RU2425687C2 - AGENT FOR DISEASES CAUSED BY MYCOBACTERIA OF Ungernia victoris UV-22 CALLUS CULTURE STRAIN - PRODUCER OF BIOLOGICALLY ACTIVE COMPLEX FOR TREATING DISEASES CAUSED BY MYCOBACTERIA AND CULTIVATION TECHNIQUE (VERSIONS) - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to an agent for prevention and treatment of the diseases caused by mycobacteria. A Ungemia victoris UV-22 callus culture strain deposited in the Cell culture collection of Institute of Molecular Biology and Genetics of National Academy of Sciences of Ukraine No. 13/2008 - a producer of a complex of biologically active substances for treating the diseases caused by mycobacteria. A aqueous-alcoholic extract of Ungernia victoris callus tissues of UV-22 strain deposited in the Cell culture collection of Institute of Molecular Biology and Genetics of National Academy of Sciences of Ukraine No. 13/2008 as an agent for treating the diseases caused by mycobacteria. A cultivation technique for Ungernia victoris callus tissues of UV-22 strain by microclonal propagation (versions).

EFFECT: agents are effective for treating the diseases caused by mycobacteria.

4 cl, 7 tbl, 10 ex

Description

The invention relates to medicine, more specifically to means for the treatment of diseases caused by mycobacteria, and the technology for their preparation, in particular a new producer strain and technology for growing plant cells using microclonal propagation.

The invention relates to the field of medicine and biotechnology, in particular to new medicines for the treatment of diseases caused by mycobacteria (MB), such as leprosy (leprosy) and tuberculosis, as well as to methods for their preparation, in particular, using cell tissue cultivation technology.

Diseases caused by mycobacteria, in particular tuberculosis and leprosy, are among the most difficult to treat. Studies by the World Health Organization (WHO) have shown that the problem of tuberculosis (TB) is extremely acute, as evidenced by the level of infection of the world's population of more than 1 billion people, of which 44% suffer from the most dangerous pulmonary form of the disease for others . About 8 million people fall ill with tuberculosis and about 2 million people die from this disease every year. The causative agent of the disease is Mycobacterium tuberculosis, mainly human, rarely bovine and, in exceptional cases, avian type. (Dye C, Scheele S, Dolin P, Pathania V and Ravinglione M C, 1999, JAMA, 282, 7, 677-686).

Leprosy (leprosy) is caused by Mycobacterium leprae and is characterized by damage to the skin, nervous system, bones and eyes and leads to disability, blindness and death. (humbio.rn / humbio / infect_har / 0032c27b.htm) It is less common than tuberculosis, but in the modern world, mainly in developing countries, there are about 2 million patients. The current clinical and epidemiological situation of leprosy in Russia is distinguished by a number of features. The incidence is sporadic, new cases are practically not recorded, the existing contingent of patients is mainly composed of elderly and elderly people suffering from concomitant somatic pathology. About 18 percent of patients have leprosy complications (neurotrophic foot ulcers, amyotrophy, finger mutations), liver damage, etc.

The main focus in the treatment of various forms of tuberculosis is chemotherapy with drugs that affect MB, inhibiting their reproduction and reducing their virulence. These include, in particular, isonicotinic acid derivatives, drugs such as isonicotinic acid hydrazide (isoniazid) and its derivatives of paraaminosalicylic acid (PASK), antibiotics, as well as kanamycin, ethionamide, protionamidthioacetosone, florimycin (Mashkovsky M.D. Medicines Part 2. - M .: Medicine, 1985, p. 314-327). As a rule, 2-3 anti-TB drugs are used for a long period (9-18 months or more), taking into account their tolerance to patients and the stability of MB. The generally accepted traditional antibacterial therapy involves the use, as a rule, of tubazide (isoniazid), rifampicin and streptomycin (Mashkovsky M.D. Medicines. - M .: Medicine, 1993, v. 2, p. 366-367; Ursov I.G. , Borovinsky A.I., Fedorova T.A., Fomintseva O.A., Zyryanova T.V. The problem of tuberculosis, 1993, N 2. - M .: Medicine, p. 34-36). It was proposed to use methyluracil in the treatment of tuberculosis in combination with generally accepted complex therapy (antibacterial, detoxification) and vitamins. (Kozlenko L.S. Problems of tuberculosis, 1993, N 6, p. 52-53). The most effective drug for treating tuberculosis is rifampicin, which is administered orally in the form of capsules in a daily dose of 150 mg. To reduce the rate of excretion of rifampicin from the body with urine, the drug is administered while taking pyrazinamide (G.B.Sokolova et al. Antibiotics and chemotherapy, 2000, 45, No. 9, p.30-37).

The disadvantages of the chemotherapy used are the relatively low effectiveness and a large number of contraindications (liver, kidney, anemia, etc.), as well as the presence of negative side effects, such as weakness, headaches, lesions, which negatively affect the state of other body systems.

Treatment of leprosy is also carried out as part of complex therapy by using 2-3 anti-leprosy drugs (diaphenylsulfone, solusulfone, diuciphon) in combination with restorative drugs for 9-18 months. (Handbook of a practical doctor. - M., Medicine, 1982, p. 464; Mashkovsky M.D. Medicines, part 2, M .: Medicine, 1985, p. 329-331).

The disadvantages of the drugs used are relatively low efficiency, a large number of contraindications (liver, kidney, anemia, epilepsy, etc.), as well as the presence of negative side effects, such as weakness, headaches, liver damage (Handbook of a medical practitioner. - M .: Medicine, 1982, p. 120-122).

In folk medicine, a significant amount of drugs is recommended for the treatment of tuberculosis and leprosy. In particular, compositions and collections based on marshmallow, stonecrop, goldenrod, initial letters, perennial daisies, pine buds, etc. are proposed (A. Aksenov. Encyclopedia of the healer. ACT, Donetsk, 1982, 482-484), and it is also advisable to use for these targets ginger, lemon with honey, aloe with honey and butter and other means (Complete Encyclopedia of Traditional Medicine, Vol. 3, Moscow, ANS, 1999, 440, 441, 454, 520). For the treatment of leprosy, the use of henna leaves is recommended (Complete Encyclopedia of Traditional Medicine, Vol. 3, Moscow, ANS, 1999, p. 532). However, this use of natural raw materials, as a rule, is not effective enough and has a significant number of contraindications.

The technical challenge faced by the authors was the creation of an effective and safer drug for the treatment of diseases caused by mycobacteria, and the technology for its production.

The technical result was achieved by using for this purpose an aqueous-alcoholic extract of the callus tissue strain of the plant Ungernia victoris UV-22.

An extract containing 0.01-0.05% dry matter, which is a set of biologically active substances (BAS), is used to prevent and treat diseases, usually in a dose of 0.1-10 drops of extract per 20 kg of body weight as an additive in water or juice after eating 3 times a day. The specific dose and regimen are established on the basis of the characteristics, stage and nature of the course of the disease.

Ungernia Victor Ungernia victoris (HC) is an endemic bulbous plant of the Pamir-Alai, which grows only on the Gissar Range and its southern spurs. Bulbs, roots and leaves of HC contain important for medicine galantamine, lycorin and other isoquinoline alkaloids, and a complex of biologically active substances with a wide spectrum of positive effects on the body has been found in callus tissue. In particular, it has a radioprotective, antimutagenic, hepatoprotective, anticarcinogenic effect, increases physical and mental performance (RU 0010088, 10.21.1998; RU 2147439, 10.28.98; RU 2215762, 28.12.01; RU 0011467, 18.06.99; RU 2221584 , 11.04.02; RU 2186577, 03.03.01; RU 2178979, 05.25.00). However, the therapeutic effect on diseases associated with mycobacteria has not been studied previously, and preliminary studies using this extract, produced under the name "Vlairin" (see examples), showed that this effect is not high enough.

