WO2009075605A1 - Hypsizygus ultramarius (bull.) redhead s-ulm 1 fungus strain used for producing an agent exhibiting antitumor activity and anti-oxidant action and a method for producing an agent exhibiting antitumor activity and anti-oxidant action based on said strain - Google Patents

Abstract

The invention relates to biotechnology and medicine and can be used for producing an agent exhibiting an antitumor activity and an anti-oxidant action by cultivating a basidiomycete strain on a liquid nutritional medium in a submerged culture. The novel Hypsizygus ultramarius (Bull.) Redhead S-ulm 1 fungus strain exhibits a high growth rate in a submerged culture (3 days in comparison with known 7-14 days), an antitumoral activity and an anti-oxidant action. The strain is stored in the Collection of microorganism cultures of GU NII for investigating novel antibiotics named after G.F: Gauze RAMH under N 01060. The method for producing the agent exhibiting an antitumor activity and an anti-oxidant action involves cultivating the above-mentioned fungus strain in a submerged culture with an aeration on a nutritional medium which contains carbon and hydrogen sources and mineral salts, and separating said agent. The agent can be separated form the submerged culture which is obtained, after the strain cultivation, by heat treatment, for example by boiling or autoclaving at 1.2-1.8 atm for 1.5-3 hours, or from a dry mycelium released from the submerged culture by water extraction or ethanol. The antitumor activity of the produced agents expressed in the retard of tumor growth ranges from 43 to 86% and the anti-oxidant action expressed in the antioxidant capacity of the agent ranges from 31 to 524 mg of quercitine per 100 ml of agent.

Description

 The strain of fungus Nursizugis itaria (BuIl.) Redhead S-ulml used to obtain funds with antitumor activity and antioxidant action and a method of obtaining funds with antitumor activity and antioxidant action, based on this strain

The technical field The invention relates to biotechnology and medicine and can be used to obtain funds with antitumor activity and antioxidant effect by cultivating a strain of basidiomycete in a liquid nutrient medium in an immersed culture.

State of the art

Basidiomycetes are known that have antitumor and antioxidant properties: Leptipids, Lysiditis, Tatesisolor, Grifola, Schizorhulitis sottype, and others. On their basis, such as onoanole, Rozanomolanum, Anthynolum, Anthraeanum, and Anthynolum. The sensitivity of tumors, depending on their nature to these drugs is different. The recommended regimen for administering anticancer drugs from mushrooms is intravenous, because with other methods of administration, for example, when administered orally, the antitumor effect of the drugs can decrease sharply until it disappears. To obtain antitumor drugs and antioxidants, basidiomycetes are grown for 10-21 days in a submerged culture or 2-5 months to obtain fruiting bodies (Wasser, 2002, Smith et al., 2003). Therefore, the search for new species and strains of basid fungi, producers of biologically active metabolites, is relevant. The task is to detect species capable of rapid growth in culture and the synthesis of biologically active substances that act when administered orally.

Known strain Gapoderta lysidit, used to obtain funds with antitumor activity, and a method of obtaining funds - proteoglycan G009, which has antitumor activity. The specified strain is cultivated on a liquid nutrient medium in an immersed culture, followed by separation of the mycelium and the allocation of funds from the mycelium, possessing antitumor activity - proteoglycan G009. As a liquid nutrient medium, a medium containing: 50 g glucose, 20 g peptone, 0.87 g KH ₂ PO ₄ , 0.5 g MgSO ₄ , 10 mg FeCl ₂ x6H ₂ O, 7 mg MnCl ₂ x4H ₂ O, 10 mg ZnSO ₃ x5H ₂ O, 4 mg ZnCl ₂ per 1 liter of water, pH 5.5. Initially, seed is prepared. For this, the strain is cultivated with stirring 180 rpm at 25 ^° C for 10 days until pellets with a diameter of about 5 mm are obtained. The resulting culture is transferred to fresh medium in an amount of 5% and re-grown for 10 days. The resulting seed in an amount of 5% is seeded with fresh medium and cultivated at 180 rpm on a shaker for 7 days. The mycelium is separated from the culture fluid and proteoglycan is isolated from it - an agent with antitumor activity, it is purified and its antitumor activity against sarcoma 180 vaccinated in male mice is checked (RU 2082 755 CI, June 27, 1997). The disadvantage of the above strain is the long preparation of seed (20 days), prolonged fermentation (7 days) and the complex process of isolating an antitumor agent from mycelium. Known strain Nursiugrit itari 812, used to obtain funds with antitumor activity and a method of obtaining funds with antitumor activity from mycelium of the above strain. The strain Nursizugs iltarite is cultivated on a nutrient medium containing: 2% glucose, OD% peptone, OD% yeast extract, OD% KH ₂ PO ₄ , 0.1% MgSO ₄ , 10 ml / l saline (18 mM iron (II) sulfate , 3.7 mM manganese sulphate, 1.5 mM zinc sulphate, 0.8 mM copper sulphate). The duration of submerged cultivation is 2-3 weeks. After cultivation, the mycelium is separated and an antitumor agent is isolated from it by extraction with various reagents, such as 70% ethanol, a mixture containing 33% ethyl acetate and 50% methanol, a mixture containing 22% ethyl acetate and 11% methanol, or a mixture consisting of 33% chloroform and 50% methanol (US 200600057157, March 16, 2006, A61K 36/07).

