RU2716590C1 - Strain of basidiomycete inonotus obliquus - producer of melanin pigment possessing antiviral and antitumoral activity - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: invention relates to the biotechnology. Strain of Basidium fungus Inonotus obliquus TV-18, having antiviral and antitumoral activity, is deposited in a collection of bacteria, bacteriophages and fungi of FBUN State Research Center of Virology and Biotechnology Vector under registration number F-1375.

EFFECT: strain of basidium fungus Inonotus obliquus can be used for production of medical preparations, creams, lotions, tonic and biologically active additives; invention allows to increase output of melanin.

1 cl, 4 dwg, 3 tbl, 6 ex

Description

The invention relates to a new strain of the basidiomycete Inonotus obliquus, a producer of melanin with antiviral and antitumor effects, and can be used in biotechnology, the pharmaceutical industry for the production of medicines, in the cosmetic industry for the manufacture of creams, lotions, tonics, in the food industry for the production of biologically active additives.

Obtaining melanin pigments from mycelium biomass and culture fluid (QL) of chaga based on effective producer strains is a very relevant area of medical biotechnology.

A number of cultures of microorganisms that synthesize melanin pigment in small quantities are known - Microsporum ganis, Trichophyton rubrum (Malama A.A. and Kasperovich TB Study of the pigments of the fungi Microsporum ganis and Trichophyton rubrum // Mycology and Phytopathology. - T. 10. - Leningrad, 1976 .-- S. 422).

For industrial production, these cultures are of no interest.

Known melanin-forming strain of the fungus Cladosporium cladosporidies-396 (A.S. USSR 1063834A, IPC C12N 15/00; C02F 3/34; C12R 1/645, publ. 1983), proposed by the authors for the binding of heavy metals in wastewater treatment. The fungus is an aerobic, optimal growth and sporulation at 25 ° C, the optimum value of water activity is 0.98, the optimum acidity of the medium is pH 4-5, highly resistant to total doses of UV and γ-radiation, high-intensity artificial sunlight. The fungus assimilates nitrate forms of nitrogen, uses glucose, sucrose, starch, pectin, cellulose, citric, oxalic, acetic acid as a carbon source, and has cellulolytic and tyrazinase activity. The biomass formed during fungal growth is separated by filtration from the culture fluid and dried at 60 ° C. The yield of fungal biomass is 15-20 g / l. Melanin pigment is extracted from the biomass of the fungus by a modified method by hydrolysis of its non-melanin components when boiled in a mixture of acetic and hydrochloric acids.

However, the strain does not find practical application, as it has a low melanin-forming ability and, being an aerobic, requires significant costs for industrial cultivation.

It is known to use the fungus Inonotus obliquus for the prevention and treatment of gastrointestinal diseases [Patent RU 2076728, IPC A61K 35/78, publ. 04/10/1997.].

A biostimulating agent is known, one of the components of which is chaga birch fungus (Inonotus obliquus) [Patent RU 2125460, IPC A61K 35/78, publ. January 27, 1999].

A known composition having antitumor, hepatoprotective and immunostimulating effects, in which chaga is used as an additive to leaf or granular tea [Patent RU 2125460, IPC A61K 35/78, publ. January 20, 2004].

Known antiviral agent based on melanin from the natural fungus Inonotus obliquus (Patent RU No. 2480227, IPC C12N 1/14; A61K 36/06, publ. 04/27/2013), in which a 2.5% aqueous solution of melanins from sclerotia chaga showed an antiviral effect against herpes simplex virus type 2 (HSV-2), human immunodeficiency virus (HIV-1), influenza virus (HS) and vaccinia virus (BOB).

However, all of the above inventions use natural raw materials from sclerotia (chaga infertile bodies) growing on trees, and not a pure culture of the chaga strain grown under sterile deep conditions.

Known strain of the fungus Aspergillus niger BMP-97/2 (KPMKB-G-1-9) for the production of melanin (patent of the Republic of Belarus No. 5057, IPC C12N 1/14, publ. 30.03.2003). The strain was obtained by multi-stage selection of culture isolated from mold plaque on the walls of domestic premises. The strain is deposited in the collection of industrial microorganisms of the Belbiopharm concern, registration number G-1-9 from 1.09.1999. The fungus is an optional anaerobic, growing in the temperature range from 18 to 40 ° C. The optimum temperature for culture growth is 28 ° C, and melanin biosynthesis is 37 ° C. The optimum pH is 6.5 ± 0.5. Synthesizes melanin inside cells and extracellularly. As a carbon source, the fungus uses the majority of sugars (glucose, sucrose, maltose, arabinose), does not absorb ethyl alcohol. Coagulates and peptones milk, liquefies gelatin. It has protease and oxidase activity. The fungus is grown on jambs (wort agar) for 14 days., A spore suspension is obtained for 14 days. Fermentation of the culture is carried out under anaerobic conditions at a temperature of 37 ± 1 ° C for 20 days. 12 g of technical melanin are obtained, after dissolution of which in 40% sodium hydroxide solution, neutralization and drying, 8 g of melanin substance are obtained. The culture is used in the production of dosage forms based on melanin in JSC Belmedpreparaty.

