RU2360960C1 - Fomitopsis Tyv-2006 BASIDIOMYCETE STRAIN APPLIED AS PRODUCER FOR MAKING ANTINEOPLASTIC DRUGS - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: Fomitopsis officinalis Tyv-2006 All Russian Collection of Industrial Microorganisms F-961 basidiomycete strain characterised with high activity to malignant cells can also be used for making antineoplastic drugs.

EFFECT: extended range of agents with anticancer activity.

2 tbl, 2 ex

Description

The invention relates to the field of microbiology, relates to a new strain Tyv-2006 Fomitopsis officinalis (Will.) Bond. et Sing and can be used to obtain antitumor drugs.

In the practice of creating antibacterial, immunomodulating and antitumor drugs, the fruiting bodies of basidiomycete fungi, in particular Fomitopsis officinalis, are used [Oorzhak U.S. Scientific and practical aspects of the rational use of fruiting bodies Fomitopsis officinalis (Will.) Bond. et Sing. / Abstract. Diss. Cand. biol. sciences. Krasnoyarsk, 2006. - 18 p.]

Recent studies have shown that aqueous and carbon dioxide extracts obtained from the fruit bodies of the medicinal tinder fungus possess antimicrobial activity against opportunistic bacteria Fcinetobacter bauvfnii, Klebsiella pneumoniae, Proteus mirabilis [Sharikov A.M. Bactericidal activity of metabolites of the fungus Fomitopsis officinalis against opportunistic bacteria / A.M. Sharikov, W. S. Oorzhak, O. V. Peryanova, V. M. Ushanova and D. A. Neshumaev // Mushrooms in natural and anthropogenic ecosystems: Tr. Int. Conf. - St. Petersburg, 2005. - p.305-307], as well as the pathogenic bacteria Mycobacterium tuberculosis [Sharicov, A.M. Metabolites of the fungus Fomitopsis officinalis (Will.) Bond. et Sing: the bactericidal activity regarding the conditionally-pathogenic bacteria // A.M.Sharicov, U.S. Oorzhac, T.I. Gromovykh, O.V. Peryanova, D.A. Neshumaev // Program & abstracts: XII Symposium of Russia-Japan medical exchange. - Krasnoyarsk, 2005. - P.659-661].

At present, immunomodulating preparations based on water-soluble components extracted from the fruit body of Fomitopsis officinalis are widely known [Bogdaev A.G. Studying the induction of immunomodulating effects by oil extracts of the fruiting bodies of basidiomycetes in mice with a tumor inoculated with a carcinoma / A.G. Bogdaev, A.V. Lavlinsky, M.A. Nakvasina // Successes in medical mycology. T.9, 2007. - S.148-150]. The biochemical composition of fruiting bodies was established depending on the places of growth [Oorzhak U.S. Protein substances and vitamins of the fungus Fomitopsis officinalis (Will.) Bond. et Sing / U.S. Oorzhak, V.M. Ushanova // Storage and processing of agricultural raw materials. 2004. - No. 11. - S. 52-53]. Methods for processing fruit bodies of Fomitopsis officinalis to obtain biologically active compounds are proposed [Pat. RU 2257222, IPC 7 A61K 35/84, declared 07/02/04, publ. 07/27/05, bull. No. 21. - 5 p.].

An analysis of the literature shows that there are no close analogues for the claimed strain of Fomitopsis officinalis, which is explained by the fact that this type of fungus is a rare, endangered species and is found only in hard-to-reach natural places. In this connection, studies of extracts from the mycelium of the fungus Fomitopsis officinalis have not been carried out, due to the difficulty of isolating a pure mycelial culture and selecting nutrient substrates for laboratory cultivation.

At present, the microbiological industry has not mastered the production of antitumor biological preparations based on active strains of basidiomycetes. The lack of work on the creation of biotechnology based on the mycelium of basidiomycetes Fomitopsis officinalis hinders the possibility of obtaining antitumor drugs based on it.

The technical result of the invention is a strain of Fomitopsis officinalis VKPM F-961 with antitumor activity.

The technical result is achieved using the strain Fomitopsis officinalis VKPM F-961 to suppress tumor cells of Ehrlich carcinoma. A feature of the strain is a unique biochemical composition. The recommended strain is characterized by the accumulation of biomass on elective media containing plant residues and the ability to form blastoconidia.

The use of this strain as a producer for creating antitumor drugs based on it is proposed. The mycelium of the strain in large quantities contains proline, histidine, lysine (table 1).

The strain isolated from the fruiting body, found in vivo in the Republic of Tuva in 2006 (figure 1).

The strain is characterized by the following features.

Macromorphological signs

On a standard nutrient medium, the wort-agar strain forms a dense gray-white mycelium. In the center of the colony, the mycelium is denser. The edge of the colony is rugged, uneven (figure 2).

The mushroom is slowly growing. On the 40th day of incubation, the size of the colony on wort agar reaches 80 mm (Fig. 3). The aerial mycelium on the wort-agar medium is plentiful white, the substrate mycelium on the wort-agar medium is gray-white, on the reverse side of the gray colony. The pigment strain does not form. With aging, the colony of the strain becomes gray-fawn.

On wort agar with the addition of sawdust (5%) on the 40th day of incubation, the strain colony size reaches 88 mm, on Saburo medium - 50 mm, on mushroom agar - 75 mm, on potato-dextrose - 50 mm (Fig. 3) .

Micromorphology

Separated mycelium, hyphae size from 3.5-5.8 microns. Buckle cells form in the mycelium. The fungus forms blastoconidia abundantly on the 10-15th day (Fig. 4). Chlamydospores and sclerotia does not form.