The specified source does not describe the cultivation conditions of the strain (the exact duration of the process, pH level, aeration), and does not describe the extraction conditions (ratio of mycelium and extractant). Testing extracts of mycelium of the indicated fungus was carried out in ip vitro experiments. Of the extracts obtained, only mycelium extract obtained by extraction with a mixture containing 33% chloroform and 50% methanol inhibited the growth of K562 cell line - human myelogenous leukemia by 56.7% and could cause apoptosis of these cells. The antioxidant effect of the extracts has not been studied.

Disclosure of invention

The problem is solved by the strain of the fungus Nursizugis itaria (BuIl.) Redhead S-ulml - 01060 (KKM State Research Institute for the search for new antibiotics named after G.F. Gauze RAMS), used to obtain funds with antitumor activity and antioxidant effect.

The problem is also solved by the method of obtaining funds with antitumor activity and antioxidant action, including the cultivation of a basidiomycete strain in a submerged culture by aeration on a nutrient medium containing carbon, nitrogen and mineral salts, and the allocation of funds with antitumor activity and antioxidant an action in which of the basidiomycetes for cultivation using the above-described strain of the fungus Nursizugs itaria.

In this case, the allocation of funds with antitumor activity and antioxidant effect is carried out from the submerged culture obtained after culturing the strain by heat treatment: boiling or autoclaving at 1.2-1.8 atm. within 1.5 to 3 hours. After cultivation of the strain, the mycelium is separated from the obtained submerged culture, dried, and an agent having antitumor activity and antioxidant action is isolated from it. In addition, the allocation of funds with antitumor activity and antioxidant effect, carried out by extraction of dry mycelium with water or ethanol.

The strain Nursizugis ilaria S-ulml was obtained by isolating the primary culture from the fruiting body of the fungus Nursizugis itaria and the implementation of selection operations. Isolation of the primary pure mycelial culture was carried out from the fetal body of Nursisugs itaria. Mushroom slices cut from the middle fruit body, under sterile conditions, were transferred to a nutrient medium - potato-glucose agar. The grown mycelium was identified as a pure basidial culture by the presence of buckles on the mycelium. Mycelium Nursizugs itariiis, stored in a test tube, was seeded in a liquid medium and grown in submerged culture in flasks on a rotary shaker. The sawdust-grain substrate was seeded with the obtained submerged mycelium, on which under sterile conditions fruiting bodies of Nursisugs itaria were obtained. Bazidiospores obtained from mature fruit bodies by the spore imprint method were subjected to alternating cooling and heating, and then plated into a liquid medium. The Nursizugs ilaria colonies obtained in the submerged culture were planted on Petri dishes with a dense agar medium (potato glucose agar). S-ulml culture was selected according to kinetic parameters of growth (high growth rate), morphological characteristics (dense, well-developed mycelium), antitumor activity and antioxidant effect. The strain Nursizugis ilaria S-ulml is stored in the Culture Collection of microorganisms of the State Research Institute for the search for new antibiotics named after G.F. Gauze RAMS (acronym INA) under N ° 01060. The strain Nursizugis ilaria S-ulml belongs to the Basidiomycetes (detached Vasidiomusota).