However, the strain has several disadvantages: the lengthy process of obtaining seed spore material of the fungus (two transfers for 14 days.) - only 28 days .; prolonged fermentation of the fungus culture (20 days); anaerobic cultivation conditions, which complicates the manufacturability of the culture growing process; melanin synthesis at high temperature - 37 ± 1 ° С; the use of a spore-forming culture that pollutes the environment and can cause allergies in workers.

The known strain I. obliquus BIM F-350 D BIM F-350 D is a producer of melanin with antimutagenic properties (Patent of the Republic of Belarus No. 10670, IPC C12N 1/14, C12P 19/00, publ. 30.06.2008). Strain I. obliquus BIM F-350 D is an aerobic, able to grow in the temperature range 18-30 ° C, the optimum temperature for growth is 24-25 ° C, the optimal pH is 6.0-7.0. It is characterized by the synthesis of exo- and endomelanins. Studies of the antimutagenic activity of melanin-containing substance in male Af mice showed that its intraperitoneal administration at a dose of 50 mg / kg of animal weight reduces the level of radio-induced translocations in spermatocytes by 2 times, which indicates high antioxidant and gene-protective properties of the melanin-containing substance of the fungus I. obliquus BIM F-350 D.

However, when cultivated on an agar medium (wort-agar) in Petri dishes at a temperature of 24-25 ° C, a full overgrowing of the cup is observed only by 20-23 days, the growth rate of the colonies is 2-2.3 mm / day., A long time for production seed material in a liquid medium (7 days), a liquid nutrient medium contains relatively expensive components that are of little use in industrial microbiological production (beer wort, thiamine, tyrosine). A relatively high amount of seed, 15%, is used. There is also a low yield of melanin-containing biomass - 12 g / l. From the culture fluid there is a low yield of melanin - 1.3 g / l.

In addition, the melanin from the strain of the prototype fungus was not tested for antiviral activity.

The inventive new strain has advantages over the above-mentioned analogue strain in the amount of biomass containing melanin accumulated in the GTS environment and in the amount of melanin released from the culture fluid. Table 1 shows the comparative characteristics of the claimed strain of Inonotus obliquus TV-18, a strain of the analogue of Inonotus obliquus BIM F-350.

The closest analogue (prototype) is the strain Inonotus obliquus F-1244, which we isolated from the sclerotia of the fungus brought from Tuva (Kosogova T.A. Strains of basidiomycetes of the south of Western Siberia are promising producers of biologically active drugs: abstract. Cand. Biol. Theses. Sciences, 03.01.06., Koltsovo, 2013 .-- p. 26).

However, the prototype strain has an insufficient amount of biomass containing melanin and the amount of melanin secreted in the GTS environment. In addition, there is no evidence of antitumor activity of the prototype strain.

The technical result of the claimed invention is to obtain a strain of the basidiomycete fungus Inonotus obliquus, a producer of melanin pigments, having higher productivity in producing biomass of the fungus containing melanin and a higher yield of melanin pigments from the culture fluid, having both antiviral and antitumor effects.

The specified technical result is achieved by obtaining a new strain of the basidiomycete fungus Inonotus obliquus - a producer of melanin pigments with antiviral and antitumor activity, deposited in the collection of bacteria, bacteriophages and fungi of the Federal State Budget Scientific Research Center of the World Bank Vector (number Koltsovo, Novosibirsk Region) under the number F-1375 (help attached).

The inventive strain has the following characteristics.

The strain Inonotus obliquus TV-18 (copyright) was isolated in 2018 from natural sclerotia chaga, found on a birch tree near the river Koltsovo, Novosibirsk Region. The strain Inonotus obliquus TV-18 has the following cultural-morphological and physiological-biochemical characteristics.

Colonies on an agar medium based on oat broth are cotton-like, white, turning into yellow-straw, sand and brown. Hyphae septate, thin-walled, without buckles, hyaline to yellowish, branching, 2-4 microns in diameter, turning into thick-walled, rusty-brown, parallel hyphae 3.5-7 microns in diameter. On agarized culture media often forms fruiting bodies from light brown to dark brown in color with an uneven surface.