The use of Ungernia tubers for the production of biologically active substances has its own limitations, since the natural raw material base of this plant is limited, and the introduction of Ungernia was unsuccessful. In this regard, the authors considered an alternative to natural reproduction biotechnological methods, in particular, a method of microclonal propagation in an in vitro culture of native cell tissue. The advantages of this method of reproduction in comparison with traditional methods are higher reproduction rates, miniaturization of the process, improvement of planting material from different types of infections.

Currently, for many plant species, various methods of microclonal propagation of plant cells are known, including callus tissue cells (Kalinin F.L., Kushnir G.P., Sarnatskaya V.V. Technology of microclonal propagation of plants. - K .: Science. Dumka, 1992. - 230 p .; RU 2052925; RU 2130709; RU 2171841; RU 2141525). The advantages of this type of reproduction in comparison with traditional methods are higher reproduction rates, miniaturization of the process, improvement of planting material from different types of infections.

The main stages in microclonal propagation of plants is to obtain regenerants from explants that are isolated from various organs and tissues of plants, and the propagation of regenerants by stimulation.

The cultivation of explants prior to the formation of regenerants and the rooting of micrograins is carried out, as a rule, on agarized nutrient media. Murashige-Skoog medium (Murashige T., Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Phisiol. Plant. 1962. v.15. N13. P. 473-497), which contains mg / 100 g:

- macro salts: NH ₄ NO ₃ 1650, KNO ₃ 1900, MgSO ₄ × 7H ₂ O 1900, CaCl ₂ × 2H ₂ O 440, KH ₂ PO ₄ 170;

- micro salts: MgSO ₄ × 4H ₂ O 22.3, H ₃ BO ₃ 6.2, ZnSO ₄ × 4H ₂ O 8.6, CoCl ₂ × 6H ₂ O 0.025, CuSO ₄ × 5H ₂ O 0.025, NaMoO ₄ × 2H ₂ O 0.025; KI 0.83;

- vitamins: thiamine 0.1, myoinositol 80, pyrodoxin 0.5, nicotinic acid 0.5;

- carbon source - sucrose - 20,000;

- agar - 7000;

- additives to increase the rate of formation of regenerants - gibberelic acid 2.0, kinetin - 1.0;

- water - the rest.

At the second stage, as a rule, β-indolylacetic acid - 0.05 mg / L or b-indolylbutyric acid - 0.1 mg / L, is usually added to this medium.

However, biologically active substances (BAS) used in this process are capable of causing genetic changes in cells that accumulate during cultivation, which leads to an increase in the number of regenerants that differ from the original type (Leonova N.S. Siberian Journal of Agricultural Sciences, 1986, No. 3, p. 18-23). In addition, almost all of these technologies are species-specific, i.e. suitable for a certain group of plants and without modification cannot be used for culturing cells of other plants. In particular, for the cultivation of calus tissue Silene vulgaris (RU 2169769), an agar medium containing macro and micro salts according to Murashige-Skoog, growth hormones, including 6-BAP and 2.4 D, folic acid, riboflavin, biotin, S-pantothenate is used and cobalamin; for the cultivation of calum tissue Lemna minor (RU 2171840) use an agar medium containing macro and micro salts according to Murasige-Skoog, vitamins, growth hormones, including 6-BAP and 2.4 D, folic acid, riboflavin, biotin, S-pantothenate and nicatinamide.

However, the cultivation technologies and plant cultivation methods proposed for cultivation of these plant strains during the cultivation of Ungernia Victor cells do not allow achieving the set objectives without modifying them, because the use of such standard nutrient media as Murashige-Skoog, Linsmeier-Skoog, Hamburg, B 5, Erickson, and White for their cultivation was ineffective - induction of regeneration of microtubes was observed in 2-3% of explants.

Currently known is used to obtain biologically active substances (BAS), a strain of cultured leaf cells of Ungernia victoris, a producer of alkaloids (galantamine and lycorin) (RU 1806188, 1993). For the cultivation of cells, a modified Murashige-Skoog medium is used, in which alpha-NAA and tyrosine are added as additives, and hydrolyzed casein is used as an additional source of carbon and nitrogen. However, this strain is used only for the production of alkaloids of the galantamine and lycorin group, which does not allow the use of biologically active substances produced by it for the prevention and treatment of diseases caused by mycobacteria.

The closest to the claimed new strain in technical essence and the achieved effect is the previously created by the authors on the basis of callus tissue of the plant - a strain of cultured Ungernia Victor cells - Ungernia victoris UV-2, which is a producer of biologically active substances that are promising for use as a radioprotective and antimutagenic drug (UA No. 39572, 2001). The aqueous-alcoholic extract of the biologically active substance of this strain, which served as the basis for the biologically active additive (BAA) “Vlairin”, as shown by the studies, also has a hepatoprotective, anticarcinogenic effect, increases physical and mental performance (RU 0010088, 1998; RU 2147439, 198; RU 2215762 , 2001; RU 0011467, 1999; RU 2221584, 2002; RU 2186577, 2001; RU No. 2178979, 2000).

The strain is cultivated in the form of callus in a one-step way in a nutrient medium having the following composition, mg / l:

macrosalts: NH ₄ NO ₃ - 400-600; KNO ₃ - 1000-1200; (NH ₄ ) ₂ SO ₄ - 200-400; MgSO ₄ × 7H ₂ O - 400-600; Ca (NO ₃ ) ₂ × 4H ₂ O - 800-1000; NH ₄ H ₂ PO ₄ - 500-700; (NH ₄ ) ₂ HPO ₄ 90-110; KCI - 60-80;

microsols: MnSO ₄ × 5H ₂ O - 23-25; KI - 0.8-0.9; Na ₂ MoO ₄ × 2H ₂ O - 0.2-0.3; CuSO ₄ × 5H ₂ O - 0.02-0.03; CoCl ₂ × 6H ₂ O — 0.02-0.03; H ₃ BO ₃ - 5-8; ZnSO ₄ × 4H ₂ O - 7-9; FeSO ₄ × 7H ₂ O - 27-28; MnSO ₄ × 5H ₂ O - 23-25;

sodium salt of ethylenediaminetetraacetic acid (Na ₂ EDTA) - 37-38;

vitamins: thiamine 0.8-1.2;

sucrose 40000-60000;

agar 6000-8000;

The pH before autoclaving was 5.8-6.2.

The yield of crude biomass from 1 l on the 65th day of growth is 400-550 g. In terms of dry biomass, the yield is 20-35 g / l.

The disadvantage of this strain is the length of the growing time (more than 60 days). The disadvantage of its complex of biologically active substances is, as shown by experiments, insufficient effectiveness in the treatment of diseases such as tuberculosis and leprosy.

The basis of the claimed group of inventions was the creation of a new strain of callus culture UNGERNIA VICTORIS UV-22, deposited in the Collection of cell cultures at the Institute of Molecular Biology and Genetics of NAS of Ukraine. The collection number of the strain 13/2008.

Strain genealogy

The raw material for obtaining callus tissue was an ungernia bulb weighing 135 g. Before being introduced into the in vitro culture, the bulb was kept for 18 months at rest at a temperature of 6-10 ° C. The basal parts of the inner bulbous scales were used as explants.

Standard growing conditions

The strain is cultivated in the form of callus (on an agarized nutrient medium) or in the form of a suspension (in a liquid medium) at a temperature of 24-26 ° C, relative humidity of 70-80% in the dark.