Characterization of Mycelium Nursisugaria and Sari ularia on dense agarized media. 1. Potato-glucose agar (KG A) composition: a potato broth - 1 l (200 g of potatoes, diced lхlхl cm, filled with 1 l of tap water, boiled for 30 minutes, The broth is cooled, brought to a liter), glucose 20 g / l, agar 20 g / l. The colony of the strain on the KGA medium:

- cotton-felt,

- white

- the intensity of the development of aerial mycelium 2-3 points on a three-point scale, - the edge of the colony is flat, slightly elevated to the edge of the colony,

- there is no concentric zoning,

- there is no radial striation,

- the growth rate of the diameter of the mycelium 15-20 mm per day at 25-28 ⁰ C.

2. Chapek-Doks environment with yeast extract (BHD) composition: Water 1 l, potassium dihydrogen phosphate 1 g, magnesium sulfate 0.5 g, sodium nitrate 2 g, yeast extract 10 g, glucose 30 g, agar 20 g. The colony of the strain on the medium BHD:

- cotton

- white

- the intensity of the development of aerial mycelium 3 points on a 3-point scale,

- the edge of the colony is even, raised, fluffy to the edge of the colony,

- there is concentric zoning,

- there is no radial striation, - the growth rate of the diameter of the mycelium is 10-15 mm per day at 25-28 ⁰ C.

3. Wheat agar with corn extract (psh + ke) pH 6.2, composition: Broth of wheat grains 1 l (100 g of wheat, filled with 1 l of tap water, boiled for 1 hour. Broth cooled, brought to a liter), corn extract 10 ml / l, agar 20 g / l. The colony of the strain on the medium psh + ke:

- felt matted

- white, - the intensity of the development of aerial mycelium 2 points on a three-point scale,

- the edge of the colony is even,

- there is no concentric zoning,

- there is no radial striation, - the growth rate of the diameter of the mycelium 19-22 mm per day at 25-28 ° C.

Characteristics of the fruit bodies Nursizugis s-ulmlari grown on wheat grain: - The hat is flat, convex-prostrated over time, open, with a curled, wavy edge. The color of the hat is from whitish through beige to fawn or pale whitish-brownish (see Figure 1). records: descending, frequent, wide, broad-grown, white, whitish yellowish with age

- the leg is eccentrically attached, thick, uneven thickness (may be extended to the base), sometimes curved. - fruiting bodies solitary or in a group of 2-5 fruiting bodies.

- the size of the fruiting body is on average 4-25 cm in diameter of the cap.

Of the carbon sources, the Nursizugs ilaria S-ulml strain absorbs glucose well, sucrose and xylose are less good. Among nitrogen sources, the S-ulml strain prefers peptone and soy flour. The optimal pH range for the growth and development of the strain S-ulml is 4.5 - 6.5. AND

The strain is stored on a slanted agar medium KGA (potato glucose agar), diluted three times with the addition of wood shavings or sawdust of hardwood. Immersed cultivation is carried out in flasks with a volume of 0.5 and 0.75 l on a rotary shaker with a rotation speed of 220 rpm. at 25 ° -28 ° C. The submerged cultivation process includes the cultivation of liquid seed and the actual fermentation. Medium for immersed cultivation contains glucose - 20 g / l, peptone - 10 g / l, potassium dihydrogen phosphate - 2.5 g / l and magnesium sulfate - 0.25 g / l. The duration of the process of obtaining liquid seed is 3 days, fermentation is 3-4 days. The final pH of the culture fluid after fermentation is 4.5 to 5.5.

Brief Description of the Drawings

In FIG. 1 depicts the fruiting body of Nursisugis ilaria S-ulml.

The best example of carrying out the invention

The invention is illustrated by the following examples, which do not limit the scope of the claims. Example 1. Obtaining an immersed culture of the fungus Nursizügis ilaria S-ulml.

Submerged cultivation of the strain Nursizugis ilaria S-ulml was carried out in 500 and 750 ml rocking flasks. The process of submerged cultivation was carried out in two stages. The first stage was the cultivation of liquid seed. At the second stage of submerged cultivation, fermentation occurred in order to obtain vegetative biomass.

Liquid seed was grown for 3 days in 500 ml flasks with 150 ml of medium containing glucose - 20 g / l, peptone - 10 g / l, potassium dihydrogen phosphate - 2.5 g / l and magnesium sulfate - 0.25 g / l The culture was grown on a circular shaker (220 rpm) at a temperature of 26 ^° C. For inoculation of flasks with inoculum, a culture grown on jambs with potato-glucose agar was used, based on the value of VA cant on the flask. The second stage of submerged cultivation - fermentation in order to obtain vegetative biomass, was carried out on a circular shaker 220 rpm, in 750 ml flasks with 200 ml of medium. The cultivation temperature was 25 ⁰ C. Medium for immersed cultivation contained glucose - 20 g / l, peptone - 10 g / l, potassium dihydrogen phosphate - 2.5 g / l and magnesium sulfate '- 0.25 g / l. The maximum yield of air-dry biomass with a water content of 5% was 15.3 g / l for 4 days. Example 2. Obtaining funds with antitumor activity and antioxidant action.

