases.

The results showed that with individual action, Pleurotus ostreatus 1137 mycelium extract (VKPM, F-819) at doses of 100 mg / kg, 200 mg / kg is a weakly expressed apoptosis inducer and causes abnormal chromosome segregation at the metaphase stage and the appearance of chromosome aberrations of the type " bridge ”at the anaphase stage.

With the combined use of the mycelium extract Pleurotus ostreatus 1137 (VKPM, F-819) and cyclophosphamide, a pronounced synergism is manifested, which, depending on the concentration of the reagents (extract 100 mg / kg, cyclophosphamide 50 mg / kg and 100 mg / kg) and their method installations, is expressed in partial suppression of proliferation and in the induction of apoptosis.

It was found that the combined use of these drugs determines the ability of cancer cells to include programs for their apoptotic death. Moreover, the anticancer effect of the extract of the mycelium Pleurotus ostreatus 1137 (VKPM, F-819) is not directly related to its cytotoxic effect, but rather leads to the activation of some enzyme systems, including superoxide dismutase. However, a similar effect does not occur in normal human fibroblasts.

According to the results of a study of the drug 4-hydroxy-17R-methylincisterol, it was found that its use in doses of 100 mg / kg, 200 mg / kg to equivalent doses of the drug, 100 mg / kg, 200 mg / kg, in cultured transformed Hela cells after 24 hours causes pronounced apoptotic effect.

The claimed preparation in applied doses is a strong inducer of apoptosis in cultured transformed cells, increasing the number of apoptotic cells by 20-30 times, compared with the control. However, it does not have an apoptotic and mitostatic effect on normal fibroblasts.

Based on the results of cytological studies of cultured cells, it was found that the main mechanism of the anti-cancer effect of 4-hydroxy-17R-methylincisterol is the stimulation of apoptosis in cancer cells, therefore, the developed drug in its pure form is an inducer of apoptosis of cancer cells.

When studying the antitumor activity of the drug 4-hydroxy-17R-methylincisterol in animals on a model of a solid tumor of melanoma B-16, 4 groups of mice took part in the experiment (8 mice in each group):

Group 1 - intact control with a B-16 melanoma tumor transplanted into mice;

Group 2 — a B-16 melanoma tumor transplanted into mice with the introduction of 4-hydroxy-17R-methylincisterol at a dose of 100 mg / kg, orally, ten times, daily, in the first 10 days. On the first day, the drug was administered 1 hour after transplantation into tumor mice and then on the next 9 days daily;

3rd group - a tumor of B-16 melanoma transplanted into mice with the introduction of cyclophosphamide (CF) at a dose of 50 mg / kg, intraperitoneally, once, on the first day;

4th group - a B-16 melanoma tumor transplanted into mice with the introduction of combined use of CF at a dose of 50 mg / kg, intraperitoneally, once, on the first day and 4-hydroxy-17R-methylincisterol at a dose of 100 mg / kg, orally, ten times daily for 1 ... 10 days.

Every day in all 4 groups, animals were examined to assess the presence and severity of the tumor (visually and by palpation), as well as signs of possible intoxication. Fixed: the average mass of the tumor (g); tumor growth inhibition (SRW) (in%); the average life expectancy of animals (day); an increase in the average life expectancy of animals of the experimental groups relative to the life expectancy of animals in the control group (in%).

The research results of this series of experiments are shown in Fig.13 and in table.5.

In particular, FIG. 13 shows the results of the antitumor activity of 4-hydroxy-17R-methylincisterol and its combined use with CF in the B-16 melanoma model, where:

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

2 - experimental group of mice with a transplanted tumor of melanoma B-16 with the introduction of the drug 4-hydroxy-17R-methylincisterol at a dose of 100 mg / kg, orally, ten times, daily, in the first 10 days;

3 - experimental group of mice with a transplanted tumor of melanoma B-16 with the introduction of CF at a dose of 50 mg / kg, intraperitoneally, once, on the first day;

4 - experimental group of mice with a transplanted tumor of melanoma B-16 with the introduction of the combined use of CF at a dose of 50 mg / kg, intraperitoneally, once, on the first day and the drug 4-hydroxy-17R-methylincisterol at a dose of 100 mg / kg, oral, tenfold daily for 1 ... 10 days.