Suspension culture is grown with constant stirring on a rocking chair at 80-100 rpm, for callus culture rocking is not used. The cultivation of the strain Ungernia UV-12 is carried out by the method in a nutrient medium composition, mg / l:

NH ₄ NO ₃ 400-600 Kno ₃ 1000-1200 (NH ₄ ) ₂ SO ₄ 200-400 MgSO ₄ x7H ₂ O 400-600 Ca (NO ₃ ) ₂ × 4H ₂ O 800-1000 NH ₄ H ₂ PO ₄ 500-700 (NH ₄ ) ₂ HPO ₄ 90-110 KCI 60-80 Kj 0.8-0.9 Na ₂ MoO ₄ × 2H ₂ O 0.2-0.3 CuSO ₄ × 5H ₂ O 0.02-0.03 CoCl ₂ × 6H ₂ O 0.02-0.03 MnSO ₄ × 5H ₂ O 23-25 H ₃ IN ₃ 5-8 ZnSO ₄ × 4H ₂ O 7-9 FeSO ₄ × 7H ₂ O 27-28 Na ₂ EDTA 37-38 Thiamine 0.8-1.2 Pyridoxine 0.5-1.0 A nicotinic acid 0.5-1.0 Glycine 1.5-2.5 1-naphthylacetic acid 1.8-2.2 Kinetin 0.8-1.2 Casein Hydrolyzate 450-500 Mesoinosine 70-100 Sucrose 50000-60000 Agar 6000-8000 pH before autoclaving 5.8-6.2.

For suspension culture using a similar medium, characterized in that agar is excluded from it.

To grow the culture using glass vessels (banks, flasks) with a volume of 250-300 ml, containing 50-60 ml of medium. Callus culture can also be grown in test tubes and Petri dishes of different sizes.

Multiplicity of sieving: 8-12.

Cultural features

The biomass of cultured cells of the strain Ungernia UV-12 consists of cells and cell aggregates of different densities. The size of the cell aggregates of the suspension culture is about 0.5 cm in diameter. When grown on a solid (agarized) nutrient medium, callus culture is more heterogeneous, it consists of aggregates from 0.1 to 0.8 cm in diameter more dense than suspension aggregates. The color of the culture is from light gray to light yellow.

The duration of the passage and the regime of reseeding suspension culture is 45-55 days, callus culture - 55-65 days.

Callus culture does not lose viability and does not noticeably change the main parameters when grown under standard conditions without transplanting for 90-95 days. The mass of the inoculum is 100-120 g of living biomass in terms of 1 liter of nutrient medium.

Growth signs

Type of growth unorganized. Growth index 3-5 for suspension culture and 4-7 for callus. The growth curve has a typical S-like character with reaching a plateau on the 45-50th day of growth for suspension and on the 55-60th day of growth for callus. Cell viability is 85-90% under standard culture conditions.

The yield of crude biomass from 1 l on the 65th day of growth is 400-550 g. In terms of dry biomass, the yield is 20-35 g / l.

The mitotic activity curve has, as a rule, one rise with a maximum on the 8-10th day of growth in suspension and on the 10-15th day of growth on an agar medium. The number of cells at different stages of mitosis (mitotic index) reaches 1.4–2.5% at the maximum of mitotic activity.

Karyological signs

Culture is a mixoploid population. The distribution of cells by the number of chromosomes has a range of variation from 16 to 92 chromosomes. The modal class is formed by cells with 22-30 chromosomes, which makes up 50% of the population. The frequency of chromosome aberrations in anaphase is about 8%.

Cytomorphological signs

Both suspension and callus culture are formed by cells of the parenchymal and meristematic type, mainly round. They make up small aggregates, from 4-8 to 25-35 cells. The size of the cells of the meristematic type ranges from 25-65 microns, the size of their nuclei is 15-25 microns. Parenchymal cells are 80-300 μm in size. Their nuclei are more heterogeneous in size and shape, the size of the nuclei is from 12 to 30 microns. More often, the nuclei of parenchymal cells have a size of 20-28 microns, sometimes (in 10-15% of cases) such cells contain several (usually 2-4) nuclei. In an aging culture (after 30-35 days of growth), the number of elements of cell differentiation increases. In particular, the number of tracheids can reach on the 50th day of growth and later 2.5% or more of all cells. Polymorphism of nuclei also increases: pyknotic, elongated, ribbon-like, flattened, dumbbell-shaped nuclei appear, large diffuse nuclei of irregular shape, nuclei with a large number (four to six) nucleoli, etc.

Marker signs

The color of the culture is from light gray to light yellow, the ability to unorganized growth type on both agar and in a liquid medium with a mineral base according to (UA No. 10338, 12/25/1996, IPC C 12 N 5/00, C 12 N 5 / 02) without growth stimulants. The increase in biomass on the 65th day of growth is 400-550 g of wet weight. In terms of dry biomass, this is 20-35 g per 1 liter of medium. Growth index 5-8.

Throughout the passage (50-60 days), the curve of mitotic activity is characterized by a single rise in mitotic activity with a peak on the 10-15th day of growth. The value of the mitotic index at the maximum is 1.4-2.5%.

Cells with 22-30 chromosomes form the modal class; the frequency of cells with this number is about 50%. The level of chromosome aberrations in anaphase is about 6%, the prevailing number of which are anaphases with bridges without fragments.

The culture is formed mainly by small cells of the meristematic type with relatively large nuclei collected in aggregates. The size of such cells is 25-65 microns, their nuclei are 15-25 microns in size. With the aging of the culture, the number of differentiation elements increases in it, in particular, the number of tracheid elements can exceed 2.5%. Under standard growing conditions, the culture is not capable of organogenesis.

The main variant of the method of cultivating Ungernia Victor cells is that the explants of fragments of bulb scales are placed on a nutrient medium of the following composition, mg / l:

NH ₄ NO ₃ 400-600 Kno ₃ 1000-1200 (NH ₄ ) ₂ SO ₄ 200-400 MgSO ₄ × 7H ₂ O 400-600 Ca (NO ₃ ) ₂ × 4H ₂ O 800-1000 NH ₄ H ₂ PO ₄ 500-700 (NH ₄ ) ₂ HPO ₄ 90-110 KCI 60-80 Kj 0.8-0.9 Na ₂ MoO ₄ × 2H ₂ O 0.2-0.3 CuSO ₄ x5H ₂ O 0.02-0.03 CoCl ₂ × 6H ₂ O 0.02-0.03 MnSO ₄ × 5H ₂ O 23-25 H ₃ IN ₃ 5-8 ZnSO ₄ × 4H ₂ O 7-9 FeSO ₄ × 7H ₂ O 27-28 Na ₂ EDTA 37-38 Thiamine 0.8-1.2 Pyridoxine 0.5-1.0 A nicotinic acid 0.5-1.0 Glycine 1.5-2.5 Acetic Acid Derivatives 0.5-2.2 Kinetin 0.8-1.2 Casein Hydrolyzate 50-500 Mesoinosine 20-100 Sucrose 50000-60000 Agar 6000-8000 pH before autoclaving 5.8-6.2,

where 1-naphthylacetic (IAA) or indolylacetic (IAA) acid is used as acetic acid derivatives, then the resulting onions are propagated on a medium of the following composition, mg / l:

NH ₄ NO ₃ 400-600 Kno ₃ 1000-1200 (NH ₄ ) ₂ SO ₄ 200-400 MgSO ₄ × 7H ₂ O 400-600 Ca (NO ₃ ) ₂ × 4H ₂ O 800-1000 NH ₄ H ₂ PO ₄ 500-700 (NH ₄ ) ₂ HPO ₄ 90-110 KCI 60-80 Kj 0.8-0.9 Na ₂ MoO ₄ × 2H ₂ O 0.2-0.3 CuSO ₄ × 5H ₂ O 0.02-0.03 CoCl ₂ × 6H ₂ O 0.02-0.03 MnSO ₄ × 5H ₂ O 23-25 H ₃ IN ₃ 5-8 ZnSO ₄ × 4H ₂ O 7-9 FeSO ₄ × 7H ₂ O 27-28 Na ₂ EDTA 37-38 Thiamine 0.8-1.2 Pyridoxine 0.5-1.0 A nicotinic acid 0.5-1.0 Glycine 1.5-2.5 Indolylacetic acid 1.8-2.2 Kinetin 0.01-0.03 Casein Hydrolyzate 40-60 Mesoinosine 15-30 Sucrose 50000-60000 Agar 6000-8000.