An immersed culture was obtained as described in Example 1, but the fermentation process was carried out at a temperature of 28 ⁰ C. The immersed culture thus obtained was autoclaved at 1.5 atm. within 2 hours. After autoclaving, the liquid phase was separated from the solid one and a preparation having antitumor activity and antioxidant effect was obtained in the form of a light yellow transparent liquid.

Example 3. Obtaining funds with antitumor activity and antioxidant action.

The submerged culture obtained in Example 1 was filtered, separating the mycelium from the culture medium. The mycelium was washed with distilled water and dried to constant weight at a temperature not exceeding 45 ° C. A weighed portion of dried and ground material at a rate of 36 g / l was filled with distilled water. Extraction was carried out in an autoclave at a rate of 1.2 atm. 3 hours. After that, the extract was separated by filtration through filter paper to obtain an agent with antitumor activity and antioxidant effect, in the form of a clear, light yellow liquid. The finished extract was stored at t = - 2O ⁰ C.

Example 4. Obtaining funds with antitumor activity and antioxidant action.

Four volumes of 96% ethanol were added to one volume of the dry mycelium aqueous extract obtained in Example 3, and the precipitate was separated by centrifugation at 3000 rpm. for 20 minutes. Then, the precipitate was dissolved in a small amount of water, the solution was filtered, lyophilized and an agent with antitumor activity and antioxidant effect was obtained in the form of a light beige powder. Example 5. Obtaining funds with antitumor activity and antioxidant action.

The submerged culture obtained in Example 1 was filtered, separating the mycelium from the culture medium. The mycelium was washed with distilled water and dried to constant weight at a temperature not exceeding 45 "C ₅ prepared three sample dried and crushed mycelium and the rate of 60 g / l was poured with ethanol: first - 40%, second - 70% and third - 96% The obtained suspensions in the flasks were put on a rocking chair and kept at 220 rpm and a temperature of 25 ⁰ C. The extract was then filtered off from the mycelium and an antitumor agent and antioxidant agent was obtained in the form of a light yellow liquid.

Example 6. Evaluation of the antitumor activity of the funds obtained in examples 1-5.

The funds obtained were tested in vivo experiments on adult male hybrid mice (C57B1 / 6 x DBA / 2) Fi (hereinafter B6D2Fi) with transplantable solid T-cell lymphoma P388.

Mice were obtained from the nursery RAMS "Krukovo". After admission, the mice were quarantined for 21 days.

Lymphoma was maintained under syngeneic conditions in ascites form by serial intraperitoneal passages on mice of the DB A / 2 line. In the experiment, the tumor was inoculated under the skin of the right side with 10 ^b cells per day "0". The effect of the studied drugs was evaluated by oral administration. Treatment was started 48 hours after tumor inoculation. Each of the agents was administered using a probe intragastric daily 1 time per day for 10 days at 0.3 ml / mouse. The tool obtained in example 4 was dissolved in distilled water at the rate of 2 and 20 mg / kg / day. The agent obtained in example 1 was administered in starch paste at 0.3 ml / mouse / day at the rate of 50 mg / kg / day. The number of mice in the control and experimental groups was 8-10 animals. During the experiments, the general condition of the mice, changes in body weight, tumor grafting, and tumor growth dynamics were monitored. Tumor growth inhibition (TPO) was calculated by the formula: TPO (%) = (M _K - M _o ) / (M _k ) xl00, where M _K and M ₀ are the average calculated tumor mass in the control and experiment, respectively. Tumor mass was calculated according to the formula M (mg) = (a x b x c) / 2, where M is the calculated mass of the tumor, and a, b, c are the 3 largest mutually perpedicular diameters of the tumor node in mm. The significance of differences in the average values of the mass of the tumor was determined by t-student test. Differences at P <0.05 were taken as significant. The tumor growth inhibition coefficient (TPO) during treatment with the agents obtained in Examples 1-5 was 43-86%. The obtained data on antitumor activity are presented in table 1. When mice were injected with the biomass of Nursisugs ilaria strain S-ulml obtained in example 1, TPO was 43%. The funds obtained in examples 2-5 had a higher antitumor activity. TPO with the introduction of funds obtained in example 2 was 60%. When mice were injected with an antitumor activity and antioxidant effect obtained in Example 3, the tumor growth inhibition rate was 63-80%. When treating animals with an antitumor activity and antioxidant effect obtained in Example 4, it was shown that at a dose of 2 mg / kg it showed a higher antitumor effect compared to the aqueous extract (72-86%). Comparison of the antitumor effect of the agent with antitumor activity and antioxidant effect obtained in Example 4 at a dose of 2 mg / kg and 20 mg / kg showed that a dose of 2 mg / kg is a much more effective oral dose. Funds received by Example 5, had an average activity with a TPO coefficient of 52-58%.