When analyzing the data on the dynamics of changes in the controlled parameters in the control and experimental groups of mice (see Fig. 13 and Table 5), it was noted that both with the use of the claimed preparation 4-hydroxy-17R-methylincisterol and with combined use with CF throughout time of observation, its antitumor effect is manifested.

For example, the use of the drug 4-hydroxy-17R-methylincisterol by the 2nd experimental group of mice increases their average life expectancy by 25% compared to the 1st group of mice of intact control, the use of CF by 10%, and their combined use by 100 %

The average life expectancy in the 1st group of intact control mice was 20.0 ± 0.6 days, in the 2nd group of mice 25.0 ± 0.9 days, in the 3rd group of mice 21.0 ± 0, 5 days, in the 4th group of mice 40.0 ± 0.5 days.

The remaining observed indicators with evidence of the effectiveness of the drug 4-hydroxy-17R-methylincisterol are given in table.5.

Summarizing the results of studying the efficacy of 4-hydroxy-17R-methylincisterol, it was found that in addition to the manifestation of the antitumor effect, the use of the drug, either alone or in combination with CF, significantly improves the clinical picture of the “quality” of animal life (activity, appearance, condition of the coat, behavioral reactions, body weight, lack of diarrhea) compared to animals that received only CF, and also compared to control tumor mice.

In the study of the hypolipidemic activity of the drug in animals on the model of an atherogenic diet (BP), 4 groups of outbred laboratory sexually mature male rats with an initial body weight of 300-350 g (8 rats in each group) took part in the experiment:

1st group - intact control;

2nd group - atherogenic diet (HELL);

3rd group - blood pressure using the well-known reference drug Zokor (simvastin) from Merck Sharp and Dome B.V., The Netherlands, NJ41170 series, with a shelf life of 08.2010 (used in March 2009) in a dose of 8 mg / kg, orally, ten times daily, after the first 4 days of blood pressure;

4th group - HELL with the claimed drug 4-hydroxy-17R-methylincisterol at a dose of 2 mg / kg, orally, tenfold, daily, after the first 4 days of using HELL.

All test substances were administered to rats orally with an interval of 3 hours after atherogenic loading during the last 10 days of blood pressure.

Throughout the experiment, an observation sheet was kept: survival, general view, condition and behavior of animals, change in body weight (rats were weighed once a week).

At the end of the experiment, animals after 18 hours of fasting took blood from the tail vein into centrifuge tubes. Blood was centrifuged and serum was taken for biochemical analyzes: total cholesterol content (cholesterol total); high density lipoprotein cholesterol (HDL cholesterol); low density lipoprotein cholesterol (LDL cholesterol); very low density lipoprotein cholesterol (VLDL cholesterol); triglycerides (TG); atherogenic index (K ather.).

The analyzes were carried out using standardized methods using a US Fax Stat-Fax 1904+ biochemical photometer using standard reagent kits.

The research results of this series of experiments are shown in Fig.14, 15 and table.6, 7.

In particular, on Fig shows the results of biochemical indicators of lipid metabolism of intact animals and animals kept on blood pressure.

On Fig shows the results of biochemical parameters of lipid metabolism in experimental groups of rats, including 4 groups using blood pressure with the claimed drug 4-hydroxy-17R-methylincisterol.

When analyzing the data of the dynamics of changes in the controlled parameters of the experimental groups of rats (Fig.14, 15 and table.6, 7), it was noted that:

- in the 2nd group of animals that are only on an atherogenic diet (Fig. 14, Table 6), significant differences in biochemical blood tests are observed with respect to intact animals (1st group);

- after 14 days of atherogenic load in rats of the 2nd group in comparison with the 1st intact group, an increase in the content of cholesterol is 1.5 times (2.9 mmol / l versus 2 mmol / l), LDL cholesterol - 2 3 times (1.8 mmol / L versus 0.8 mmol / L), HDL cholesterol by 25%. The content of TG is 1.2 times higher. Due to the increase in cholesterol and its reduction in antiatherogenic fractions of lipoproteins (HDL cholesterol), the atherogenic index (K ater.) Increased by more than 2 times (1.8 cu against 3.7 cu) in the group of animals, receiving atherogenic load.