An additional option, used in case of need to achieve accelerated growth of microwells, is that the explants of fragments of bulb scales are placed on a nutrient medium of the following composition, mg / l:

NH ₄ NO ₃ 400-600 Kno ₃ 1000-1200 (NH ₄ ) ₂ SO ₄ 200-400 MgSO ₄ × 7H ₂ O 400-600 Ca (NO ₃ ) ₂ × 4H ₂ O 800-1000 NH ₄ H ₂ PO ₄ 500-700 (NH ₄ ) ₂ HPO ₄ 90-110 KCI 60-80 Kj 0.8-0.9 Na ₂ MoO ₄ × 2H ₂ O 0.2-0.3 CuSO ₄ × 5H ₂ O 0.02-0.03 CoCl ₂ × 6H ₂ O 0.02-0.03 MnSO ₄ × 5H ₂ O 23-25 H ₃ IN ₃ 5-8 ZnSO ₄ × 4H ₂ O 7-9 FeSO ₄ × 7H ₂ O 27-28 Na ₂ EDTA 37-38 Thiamine 0.8-1.2 Pyridoxine 0.5-1.0 A nicotinic acid 0.5-1.0 Glycine 1.5-2.5 Acetic Acid Derivatives 0.5-2.2 Kinetin 0.8-1.2 Casein Hydrolyzate 50-500 Mesoinosine 20-100 Sucrose 50000-60000 Agar 6000-8000 pH before autoclaving 5.8-6.2,

where, as acetic acid derivatives, 1-naphthylacetic (IAA) or indolylacetic (IAA) acid is used, and the obtained microwells are grown on a medium of the following composition, mg / l:

NH ₄ NO ₃ 400-600 Kno ₃ 1000-1200 (NH ₄ ) ₂ SO ₄ 200-400 MgSO ₄ × 7H ₂ O 400-600 Ca (NO ₃ ) ₂ × 4H ₂ O 800-1000 NH ₄ H ₂ PO ₄ 500-700 (NH ₄ ) ₂ HPO ₄ 90-110 KCI 60-80 Kj 0.8-0.9 Na ₂ MoO ₄ × 2H ₂ O 0.2-0.3 CuSO ₄ × 5H ₂ O 0.02-0.03 CoCl ₂ × 6H ₂ O 0.02-0.03 MnSO ₄ × 5H ₂ O 23-25 H ₃ IN ₃ 5-8 ZnSO ₄ × 4H ₂ O 7-9 FeSO ₄ × 7H ₂ O 27-28 Na ₂ EDTA 37-38 Thiamine 0.8-1.2 Pyridoxine 0.5-1.0 A nicotinic acid 0.5-1.0 Glycine 1.5-2.5 1-naphthylacetic acid 0.2-0.5 Kinetin 0.02-0.1 Casein Hydrolyzate 40-60 Mesoinosine 15-25 Sucrose 50000-60000 Agar 6000-8000.

As the experiments showed, from one bulb in 1.5-2 years it is possible to get more than 1 million planting micropulses, the stage of which corresponds to natural bulbs of 5-7 years of age, capable of producing biologically active substances with a wide range of biological activity, in particular, promising treatment of diseases caused by mycobacteria.

The essence of the claimed group of inventions is illustrated by the following examples.

Example 1. The bulb of Ungernia Victor, which is at rest, is cleaned of external dry scales, after which an additional 1-2 layers of external living scales are removed, sterilized and, under sterile conditions, divided into separate scales. For further work, the basal part of the scale is used, for which the upper third is cut from each scale, which is removed. The basal parts of the scales are cut with a scalpel into pieces (fragments, explants) of 0.5-1 × 0.5-1 cm in size and planted on a nutrient medium. Use a nutrient medium of the following composition, mg / l:

NH ₄ NO ₃ 500 CCW ₃ 1100 (NH ₄ ) ₂ SO ₄ 300 MgSO ₄ × 7H ₂ O 500 Ca (NO ₃ ) ₂ × 4H ₂ O 900 NH ₄ H ₂ PO ₄ 600 (NH ₄ ) ₂ HPO ₄ one hundred KCI 70 Kj 0.85 Na ₂ MoO ₄ × 2H ₂ O 025 CuSO ₄ × 5H ₂ O 0.024 CoCl ₂ × 6H ₂ O 0,025 MnSO ₄ × 5H ₂ O 24 H ₃ IN ₃ 6 ZnSO ₄ × 4H ₂ O 8 FeSO ₄ × 7H ₂ O 27.5 Na ₂ EDTA 37.5 Thiamine 1,0 Pyridoxine 0.8 A nicotinic acid 0.8 Glycine 2.0 NUK 2.0 Kinetin 1,0 Casein Hydrolyzate 480 Mesoinosine 80 Sucrose 55,000 Agar 7000 pH before autoclaving 6.0.

The medium was poured into flasks and sterilized with hot steam for 20 min at 1 atm.

After disembarkation of the explants, the flasks were placed in a thermostatic room at a temperature of 24-26 ° C with a relative humidity of 79-80%. After 45 days, out of 425 bulbs scanned, 127 (30%) on average 3.1 microteks each formed.

The resulting regenerates for animation are transferred to a medium of the following composition, mg / L:

NH ₄ NO ₃ 500 Kno ₃ 1100 (NH ₄ ) ₂ SO ₄ 300 MgSO ₄ × 7H ₂ O 500 Ca (NO ₃ ) ₂ × 4H ₂ O 900 NH ₄ H ₂ PO ₄ 600 (NH ₄ ) ₂ HPO ₄ one hundred KCI 70 Kj 0.85 Na ₂ MoO ₄ × 2H ₂ O 025 CuSO ₄ × 5H ₂ O 0.024 CoCl ₂ × 6H ₂ O 0,025 MnSO ₄ × 5H ₂ O 24 H ₃ IN ₃ 6 ZnSO ₄ × 4H ₂ O 8 FeSO ₄ × 7H ₂ O 27.5 Na ₂ EDTA 37.5 Thiamine 1,0 Pyridoxine 0.8 A nicotinic acid 0.8 Glycine 2.0 IAA 2.0 Kinetin 0.02 Casein Hydrolyzate fifty Mesoinosine twenty Sucrose 55,000 Agar 7000

The results of reproduction are shown in table 1.

Example 2. Grow explants in the conditions of example 1 on a medium of the following composition, mg / l:

NH ₄ NO ₃ 600 Kno ₃ 1000 (NH ₄ ) ₂ SO ₄ 400 MgSO ₄ × 7H ₂ O 400 Ca (NO ₃ ) ₂ × 4H ₂ O 1000 NH ₄ H ₂ PO ₄ 500 (NH ₄ ) ₂ HPO ₄ 110 KCI 60 Kj 0.9 Na ₂ MoO ₄ × 2H ₂ O 0.2 CuSO ₄ × 5H ₂ O 0,03 CoCl ₂ × 6H ₂ O 0.02 MnSO ₄ × 5H ₂ O 25 H ₃ IN ₃ 5 ZnSO ₄ × 4H ₂ O 9 FeSO ₄ × 7H ₂ O 27 Na ₂ EDTA 38 Thiamine 0.8 Pyridoxine 1,0 A nicotinic acid 0.5 Glycine 2.5 NUK 2.2 Kinetin 0.8 Casein Hydrolyzate 500 Mesoinosine 70 Sucrose 60,000 Agar 6000 pH before autoclaving 5.8.