Example 7. Evaluation of the antioxidant effect of the funds obtained in examples 2-5. The total content of antioxidants in the agent with antitumor activity and antioxidant effect obtained in Examples 2-5 was determined by coulometric method using electro-generated bromine on the analyzer “Expert-006” NPK Ekoniks-Expert. The instrument was calibrated with a standard quercetin solution. Total antioxidant capacity (AOE) was expressed in mg of quercetin per 100 ml of product. When using the funds obtained in example 4, it was previously dissolved in a ratio of 2 mg in 10 ml of distilled water.

The antioxidant effect of the funds obtained in examples 2-5 are presented in table 2.

AOE funds obtained in example 2, amounted to 35 mg of quercetin per 100 ml of funds. The AOE of the agent obtained in Example 3 was 31 mg of quercetin per 100 ml of agent. The AOE of the agent obtained in Example 4 was 42 mg of quercetin per 100 ml of agent. AOE funds obtained in example 5, for 40% ethanol - 114 mg of quercetin per 100 ml of the drug, 70% - 440 mg of quercetin per 100 ml of the drug, 96% - 524 mg of quercetin per 100 ml of the drug.

The results showed that the AOE in the products obtained in Example 5 is several times higher than in other products.

Thus, the strain of the fungus Nursizugis ilaria (BuIl.) Redhead. S-ulml has a higher growth rate and not only high antitumor activity, but also an antioxidant effect.

Table 1.

Antitumor activity of funds obtained from the submerged culture of the strain Nursizugis ilaria S-ulml.

* Среднее значение расчетной массы опухоли ± стандартная ошибка.

* The average value of the estimated tumor mass ± standard error.

** The difference from the control was significant at P <0.05.

Table 2.

Antioxidant capacity of funds obtained from the submerged culture of the strain Nursizugs ilaria S-ulml.

Промышленная применимость

Industrial applicability

The present invention allows to obtain a new strain of fungus Nursizugis ilaria S-ulml, which has a higher growth rate in a submerged culture (3 days compared to 7-14 days). In addition, the resulting product has not only antitumor activity, but also an antioxidant effect.

The tool can be obtained in the form of a clear liquid of light yellow color or in the form of a light beige powder. On the basis of the agent, preparations in the form of tablets, capsules, tinctures with the addition of fillers, vitamins and other agents that increase the biological activity of the agent can be obtained.

Claims

CLAIM 1. The strain of fungus Nursizugis itaria (BuIl.) Redhead S-ulml - 01060 (KKM Research Institute for the search for new antibiotics named after G.F. Gauze PAMH) ₅ used to obtain funds with antitumor activity and antioxidant effect. 2. A method of obtaining funds with antitumor activity and antioxidant action, comprising cultivating a basidiomycete strain in a submerged culture by aeration on a nutrient medium containing carbon, nitrogen and mineral salts, and isolating an agent having antitumor activity and antioxidant effect, characterized in that from basidiomycetes for cultivation use the strain according to claim 1. 3. The method according to p. 2, characterized in that the allocation of funds with antitumor activity and antioxidant action is carried out from a submerged culture obtained after cultivation of the strain, by heat treatment: boiling or autoclaving at 1.2-1.8 atm. within 1.5 to 3 hours. 4. The method according to p. 2, characterized in that after culturing the strain from the obtained submerged culture, mycelium is separated, dried, and an agent having antitumor activity and antioxidant action is isolated from it. 5. The method according to p. 4, characterized in that the allocation of funds with antitumor activity and antioxidant effect, carried out by extraction of dry mycelium with water or ethanol.