Comparing the readings of lipid metabolism of the 3rd and 4th experimental groups of animals (see Fig. 15, Table 6), we can conclude that the introduction of the drug "Zokor" and the claimed drug 4-hydroxy-17R-methylincisterol cause a decrease the content of cholesterol, although these substances do not equally affect its decrease. For the drug "Zokor" there is a slight decrease in this indicator compared with the group of blood pressure. The claimed drug in the 4th group had the greatest impact on the decrease in cholesterol in animals - its level was almost the same as in the 1st group of intact rats - 1.9 mmol / L.

The content of antiatherogenic fraction of cholesterol (HDL cholesterol) was significantly higher in the 3rd and 4th groups receiving Zokor and the claimed drug. Moreover, the average indicator of this fraction turned out to be the same for the groups receiving drugs, and intact animals - 0.7 mmol / L.

In these groups, the content of TG in serum was lower than in the group of intact animals.

In the blood of rats, when Zokor was added to blood pressure, there was no such decrease in cholesterol in the atherogenic fraction of lipoproteins (LDL cholesterol - 1.8 mmol / L) as in the 4th group receiving the claimed preparation 4-hydroxy-17R-methylincisterol, which was significant for the group of intact animals (0.8 mmol / l). In animals treated with the claimed drug, a significantly low content of LDL cholesterol (1.1 mmol / L) is observed in relation to animals with blood pressure. This was reflected in the atherogenic index: it turned out to be quite low - 1.9 cu for the claimed drug, which exceeds only 0.1 cu atherogenic index of the 1st group of intact animals.

According to the results of pathomorphological studies of the state of the internal organs of experimental groups of animals, it was found that the studied organs (chest and abdominal cavities of rats, lungs, thyroid gland, thymus, heart, esophageal mucosa, stomach, large and small intestines, pancreas, spleen, kidneys, bladder , liver) in experimental animals of the 3rd and 4th groups do not differ from the 1st group of intact control of rats.

The average body weight of animals is shown in table.7.

Thus, the claimed drug 4-hydroxy-17R-methylincisterol restores the biochemical parameters of the lipid spectrum of the blood and has a lipid-regulating effect in experimental hyperlipidemia caused by an atherogenic diet.

The inventive preparation 4-hydroxy-17R-methylincisterol can be obtained biotechnologically using a culture of the fungus Pleurotus ostreatus 1137.

The specified culture is grown in a deep way in a sterile liquid nutrient medium.

For the production of 4-hydroxy-17R-methylincisterol, use:

- microbiological agar according to GOST 17206-84;

- food beer wort GOST 51-174-98;

- oyster mushroom mycelium (strain 1137, BKPM-F819) grown under submerged conditions;

- ethyl rectified alcohol according to GOST R51652-2000;

- hydrochloric acid according to GOST 3118-77;

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

- crystalline fructose, CF4118136AG, Pharmacopoeia Germany DAB;

- food citric acid, GOST 908-79, premium;

- sodium benzoate, 3382SE023494C4, Pharmacopoeia Germany DAB;

- dichloromethane KHCH, TU 2631-019-44493179-98, Russia.

The technological process for the preparation of the drug in an industrial way using the Pleurotus ostreatus 1137 fungus culture for its preparation is illustrated by the following example.

As indicated above in this description, the manufacturing process of the drug includes 4 stages and consists of the following operations.

At the first stage of growing mycelium biomass, selection work with the strain (maintenance of the strain) is carried out by reseeding on fresh agar medium. To carry out breeding work, the spore is sieved, monospore variants are selected and their properties evaluated, which helps to maintain the target culture characteristics.

The seed is grown in test tubes with a mowed agar medium, which contains beer wort, agar and water. After seeding, the tubes are placed for 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 beer wort (10%) and drinking water. Sowing mycelium from mowed agar is introduced into the medium at the rate of 1 tube of seed per 1 liter of medium. The duration of cultivation is 12-14 days with constant aeration. Cultivation is carried out in a thermostatically controlled room equipped with control sensors with recorders at an air temperature of t = 28 ° C.

The separation of the grown mycelium biomass from the culture fluid is carried out by filtration through, for example, cheesecloth until the fluid has completely drained from the biomass. The resulting wet mycelium is subjected to extraction.

At the second stage of preparation of the mycelium extract, the wet mycelial mass separated from the culture fluid is mechanically crushed to a minimum fine fraction and poured with ethyl alcohol with added hydrochloric acid at a concentration of 0.1%. Extraction is carried out for 20 hours at a temperature of 4 ° C in a refrigerator.