The medium was poured into flasks and sterilized with hot steam for 25 min at 1 atm.

After disembarkation of the explants, the flasks were placed in a thermostatic room at a temperature of 24-26 ° C with a relative humidity of 79-80%. After 60 days, out of 346 examined bulbs, 88 (25%) on average began to form 2.9 microtubes.

The resulting regenerates for animation are transferred to a medium of the following composition, mg / L:

NH ₄ NO ₃ 600 Kno ₃ 1000 (NH ₄ ) ₂ SO ₄ 400 MgSO ₄ × 7H ₂ O 400 Ca (NO ₃ ) ₂ × 4H ₂ O 1000 NH ₄ H ₂ PO ₄ 500 (NH ₄ ) ₂ HPO ₄ 110 KCI 60 Kj 0.9 Na ₂ MoO ₄ × 2H ₂ O 0.2 CuSO ₄ × 5H ₂ O 0,03 CoCl ₂ × 6H ₂ O 0.02 MnSO ₄ × 5H ₂ O 25 H ₃ IN ₃ 5 ZnSO ₄ × 4H ₂ O 9 FeSO ₄ × 7H ₂ O 27 Na ₂ EDTA 38 Thiamine 0.8 Pyridoxine 1,0 A nicotinic acid 0.5 Glycine 2.5 IAA 2.2 Kinetin 0.03 Casein Hydrolyzate 60 Mesoinosine fifteen Sucrose 60,000 Agar 6000

The results of reproduction are shown in table 1.

Example 3. Grow the explants in the conditions of example 1 on a medium of the following composition, mg / l:

NH ₄ NO ₃ 400 Kno ₃ 1200 (NH ₄ ) ₂ SO ₄ 200 MgSO ₄ × 7H ₂ O 600 Ca (NO ₃ ) ₂ × 4H ₂ O 800 NH ₄ H ₂ PO ₄ 700 (NH ₄ ) ₂ HPO ₄ 90 KCI 80 Kj 0.8 Na ₂ MoO ₄ × 2H ₂ O 0.3 CuSO ₄ × 5H ₂ O 0.02 CoCl ₂ × 6H ₂ O 0,03 MnSO ₄ × 5H ₂ O 23 H ₃ IN ₃ 8 ZnSO ₄ × 4H ₂ O 7 FeSO ₄ × 7H ₂ O 28 Na ₂ EDTA 37 Thiamine 1,2 Pyridoxine 0.5 A nicotinic acid 1,0 Glycine 1,5 IAA 1.8 Kinetin 1,2 Casein Hydrolyzate 450 Mesoinosine one hundred Sucrose 50,000 Agar 8000 pH before autoclaving 6.2.

The medium was poured into flasks and sterilized with hot steam for 30 min at 1 atm.

After disembarkation of the explants, the flasks were placed in a thermostatic room at a temperature of 24-26 ° C with a relative humidity of 79-80%. After 60 days, out of 450 scanned bulbs, an average of 2.0 microtubes began to form in 200 (45%).

The resulting regenerates for animation are transferred to a medium of the following composition, mg / L:

NH ₄ NO ₃ 400 Kno ₃ 1200 (NH ₄ ) ₂ SO ₄ 200 MgSO ₄ × 7H ₂ O 600 Ca (NO ₃ ) ₂ × 4H ₂ O 800 NH ₄ H ₂ PO ₄ 700 (NH ₄ ) ₂ HPO ₄ 90 KCI 80 Kj 0.8 Na ₂ MoO ₄ × 2H ₂ O 0.3 CuSO ₄ × 5H ₂ O 0.02 CoCl ₂ × 6H ₂ O 0,03 MnSO ₄ × 5H ₂ O 23 H ₃ IN ₃ 8 ZnSO ₄ × 4H ₂ O 7 FeSO ₄ × 7H ₂ O 28 Na ₂ EDTA 37 Thiamine 1,2 Pyridoxine 0.5 A nicotinic acid 1,0 Glycine 1,5 IAA 1.8 Kinetin 0.01 Casein Hydrolyzate 40 Mesoinosine thirty Sucrose 50,000 Agar 8000

The results of reproduction are shown in table 1.

Example 4. Prepared a nutrient medium according to example 1, in which the amount of 1-naphthylacetic acid was reduced to 0.5 mg / l, kinetin to 0.02 mg / l, mesoinositol to 20 mg / l, casein hydrolyzate to 50 mg / l (Wednesday 5СЗН). Under the same cultivation conditions, on isolated fragments of scales after 60 days of cultivation, microtubules are formed. For the accelerated development of individual bulbs, they are transferred to 5СЗН medium (medium composition is given in Example 2), in which the amount of NAA is reduced to 0.2 mg / L. The microwells were placed on the medium of the following composition, mg / l:

NH ₄ NO ₃ 500 Kno ₃ 1100 (NH ₄ ) ₂ SO ₄ 300 MgSO ₄ × 7H ₂ O 500 Ca (NO ₃ ) ₂ × 4H ₂ O 900 NH ₄ H ₂ PO ₄ 600 (NH ₄ ) ₂ HPO ₄ one hundred KCI 70 Kj 0.85 Na ₂ MoO ₄ × 2H ₂ O 0.25 CuSO ₄ × 5H ₂ O 0.02 CoCl ₂ × 6H ₂ O 0,03 MnSO ₄ × 5H ₂ O 24 H ₃ IN ₃ 7 ZnSO ₄ × 4H ₂ O 8 FeSO ₄ × 7H ₂ O 27.5 Na ₂ EDTA 37.5 Thiamine 1,0 Pyridoxine 0.8 A nicotinic acid 0.8 Glycine 2.0 1-naphthylacetic acid 0.4 Kinetin 0.5 Casein Hydrolyzate fifty Mesoinosine twenty Sucrose 55,000 Agar 7000.

Test tubes with onion bulbs are placed in thermostated conditions (22-26 ° C) with a 14-16-hour daylight and with illumination of 1000-2500 lux. After 20-40 days, the bulbs form the roots and leaves and grow, and after 50-60 days go into a dormant state, while the leaf dries out. When the dormant bulb is transferred to fresh medium, it again forms 1-2 leaflets (depending on the age of the bulb) and the whole cycle repeats.

Example 5. In the nutrient medium according to example 4, instead of NAA, 2 mg / l of indolylacetic acid was added (5СЗИ medium). Under the same growing conditions, after 60 days of cultivation, about 29% of the explants form microtubes, on average 4 microtubes form on each of these explants.

the resulting microwells are grown on a medium of the following composition, mg / l:

NH ₄ NO ₃ 600 Kno ₃ 1000 (NH ₄ ) ₂ SO ₄ 400 MgSO ₄ × 7H ₂ O 400 Ca (NO ₃ ) ₂ × 4H ₂ O 1000 NH ₄ H ₂ PO ₄ 500 (NH ₄ ) ₂ HPO ₄ 110 KCI 60 Kj 0.9 Na ₂ MoO ₄ × 2H ₂ O 0.2 CuSO ₄ × 3H ₂ O 0,03 CoCl ₂ × 6H ₂ O 0.02 MnSO ₄ × 5H ₂ O 25 H ₃ IN ₃ 5 ZnSO ₄ × 4H ₂ O 9 FeSO ₄ × 7H ₂ O 27 Na ₂ EDTA 38 Thiamine 0.8 Pyridoxine 1,0 A nicotinic acid 0.5 Glycine 2.5 1-naphthylacetic acid 0.2 Kinetin 0.1 Casein Hydrolyzate 40 Mesoinosine 25 Sucrose 50,000 Agar 8000

Test tubes with onion bulbs are placed in thermostated conditions (22-26 ° C) with a 14-16-hour daylight and with illumination of 1000-2500 lux. After 30-40 days, the bulbs form the roots and leaves and grow, and after 50-60 days go into a dormant state, while the leaf dries out.