Cultural signs

A study of the productivity of the strain during surface liquid-phase cultivation in beer wort showed that the increase in mycelial biomass on the 10th day is 1.56 g / l, on the 30th it is 4.14 g / l. The strain remains viable on mowed wort agar with the addition of larch sawdust for 6 months. For the propagation of the strain, the optimal medium is wort agar with the addition of larch sawdust (5%), pH 4.7-5.0, incubation time of 15 days, the strain does not need lighting for growth.

Other media used for cultivation: mushroom agar, potato dextrose agar, Saburo agar. For fermentation, the wort medium (4 ° B) with the addition of larch sawdust, pH 4.8-5.0, is optimal.

According to experts of the Krasnoyarsk Regional Veterinary Laboratory, this strain is avirulent, non-toxic, non-toxic against warm-blooded organisms and does not cause infectious diseases.

Figure 1-5 presents the following illustrations.

Figure 1. Morphology of the fruiting body from which the strain Fomitopsis officinalis VKPM F-961 was isolated.

Figure 2. The morphology of the colonies of the strain Fomitopsis officinalis VKPM F-961 on various nutrient media:

1 - wort agar;

2 - wort agar with the addition of sawdust;

3 - mushroom agar;

4 - potato dextrose agar;

5 - Wednesday Saburo.

Figure 3. The growth dynamics of the strain Fomitopsis officinalis VKPM F-961 in dense standard nutrient media.

Fig 4. The morphology of the mycelium of the strain Fomitopsis officinalis VKPM F-961: A - mycelium forming buckles; B - the formation of blastoconidia.

Fig 5. The cytotoxic effect of extracts on Ehrlich carcinoma cells: A - the effect of extracts on Ehrlich ascites carcinoma cells (agglutination); B - control.

Example 1. The technology of deep periodic cultivation includes several stages:

Sowing is carried out by culture of the strain Fomitopsis officinalis VKPM F-961 at the rate of 1.5-2 × 10 ⁶ blastoconidia per 1 ml of culture medium. As a seed, a strain culture grown on beveled wort agar with sawdust (5%) is used. Flush out the tubes into flasks with sterile water, then determine the titer of propagules in the resulting suspension by microscopic means in the Goryaev chamber and use the necessary amount of suspension for inoculation using the method of recalculation for the entire volume of the nutrient medium. Inoculation is carried out in Erlenmeyer flasks with a volume of 250 ml per 100 ml of culture medium. The strain is grown for 25 days in a liquid wort with the addition of sawdust (5%).

After cultivation, the biomass is separated from the nutrient medium, dried to constant weight and ground on a homogenizer; with this form of the drug, there is no nutrient medium on which the basidiomycete strain Fomitopsis officinalis VKPM F-961 was grown.

The resulting biological product is a dry powder of light yellow color.

Table 1 Amino acid composition of the mycelium of the strain Fomitopsis officinalis VKPM F-961 Essential Amino Acids The content in the mycelium,% dry weight Essential Amino Acids The content in the mycelium,% dry weight Arginine 3.9 Alanine 3.3 Valine 1.5 Asparagine 2.0 Histidine 18.6 Glycine 3.9 Isoleucine 3.2 Proline 37.4 Leucine 4.1 Serine 1.7 Lysine 16.9 Tyrosine 2.0 Methionine 0.6 Threonine 0.9

Example 2. The mycelium of the culture of the strain Fomitopsis officinalis VKPM F-961 is grown for 25 days on wort agar with sawdust (5%). After cultivation, the biomass is separated from the nutrient medium, dried to constant weight and ground on a homogenizer; with this form of the drug, there is no nutrient medium on which the strain was grown.

The antitumor effect of the aqueous extract from mycelium is evaluated against tumor cells of Ehrlich ascites carcinoma in vitro. A suspension of tumor cells (with a titer of 4.02 × 10 ⁶ (dilutions 1:10, 1:50, 1: 100, 1: 200) is added to the aqueous extract from mycelium. Cultivation is carried out in a water bath at a temperature of 37 ° C for the entire duration of the study. The antitumor effect is assessed by the physiological state of Ehrlich carcinoma cells stained with 1% methylene blue solution: living cells, cells with signs of blebbing (partial destruction of the cell wall and exit of the contents of the cytoplasm to the outside) and dead cells (Fig. 5). Cell counting was carried out using a Goryaev camera with a magnification of 640 (8 × 40 × 2 prism) using a SK 14 26183 microscope, Poland.

table 2 The effect of the aqueous extract of the mycelium of the strain Fomitopsis officinalis VKPM F-961 on Ehrlich ascites carcinoma cells in vitro,% Dilution of extract 5 min after application 1 hour 2 h 3 h 1:10 living 90.9 97.69 83.85 100% dead agglutination the dead 2.18 1.8 9.56 blebbing 9.31 0.51 6.59 1:50 living 93.59 73.9 65,2 100% dead agglutination the dead 4.23 8.58 9.36 blebbing 2.18 17.52 25.44 1: 100 living 91.97 76.76 84.35 100% blebbing the dead 7.73 5.89 2.16 blebbing 0.29 17.35 13.49 1: 200 living 97.32 90.89 3.2 44.29 the dead 2.68 2.67 75.3 16.35 blebbing 0 6.44 21.5 39.36

Thus, the strain of basidiomycete Fomitopsis officinalis VKPM F-961 can be used to obtain antitumor drugs based on it.

Claims (1)

The strain of basidiomycete Fomitopsis officinalis VKPM F-961 (All-Russian Collection of Industrial Microorganisms) used to obtain antitumor drugs.

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