The strain is grown (cultivated) on agarized and liquid nutrient media at a temperature of 24-27 ° C. Colony growth rate - Petri dish overgrows in 8-9 days.

The composition of the liquid medium. Glucose-tryptone medium (GTS), g / l: glucose - 30; tryptone - 10.0; yeast extract - 5.0; KH ₂ RO ₄ - 1.1; K ₂ HPO ₄ 4.4; MgSO ₄ x 7 H ₂ O - 0.25 g / l; water - 1 l, pH value 7-8. When cultivated on glucose-tryptone medium by the 9th day, the nutrient medium acquires a dark brown color due to the release of melanin into it.

The composition of the agar medium. GTS liquid medium, 20 g of agar-agar per 1 liter of solution.

The invention is illustrated by the following graphic materials.

In FIG. Figure 1 shows the results of studies of the sensitivity of the A431 human epidermoid carcinoma cell population to melanin. In FIG. 2 presents data on the evaluation of the antitumor activity of melanin from the deep culture of the Chaga Inonotus obliquus TV-18 on the tumor model of xenografts of human epidermoid carcinoma A-431 with repeated administration. In FIG. 3 shows colonies of a strain of chaga I. obliquus F-1375 (TV-18), and FIG. 4 - the fruiting body of a strain of chaga I. obliquus F-1375 (TV-18) on a GTS environment.

Example 1. Growing biomass of mycelium based on a strain of Inonotus obliquus TV-18 in deep conditions on a rocking chair.

Chaga cultivation was carried out in a glucose-tryptone medium (GTS) under deep conditions on a circular rocking chair (rotation speed 180-200 rpm / min, 25 ± 2 ° С). Sowing flasks was carried out by culture from the same medium, only agarized, including 20 g of agar-agar per 1 liter. Blocks were cut from the colonies in Petri dishes with a sterile cork drill with a diameter of 10 mm and 10 pieces were introduced into flasks (500 ml) with a liquid medium of 100 ml volume. After 3 days of cultivation, a liquid culture was used to inoculate new flasks at a dose of 10% (10 ml per 100 ml of liquid medium). Starting from 8-9 days of growth on a rocking chair, the nutrient medium acquired a dark brown color due to the melanin released into it.

During this period, numerous brownish hyphae fragments were observed in the culture.

To obtain melanin, fermentation on a rocking chair was completed on day 10. Table 1 presents data on the accumulation of biomass and melanin in the deep culture of the chaga strain of the Inonotus analogue. obliquus BIM F-350 D, Inonotus prototype strain. obliquus F-1244 and the new strain I. obliquus TV-18.

The yield of biomass of the strain of Chaga I. obliquus TV-18 on dry matter is an average of 18.2 g / l. Compared with the prototype strain Inonotus obliquus F-1244, this is 1 g / l more (table 1).

The total yield of melanin of the claimed strain I. obliquus TV-18 is 7.5 g / l, and the prototype I. obliquus F-1244 has a yield of melanin 6.0 g / l.

Thus, the inventive strain has advantages in terms of cultivation time, in the amount of biomass containing melanin accumulated in the GTS environment and in the amount of melanin released from the culture fluid.

Example 2. The selection of melanin from the culture fluid

After cultivation of the fungus on a rocking chair, the mycelium was separated from the dark brown culture liquid by filtration through a nylon filter. After separation of the fungus bimass, the culture fluid was adjusted with HCl to pH = 2.0 and centrifuged. The precipitate containing melanin was adjusted to 0.01 N. NaOH to pH = 7.0 (before dissolution of melanin) and was purified. The purification of melanin included a sequential 3-fold reprecipitation followed by a 3-fold washing of the melanin precipitate in a 0.1 N hydrochloric acid solution. Received, after a 3-fold reprecipitation, the precipitate of melanin was washed with acidified water. The purified melanins were resuspended in distilled water, transferred to a measuring beaker, the pH was adjusted to a value of 7-8 units with a 40% NaOH solution. The output of melanin from the culture fluid is 2.5-3.0 g / L. From the total mass containing melanin, the yield of dry melanins is 7.0-7.5 g / l. Dried melanin is a brilliant crystals, and after grinding - a brownish matte powder.

Example 3. Monitoring the qualitative and quantitative indicators of melanin

To verify the belonging of the extracted pigments to melanins, three qualitative tests were performed for the presence of quinoid and phenolic structures in their molecule. To carry out high-quality reactions, a 0.1% pigment solution was prepared (15 mg of the pigment was dissolved in 15 ml of a 0.1 N NaOH solution). Then, the following procedures were performed:

- 5 ml of the pigment solution was poured into a test tube with a capacity of 10 ml and poured 0.5 ml of 10% hydrogen peroxide. A positive reaction is discoloration of the solution.