Example 6. The cultivation of the explants was carried out under the conditions of example 5. Then, the obtained microwells are grown on a medium of the following composition, mg / l:

NH ₄ NO ₃ 400 Kno ₃ 1200 (NH ₄ ) ₂ SO ₄ 200 MgSO ₄ × 7H ₂ O 600 Ca (NO ₃ ) ₂ × 4H ₂ O 800 NH ₄ H ₂ PO ₄ 700 (NH ₄ ) ₂ HPO ₄ 90 KCI 80 Kj 0.8 Na ₂ MoO ₄ × 2H ₂ O 0.3 CuSO ₄ × 5H ₂ O 0.02 CoCl ₂ × 6H ₂ O 0,03 MnSO ₄ × 5H ₂ O 23 H ₃ IN ₃ 8 ZnSO ₄ × 4H ₂ O 7 FeSO ₄ × 7H ₂ O 28 Na ₂ EDTA 37 Thiamine 1,2 Pyridoxine 0.5 A nicotinic acid 1,0 Glycine 1,5 1-naphthylacetic acid 0.5 Kinetin 0.02 Casein Hydrolyzate 60 Mesoinosine fifteen Sucrose 60,000 Agar 6000.

Test tubes with onion bulbs are placed in thermostated conditions (22-26 ° C) with a 14-16-hour daylight and with illumination of 1000-2500 lux. After 30-40 days, the bulbs form roots and leaves and grow, and after 50-55 days go into a dormant state, while the leaf dries up.

Example 7. Compared the efficiency of the animation of the bulbs obtained in examples 1-3, and the cellular material of strains of analogs on the medium prepared in example 1.

For this, each regenerant onion was separated from the explant on which it was formed and transferred to 5SZI medium. The results are shown in table 1.

Table 1 The dependence of the cultivation efficiency of Ungernia Victor on the technology of obtaining the source material Raw material Explants explored The explants, regenerating microwells,% The average number of microwells per regenerating Example 1 UV-22 190 57.5 ± 4.7 5.9 Example 2 UV-22 108 47.8 ± 4.3 4.8 Example 3 UV-22 127 43.1 ± 4.2 4.9 Example 4 UV-22 225 40.1 ± 2.2 4.0 Strain UV -2 175 21.2 ± 3.7 4.3 Strain UV -12 113 37.5 ± 4.7 3.9

The data obtained indicate that the claimed technology allows you to receive 3-6 or more new microtubes from an individual bulb in 40 days. If necessary, further propagation of the formed bunch of bulbs is divided into individual bulbs and again transferred to the same medium. The ability of regenerated bulbs to micropropagation (animation) remains the smallest for 5 years.

Example 8. Obtained in examples 1-3, the cell material was crushed and extracted with a 40% aqueous-alcoholic solution, after which the biological activity of the extracted biologically active substances was tested.

Evaluation of the effectiveness of the proposed drug was performed on 140 white outbred male mice with generalized tuberculosis caused by the introduction of M. Bovis 8 culture in a lateral tail vein at a dose of 0.1 mg in 2 ml of physiological saline.

Mice were divided into 6 groups of 20 animals:

1 group - control - mice that did not receive the drug;

Group 2 - control — mice treated with rifampicin (RF) orally at a dose of 10 mg / kg;

Group 3 - control — mice receiving oral supplement “Vlairin” 3 times a day in the form of a solution at a dose of 2 drops per mouse;

4-6 groups - mice that received the claimed drug according to examples 1-3.3 times a day orally in the form of a solution at a dose of 2 drops per mouse;

Group 7 — mice who received daily rifampicin orally at a dose of 10 mg / kg, and the agent obtained in Example 1 was orally administered as a solution at a dose of 4 drops per mouse.

The effectiveness of treatment of the tuberculosis process was evaluated according to the results: dynamics of the body weight of mice; animal survival; lung lesion index; sowing mycobacteria from the spleen. The lung lesion index was calculated in accordance with the number and severity of foci of specific inflammation detected by macroscopic examination. Moreover, single submiliary foci were estimated at 0.5 points; numerous (no more than 20) - 1.5 points; single miliary - in 1.75 points; numerous merging submillion single millionths - in 2.0 points, millionths (no more than 10) and 2.25 points, numerous millionths merging - 2.75 points, the presence of small caseous necrotic tricks - 3.0 points; extensive caseosis - 4.0 points; continuous lung damage - 5.0 points. In cases of serous impregnation of lung tissue, 0.25 to 1.0 points were added to the calculated lesion index, depending on the area of the lesion.

For bacteriological studies, dosed inoculation of the spleen tissue homogenate was carried out on a dense Levenshtein-Jensen nutrient medium. Growth massiveness of Mycobacterium tuberculosis (MBT) was evaluated after three weeks of incubation of spleen cultures (37 ° C) and expressed in colony forming units — CFU. The following colony counting technique was used: when registering up to 50 colonies, their total number was given; with growth by 1/3 of the test tube, the number of colonies was taken as 100 CFU; with an increase of 2/3 tubes - for 200 CFU; with continuous growth of colonies - for 300 CFU.

Statistical processing of the results was carried out according to the Student-Fisher parametric test and the non-parametric Wilkinson-Mann-Whitney test using the U criterion. Comparative data on the effectiveness of different drugs are shown in Table 2.

table 2 Comparison of the effectiveness of the effects of drugs on protecting the body from the effects of mycobacterium tuberculosis Group A drug Number of mice Survival rate,% Weight gain,% Lung lesion index Inoculation of MBT from the spleen, CFU one No twenty 20,0 -2.4 3.1 ± 0.2 300 2 RF twenty 90.0 10.6 2.3 ± 0.2 46.1 ± 4.2 3 Vlairin twenty 60.0 7.8 2.9 ± 0.1 one hundred* four Project 1 twenty 75.0 ** 10.0 * 2.7 ± 0.1 * one hundred* 5 Project 2 twenty 65.0 * 8.7 * 2.9 ± 0.1 one hundred* 6 Project 3 twenty 65.0 * 9.2 * 2.9 ± 0.1 one hundred* 7 RF + approx. 1 twenty 100.0 33.8 ** 1.9 ± 0.1 ** 21.9 ± 6.1 **

The research results indicate a sufficiently high efficiency of the claimed drug, comparable with the use of rifampicin, and higher results were obtained when using the means of example 1 on the background of standard therapy using rifampicin.

Example 9. A clinical study of the drug obtained according to example 1 (HC extract) was carried out on the basis of the 1st pulmonary department of the regional tuberculosis dispensary in Astrakhan.

20 patients (men aged 19-50 years) with pulmonary tuberculosis were examined. Among the examined - 2 patients with disseminated, 6 - infiltrative tuberculosis, 1 - pleurisy of tuberculous etiology, 1 patient was diagnosed with tuberculosis. Of these, 3 were bacterial isolators; 5 had destruction of the lung tissue. Concomitant pathology in patients was not identified.

To assess the effectiveness of the drug, two groups were identified that were identical in gender and age composition, clinical forms and phases of the specific process:

Group 1 (control) - 10 patients who received standard complex therapy;

Group 2 - 10 patients, in the complex therapy of which the drug was included according to example 1.

Standard complex therapy, due to the nature of the specific process, included anti-TB drugs (isoniazid, rifampicin, pyrazinamide, ethambutol, streptomycin), hepatoprotectors (LIV-52), pathogenetic measures (sodium thiosulfate, autohemonocaine therapy, prednisone, vitamin therapy).

Patients of the experimental group against the background of standard therapy were given the Ungernia preparation according to Example 1, 1 drop per 20 kg of body weight per 100 ml of water after meals 3 times a day.