After the extraction period, the mycelium is separated by filtration through cheesecloth, and the resulting alcoholic extract is evaporated in a vacuum unit to a state of a viscous homogeneous concentrate in the form of a condensed extract (gel) of constant weight with a water content of up to 30%. For compliance control, a sample of the obtained extract is taken and the content of carbohydrates, amino acids, and water is determined (in accordance with clause 4.6., GF XI, issue 1, p. 177).

At the third stage, from the obtained condensed oyster mushroom mycelium extract in the form of a gel, the 4-hydroxy-17R-methylincisterol sterol derivative is isolated using dichloromethane.

An example of the isolation of a sterol derivative from an extract is carried out as described above.

In the fourth stage, the preparation of the dosage form of the 4-hydroxy-17R-methylincisterol preparation is carried out in the form of dehydrated granules or tablets, or in the form of an aqueous-alcoholic solution, or in the form of a syrup.

After quality analysis, the drug is packaged and packaged.

Thus, the conducted complex of studies of the claimed drug confirmed its high physiological activity with effective multifunctional biomedical activity.

The physiological effect of the drug lies in its ability to influence tissue metabolism without the occurrence of adverse side effects, including toxicity on the human body and modulate the manifestation of antitumor and lipid-lowering activities.

The use of the claimed preparation 4-hydroxy-17R-methylincisterol with such a physical manifestation of properties will expand the arsenal of the drug isolated from the biologically active substances of the mycelium extract Pleurotus ostreatus 1137 and reduce toxicity when used as a drug with antitumor and lipid-lowering activities.

Table 4 The body weight of mice Group No. Groups of animals Body weight g Before the experiment After the experiment one 100 mg / kg 40.1 ± 0.3 38.2 ± 3.8 2 200 mg / kg 40.9 ± 0.9 39.2 ± 2.1 R p> 0.05 p> 0.05

Table 5 The results of the antitumor activity of the drug 4-hydroxy-17R-methylincisterol with its individual and combined use with cyclophosphamide Group No. Nature of groups Daily Dose (mg / kg) The average tumor volume (cm ³ ) SRW (%) The average life expectancy of animals (day) The increase in the average life expectancy of animals (%) one Intact control - 5.8 ± 0.5 ** - 20.0 ± 0.6 - 2 The inventive drug one hundred 4.5 ± 0.55 ** twenty* 25.0 ± 0.9 25 3 CF fifty 2.0 ± 0.25 ** fifty* 21.0 ± 0.5 10 four The inventive drug + CF 100 + 50 1.4 ± 0.15 ** 70 * 40.0 ± 0.5 one hundred * assessment of SRW on the 18th day after tumor inoculation; ** assessment of the average tumor volume on the 20th day after tumor inoculation.

Table 7 Average weight of animals Group No. Nature of groups Daily dose The mass of animals (g) (M ± m) Start of experiment The beginning of drug administration End of experiment one Intact control - 338.9 ± 7.64 339.7 ± 9.57 328.3 ± 8.41 2 HELL 5 mg / kg 334.9 ± 7.53 335.7 ± 7.51 337.3 ± 10.68 3 HELL + drug comparison Zokor 8 mg / kg 338.4 ± 7.35 348.1 ± 8.87 340.7 ± 15.60 four HELL + the claimed drug 2 mcg / g 330.0 ± 6.90 337.9 ± 6.79 337.7 ± 8.12

Claims (3)

1. A drug that affects tissue metabolism, including deep cultivation of the fungus Pleurotus 1137 (VKPM, F-819), followed by separation of the mycelium from the culture fluid and isolation from the mycelium by extraction with ethanol at an acidic pH of low molecular weight from 100 to 500 daltons of biologically active substances with subsequent isolation of dichloromethane from the sterol derivative 4-hydroxy-17R-methylincisterol with an established molecular weight of 332.2452, the gross formula C ₂₁ H ₃₂ O ₃ and the structural formula

and having the ability to inhibit the proliferation of cancer cells and regulate the lipid metabolism of the body. 2. The drug according to claim 1, made in the form of dehydrated granules, or tablets, or in the form of a water-alcohol solution, or in the form of a syrup. 3. The use of the strain Pleurotus ostreatus 1137 (VKPM, F-819) as a producer of the biologically active substance of the sterol derivative 4-hydroxy-17R-methylincisterol.

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