- 5 ml of the pigment solution was poured into a test tube with a capacity of 10 ml and 0.1 ml of 0.1 M KMnO ₄ solution was added. Positive reaction - the solution turns green.

- 5 ml of the pigment solution was poured into a test tube with a capacity of 10 ml and 0.1 ml of a 5% solution of FeCl _{3 was} added. A positive reaction - large brown flakes are formed, in a solution with an excess of iron chloride, the precipitate dissolves.

A 0.001% melanin solution was prepared: for this, 10 mg of melanin powder was dissolved in 100 ml of a 0.1 N NaOH solution. Then, the optical density of the solution was measured using a spectrophotometer at a wavelength of 465 nm (OFS.1.2.1.0003.15), using a 0.1 N NaOH solution as a control. The optical density of a 0.001%) solution of melanin was not less than 0.040 units.

Example 4. Determination of toxicity and antiviral activity of melanin isolated from a liquid culture of a strain of chaga Inonotus obliquus TV-18

Work with influenza viruses was carried out on an MDCK cell culture using virus A / California / 07/09 (H1N1 pdm09) from the collection of FBSI SSC WB Vector. To work with the human immunodeficiency virus, the human cell line MT-4 and the HIV-1 strain MTZN, subtype A.

The toxicity and antiviral activity of melanin against herpes simplex virus type 2 was determined in a Vero cell culture. A strain of herpes simplex virus type 2 virus (obtained from the American Type Culture Collection) was used in the work.

The studies were carried out in virological laboratories in accordance with the rules and methods of working with cell cultures and viruses (Manual on the experimental (preclinical) study of new pharmacological substances / Edited by the corresponding member of the Russian Academy of Medical Sciences, Professor R.U. Khabriev. - 2-ed., revised and add. - M.: Publishing House "Medicine", 2005. - 832 p.). Data on the toxicity and antiviral activity of melanin from the Inonotus obliquus TV = 18 deep culture against influenza virus, strain A / California / 07/09 (H1N1 pdm09), HIV-1, herpes simplex virus type 2 are shown in table 2.

As can be seen from table 2, a sample of a new strain of Inonotus obliquus TV-18 on all viruses in independent studies proved to be the best.

Notes: TC50 - 50% toxic concentration of melanin, a dose of melanin that inhibits cell growth by 50%; IC ₅₀ - 50% inhibitory concentration of melanin, a dose of melanin inhibiting the reproduction of the virus by 50%; IS - selectivity index, or therapeutic index: equal to the ratio of a 50% toxic dose of a drug to a 50% inhibitory dose (TC ₅₀ / IC ₅₀ ).

With low toxicity, melanin from the deep culture of the chaga Inonotus obliquus TV-18 showed an antiviral effect against influenza virus A / California / 07/09 (H1N1 pdm09), IC ₅₀ = 10.0 μg / ml, IS = 200, human immunodeficiency virus ( HIV-1, subtype A, MTZN), IC ₅₀ = 13.9 μg / ml, IS = 143.9), herpes (HSV-2, MS strain), IC ₅₀ = 72 μg / ml, IS = 69.4 )

The data obtained indicate the promise of obtaining melanin from the culture fluid and biomass of the chaga strain Inonotus obliquus TV-18, which has antiviral activity.

Example 5. Evaluation of the cytotoxic effect of the drug melanin on a cell culture of human epidermoid carcinoma A 431.

Analysis of the cytotoxic effect of the melanin preparation in relation to A431 human epidermoid carcinoma cells was performed using the MTT test, which allows us to assess the level of cell viability by their ability to restore 3- (4,5-dimethylthiazolyl-2) -2,5 - diphenylbromidetetrazolium (MTT) [Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983 Dec 16; 65 (l-2): 55-63].

Carcinoma cells were placed at 10 ⁴ / well in 96-well microplates (TPP, Switzerland) in complete DMEM medium (Biolot) with 10% fetal calf serum (HyClone, USA) and 0.04 mg / ml gentamicin. A day after the start of cultivation, an aqueous solution of melanin was added to the wells in decreasing concentrations (from 95.7 to 0.37 μg / ml), each dilution was examined in 6 repetitions, and physiological saline was added to the control wells. Cells were incubated in a CO ₂ incubator (SHEL-LAB, USA) at a temperature of 37 ° C for 72 hours, then the optical density of the reduced MTT reagent (Sigma - Aldrich) was measured on a Multiskan EX reader (Thermo, Finland) at a wavelength of 540 nm The ratio of the optical density of the experimental and control wells was calculated the percentage of living cells for each dilution of the test drug (MTT index,% = OD _experience / OD _control × 100). The experimental data were processed by methods of variation statistics using the software package "Statgraphics, Vers.5.0". To assess the significance of intergroup differences, the Mann-Whitney U-test was used; the critical level of significance in testing statistical hypotheses (p) was taken to be 0.05.