Before using the drug and in dynamics, a clinical and radiological examination, a general blood test, a biochemical blood test (bilirubin, ALT, sialic and thymol tests, CRP) were carried out, an immunogram was determined, blood serum was studied by wedge-shaped dehydration and polarization microscopy, followed by analysis of the obtained data. The criteria for evaluating the effectiveness were indicators of clinical, laboratory and radiological research methods, analyzed before treatment and in dynamics (within 2.5 months). The results of the study are presented in table.3-6.

Table 3 Biochemical parameters of patients with pulmonary tuberculosis who received treatment with HC extract and standard methods before treatment and in dynamics. Indicators Experimental group (n = 10) Control group (n = 10) before + LIV 2 weeks. 1 month 2 months before 1 month 2 months Transamylases (ALT, ACT) reduced - one 3 2 - 2 one promoted four 6 2 - 5 7 5 norm 6 3 one 8 5 one four Thymol test reduced - one - - - one one promoted four 5 one one 7 four 3 norm 6 four 9 9 3 5 6 Sialic test reduced - - 2 - - - - promoted 10 8 3 one 8 6 four norm - 2. 5 9 2 four 6 "C" reagent denied. 6 8 10 10 5 7 10 ny will put. four 2 - - 5 3 -

Table 4 Indicators of a general blood test for patients with pulmonary tuberculosis who received treatment with HC extract and standard methods before treatment and in dynamics. Indicators Experimental group (n = 10) Control group (n = 10) before 1 month 2 months before 1 month 2 months Hemoglobin norm 6 four 5 5 one 6 promoted 3 5 - 3 one reduced 4 one - 5 S 2 White blood cells increased 7 3 6 8 6 four norm 2 2 - 2 one one reduced 1 5 four - J 5 Lymphocytes norm 6 one 6 6 :one '' promoted 8 J :one 6 3 reduced 4 - one one 2 2 Monocytes norm 9 9 8 10 7 four increased 1 one 2 - 3 four reduced - - - four 2 ESR norm 2 2 3 3 2 one increased 8 - one 7 one 3 reduced 8 6 - 7 6

Table 5 Immunograms of patients with pulmonary tuberculosis who received treatment with HC extract and standard methods before treatment and in dynamics. Indicators Experimental group (n = 10) Control group (n = 10) before 1 month 2 months before 1 month 2 months T-lymphocytes rel.% reduced 2 3 2 one 2 2 promoted 8 6 7 5 four 3 norm - one one four four 5 p-lymphocytes rel.% reduced 3 2 one 2 2 one promoted 6 8 2 four 3 four norm one - 8 four 5 5 The percentage of phagocytosis,% reduced 3 5 - 8 6 5 promoted - one 5 - - - norm 7 four one 2 four 5 Phagocytic number reduced - 8 four 3 3 four promoted 3 one 8 - - one norm 7 one one 7 7 5 The number of active phagocytes reduced 2 6 one four 5 5 promoted - 3 6 one one 2 norm 8 one 3 5 four 3 IgM reduced four 2 one 6 5 6 promoted one 8 2 - one 2 norm 5 - 8 four four 2 IgG reduced 7 four 2 one 2 2 promoted 2 3 four one - one norm one 3 four 8 8 7 IgA reduced 22 7 5 5 5 four promoted 5 2 2 four 3 four norm 3 one 3 four 2 2

Table 6 Indicators of the structural organization of blood serum of patients with pulmonary tuberculosis who received treatment with HC extract and standard methods before treatment and in dynamics. Terms of treatment Groups Types of Fascia Pathological elements Order Depress React bl skl burn jaz foxes ser. Pig 2nd fac. Before treatment Counter. Group 5 four one 8 5 2 2 one - - one Experience. Group 3 6 one 6 3 - 2 2 3 one 3 2 weeks Counter. Group 5 four one 8 6 2 2 one 2 - 2 Experience. Group four 6 - 8 6 2 four 3 2 four 2 4 weeks Counter. Group 5 5 - 7 5 2 3 one 2 one 2 Experience. Group 3 7 - 9 6 four 2 3 3 3 2 6 weeks Counter. Group four 6 - 7 5 one 2 one 2 - one Experience. Group 3 7 - 7 four 3 2 four one four one 8 weeks Counter. Group four 6 - 7 four - 2 2 one - one Experience. Group four 6 - 7 four one - four 2 2 2 10 weeks Counter. Group 5 5 - 6 3 one 2 2 one - - Experience. Group 6 four - 5 four - - four one one one

Patients showed good tolerance to the drug, no side effects and allergic reactions were observed with its use; compared with the control, the positive dynamics of the analyzed clinical indicators (increase in appetite, decrease in the number of wet and dry rales, the disappearance of heaviness in the right hypochondrium), the disappearance of signs of intoxication (weakness, sweating, low-grade temperature), weight gain per a shorter period by an average of 1.5 kg, as well as improved laboratory tests (normalization of liver samples and transaminases, decreased ESR, increased hemoglobin ins and the number of peripheral blood lymphocytes).

According to immunograms, normalization of the absolute and relative indicators of B- and T-lymphocytes was noted, but a decrease in the percentage of phagocytosis, phagocytic number and number of active phagocytes, an increase in the level of IgM and IgG, a decrease in IgA.

The study of the structural organization of blood serum by the method of wedge-shaped dehydration showed an increase in the level of structuring and ordering of the main elements of the facies in the experimental group at an earlier date compared to the control.

Example 10. At the Federal State Institution Research Institute for the Study of Leprosy of Roszdrav during 2006 clinical trials were conducted on the possibility of using the extract of callus tissue obtained according to Example 1 in the complex treatment of patients with leprosy. Under observation were 20 patients with leprosy aged 46 to 72 years, including 12 women, 8 men.

Patients were divided into two groups of 10 people (experimental and control), comparable in age, gender and clinical manifestations. In the experimental group, against the background of standard complex therapy, the extract was prescribed according to Example 1, 1 drop per 20 kg of body weight diluted in 100 ml of water after meals three times a day, in the control - “carlsil”.

Standard chemotherapy included the use of combined anti-leprosy chemotherapy according to WHO regimens, antihistamines, vascular, and vitamin preparations. The therapy was carried out for three months under clinical and laboratory control, including dynamic clinical observation, the study of hemograms, liver samples, protein fractions and structural organization of blood serum, liver ultrasound.

Over the period of observation, we noted good tolerability of the studied drug, no side effects and allergic reactions were observed against the background of its use. An improvement in the general condition (sleep, appetite, general tone), a decrease or disappearance of subjective complaints of a feeling of heaviness in the right hypochondrium, bitterness in the mouth were noted. An improvement in a number of laboratory indicators was noted (table 7).

Table 7 The dynamics of the hemogram indices of patients with leprosy treated with HC extract Indicators Experienced group Before treatment After treatment Hemoglobin (g / l) 121.41 ± 3.62 130.82 ± 4.15 White blood cells (10 ⁹ / L) 4.23 ± 0.21 6.51 ± 0.34 Lymphocytes (%) 21.42 ± 3.63 31.22 ± 1.91 Monocytes (%) 2.12 ± 0.34 4.44 ± 0.15 Erythrocyte sedimentation rate (mm / h) 11.45 ± 0.18 9.53 ± 0.21 Total protein (g / l) 83.02 ± 3.15 82.19 ± 2.95 Albumin (g / l) 39.91 ± 2.78 44.61 ± 3.25 Total bilirubin (μmol / L) 11.16 ± 0.41 12.31 ± 0.12 Conjugated bilirubin (μmol / L) 2.78 ± 0.22 2.84 ± 0.61 Creatinine (μmol / L) 83.88 ± 4.17 82.11 ± 3.64 Urea (mmol / L) 6.48 ± 0.31 6.56 ± 0.61 Residual nitrogen (mmol / l) 18.64 ± 1.12 17.87 ± 2.11 Thymol test (Unit) 4.11 ± 0.52 3.56 ± 0.45 AcAT (μmol / L) 0.65 ± 0.01 0.51 ± 0.22 AlAT (μmol / L) 0.45 ± 0.04 0.53 ± 0.18

As follows from the above tables, against the background of the use of HC extract, there was a significant decrease in ESR (p <0.05), a tendency to a decrease in thymol test, transaminase levels, a decrease in inflammatory phenomena, and stabilization of degenerative changes.