In FIG. 1 shows the percentage of live cells of A431 epidermoid carcinoma after incubation with a melanin preparation for 72 hours. * - significant difference from control, p≤0.05.

The study showed that up to 50% of the A431 carcinoma cell population was sensitive to the action of melanin (Fig. 1).

The cytotoxic effect of the drug was dose-dependent. Significant differences in the MTT index of tumor cells incubated with melanin and control well cells were observed in the concentration range from 95.7 to 1.5 μg / ml; the most pronounced changes were noted during incubation with the drug in the maximum dose used. The data obtained confirm the presence of a direct antitumor effect of melanin in the drug.

Example 6. Evaluation of the antitumor activity of melanin from the deep culture of the chaga Inonotus obliquus TV-18 in a tumor model of xenograft human epidermoid carcinoma A-431 with repeated administration.

We used female nude mice aged 6–8 weeks, weighing 18–20 g. For the formation of tumors, mice were injected with a suspension of human epidermoid carcinoma A-431 cells subcutaneously at a dose of 5.5 * 10 ⁶ cells / mouse in a volume of 0.1 ml of physiological saline (0.9% NaCl) in the left barrel of the mouse closer to the thigh. The formation of tumors (xenografts) was 8 days, after which 18 mice with tumors of 60-100 mm ³ were selected for the experiment. Mice were divided into two groups: experimental and control (n = 8 and n = 10). The mice of the experimental group 3 times with an interval of 2-4 days (on the 11th, 13th, 17th day after tumor inoculation) were injected with a melanin preparation from a deep culture of the chaga Inonotus obliquus TV-18 at a dose of 0.4-0.2 mg per mouse into the tumor area in a volume of 0.1 ml. Mice in the control group were similarly injected with 100 μl of 0.9% NaCl. The total observation period was 27 days. Every 2-3 days before injection of the drug and then until the end of the experiment, linear dimensions of the tumor were measured in two mutually perpendicular directions. The antitumor effect of the drug was evaluated by the change in the volume of the tumor node and the percentage inhibition of tumor growth. Tumor volume was calculated by the formula: V (mm) = 0.5 * a * b * b, where a, b are length, width, mm, respectively. The percentage of inhibition of tumor growth was calculated by the formula: TPO (%) = (V _to -V _o ) / V _to ⋅100, where V _{to is the} average tumor volume in the control group; V _about - the average volume of the tumor of the experimental group. The results of the experiment showed (Fig. 2) that in the control group of animals there was a constant tumor growth, which reached the size of (1801.6 ± 384.1) mm ^{3 by} day 27. Four out of eight animals died at different times of observation. In the experimental group of animals, three animals out of 10 died in the first days of the experiment, the average tumor size by day 27 was (1092.0 ± 792.8) mm ³ . The inhibition effect was observed 2 days after the first injection of the drug and persisted until the end of the experiment (7 days after the end of administration), reaching a maximum TPO of 44% 7 days after the last injection of the drug. 10 days after the last injection of the drug, the effect of TPO decreased and amounted to 39%. It was shown that a triple, with an interval of 2-4 days, intratumoral administration of melanin at a dose of 0.2-0.4 mg / mouse in the tumor model of xenografts of human epidermoid carcinoma A-431 had an inhibitory effect on tumor development. The magnitude of the inhibition of tumor growth upon administration of the drug was 44%, the inhibition effect persisted for 10 days after administration (table 3).

The examples 1-6 confirm the achievement of the claimed technical result, which consists in obtaining a strain of the basidiomycete fungus Inonotus obliquus TV-18 (deposited under the number F-1375), a producer of melanin pigments, having more than the prototype (strain Inonotus obliquus F-1244) more high productivity in the production of fungal biomass containing melanin in the GTS environment and a higher yield of melanin pigments from the culture fluid, with both antiviral and antitumor effects ^.

Claims (1)

The strain of the basidiomycete Inonotus obliquus is the producer of the melanin pigment, which has antiviral and antitumor activity, deposited in the collection of bacteria, bacteriophages and fungi of the Federal State Biological Research Center of the World Bank of Russia Vector Rospotrebnadzor under the number F-1375.

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