The study of the structural organization of blood serum by the method of wedge-shaped dehydration showed an increase in the level of structuring and ordering of the main elements of the facies in the experimental group of patients, which testified to the harmonization of adaptive reactions of the body. In the control group of patients, such positive dynamics were not observed.

Analysis of the results showed that the extract of Ungernia Victor is effective for use in the course of treatment of diseases caused by mycobacteria. Moreover, the extract obtained from callus strain UV-22 is most effective. The proposed cultivation technology, along with the production of more effective biologically active substances, can significantly reduce the cultivation time of Ungernia bulbs and solve the problems of their reproduction.

Claims (4)

1. Callus culture strain Ungernia victoris UV-22, deposited in the Cell Culture Collection of the Institute of Molecular Biology and Genetics of the Academy of Sciences of Ukraine No. 13/2008, is a producer of a complex of biologically active substances for the treatment of diseases caused by mycobacteria. 2. Aqueous-alcoholic extract of callus tissue Ungernia victoris strain UV-22, deposited in the Cell Culture Collection of the Institute of Molecular Biology and Genetics of the Academy of Sciences of Ukraine No. 13/2008 as a means for the treatment of diseases caused by mycobacteria. 3. A method of cultivating callus tissue of Ungernia victoris strain UV-22 by microclonal propagation, characterized in that the cultivation is carried out in two stages, and the explant is first placed on a medium containing additional pyrodoxin, nicotinic acid, glycine, derivatives of acetic acid - 1-naphthylacetic acid or indolylacetic acid, kinetin, casein hydrolyzate and mesoinositol in the following ratio of ingredients, mg / l:
NH ₄ NO ₃ 400-600 Kno ₃ 1000-1200 (NH ₄ ) ₂ SO ₄ 200-400 MgSO ₄ · 7H ₂ O 400-600 Ca (NO ₃ ) ₂ · 4H ₂ O 800-1000 NH ₄ H ₂ PO ₄ 500-700 (NH ₄ ) ₂ HPO ₄ 90-110 Kcl 60-80 Kj 0.8-0.9 Na ₂ MoO ₄ · 2H ₂ O 0.2-0.3 CuSO ₄ · 5H ₂ O 0.02-0.03 CoCl ₂ · 6H ₂ O 0.02-0.03 MnSO ₄ · 5H ₂ O 23-25 H ₃ BO ₃ 5-8 ZnSO ₄ · 4H ₂ O 7-9 FeSO ₄ · 7H ₂ O 27-28 Na ₂ EDTA 37-38 Thiamine 0.8-1.2 Pyridoxine 0.5-1.0 A nicotinic acid 0.5-1.0 Glycine 1.5-2.5 Acetic Acid Derivatives 0.5-2.2 Kinetin 0.8-1.2 Casein Hydrolyzate 50-500 Mesoinosine 20-100 Sucrose 50000-60000 Agar 6000-8000 pH before autoclaving 5.8-6.2,
and then the resulting onions are propagated on the medium of the following composition, mg / l:
NH ₄ NO ₃ 400-600 Kno ₃ 1000-1200 (NH ₄ ) ₂ SO ₄ 200-400 MgSO ₄ · 7H ₂ O 400-600 Ca (NO ₃ ) ₂ · 4H ₂ O 800-1000 NH ₄ H ₂ PO ₄ 500-700 (NH ₄ ) ₂ HPO ₄ 90-110 Kcl 60-80 Kj 0.8-0.9 Na ₂ MoO ₄ · 2H ₂ O 0.2-0.3 CuSO ₄ · 5H ₂ O 0.02-0.03 CoCl ₂ · 6H ₂ O 0.02-0.03 MnSO ₄ · 5H ₂ O 23-25 H ₃ BO ₃ 5-8 ZnSO ₄ · 4H ₂ O 7-9 FeSO ₄ · 7H ₂ O 27-28 Na ₂ EDTA 37-38 Thiamine 0.8-1.2 Pyridoxine 0.5-1.0 A nicotinic acid 0.5-1.0 Glycine 1.5-2.5 Indolylacetic acid 1.8-2.2 Kinetin 0.01-0.03 Casein Hydrolyzate 40-60 Mesoinosine 15-30 Sucrose 50000-60000 Agar 6000-8000 4. A method of cultivating callus tissue of Ungernia victoris strain UV-22 by microclonal propagation, characterized in that the cultivation is carried out in two stages, the explant being first placed on a medium containing additional pyrodoxin, nicotinic acid, glycine, acetic acid derivatives - 1-naphthylacetic acid or indolylacetic acid, kinetin, casein hydrolyzate and mesoinositol in the following ratio of ingredients, mg / l:
NH ₄ NO ₃ 400-600 Kno ₃ 1000-1200 (NH ₄ ) ₂ SO ₄ 200-400 MgSO ₄ · 7H ₂ O 400-600 Ca (NO ₃ ) ₂ · 4H ₂ O 800-1000 NH ₄ H ₂ PO ₄ 500-700 (NH ₄ ) ₂ HPO ₄ 90-110 Kcl 60-80 Kj 0.8-0.9 Na ₂ MoO ₄ · 2H ₂ O 0.2-0.3 CuSO ₄ · 5H ₂ O 0.02-0.03 CoCl ₂ · 6H ₂ O 0.02-0.03 MnSO ₄ · 5H ₂ O 23-25 H ₃ BO ₃ 5-8 ZnSO ₄ · 4H ₂ O 7-9 FeSO ₄ · 7H ₂ O 27-28 Na ₂ EDTA 37-38 Thiamine 0.8-1.2 Pyridoxine 0.5-1.0 A nicotinic acid 0.5-1.0 Glycine 1.5-2.5 Acetic Acid Derivatives 0.5-2.2 Kinetin 0.8-1.2 Casein Hydrolyzate 50-500 Mesoinosine 20-100 Sucrose 50000-60000 Agar 6000-8000 pH before autoclaving 5.8-6.2,
and the resulting microwells are grown on the medium of the following composition, mg / l:
NH ₄ NO ₃ 400-600 Kno ₃ 1000-1200 (NH ₄ ) ₂ SO ₄ 200-400 MgSO ₄ · 7H ₂ O 400-600 Ca (NO ₃ ) ₂ · 4H ₂ O 800-1000 NH ₄ H ₂ PO ₄ 500-700 (NH ₄ ) ₂ HPO ₄ 90-110 Kcl 60-80 Kj 0.8-0.9 Na ₂ MoO ₄ · 2H ₂ O 0.2-0.3 CuSO ₄ · 5H ₂ O 0.02-0.03 CoCl ₂ · 6H ₂ O 0.02-0.03 MnSO ₄ · 5H ₂ O 23-25 H ₃ BO ₃ 5-8 ZnSO ₄ · 4H ₂ O 7-9 FeSO ₄ · 7H ₂ O 27-28 Na ₂ EDTA 37-38 Thiamine 0.8-1.2 Pyridoxine 0.5-1.0 A nicotinic acid 0.5-1.0 Glycine 1.5-2.5 1-Naphthylacetic acid 0.2-0.5 Kinetin 0.02-0.1 Casein Hydrolyzate 40-60 Mesoinosine 15-25 Sucrose 50000-60000 Agar 6000-8000