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WO2009075605A1 - Souche du champignon hypsizygus ultramarius (bull.) redhead s-ulm 1 utilisée pour fabriquer un produit possédant une activité antitumorale et une action antioxydante et procédé pour fabriquer un produit possédant une activité antitumorale et une action antioxydante sur la base de cette souche - Google Patents

Souche du champignon hypsizygus ultramarius (bull.) redhead s-ulm 1 utilisée pour fabriquer un produit possédant une activité antitumorale et une action antioxydante et procédé pour fabriquer un produit possédant une activité antitumorale et une action antioxydante sur la base de cette souche Download PDF

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Publication number
WO2009075605A1
WO2009075605A1 PCT/RU2008/000563 RU2008000563W WO2009075605A1 WO 2009075605 A1 WO2009075605 A1 WO 2009075605A1 RU 2008000563 W RU2008000563 W RU 2008000563W WO 2009075605 A1 WO2009075605 A1 WO 2009075605A1
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Prior art keywords
antitumor activity
strain
agent
submerged culture
producing
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Ceased
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PCT/RU2008/000563
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English (en)
Russian (ru)
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WO2009075605A8 (fr
Inventor
Larisa Mikhailovna Krasnopolskaya
Maria Ilyinichna Leontieva
Anastasia Vitalievna Avtonomova
Igor Vladimirovich Belitsky
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OTKRYTOE AKTSIONERNOE OBSCHESTVO ZAVOD EKOLOGICHESKOY TEKHNIKI I EKOPITANIYA 'DIOD'
Borovichsky Kombinat Ogneuporov OAO
Otkrytoe Aktsionernoe Obschestvo Zavod Ekologicheskoy Tekhniki I Ekopitaniya "Diod"
Original Assignee
OTKRYTOE AKTSIONERNOE OBSCHESTVO ZAVOD EKOLOGICHESKOY TEKHNIKI I EKOPITANIYA 'DIOD'
Borovichsky Kombinat Ogneuporov OAO
Otkrytoe Aktsionernoe Obschestvo Zavod Ekologicheskoy Tekhniki I Ekopitaniya "Diod"
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Publication of WO2009075605A1 publication Critical patent/WO2009075605A1/fr
Publication of WO2009075605A8 publication Critical patent/WO2009075605A8/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/06—Fungi, e.g. yeasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungal isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
    • C12P1/02—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using fungi
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi

Definitions

  • 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.
  • 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.
  • 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.
  • seed is prepared.
  • 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.
  • the mycelium 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).
  • 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.
  • 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.
  • 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).
  • Potato-glucose agar (KG A) composition a potato broth - 1 l (200 g of potatoes, diced l ⁇ l ⁇ l 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 :
  • BHD Chapek-Doks environment with yeast extract
  • the growth rate of the diameter of the mycelium is 10-15 mm per day at 25-28 0 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.
  • the Nursizugs ilaria S-ulml strain absorbs glucose well, sucrose and xylose are less good.
  • 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.
  • 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.
  • FIG. 1 depicts the fruiting body of Nursisugis ilaria S-ulml.
  • 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.
  • 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.
  • Example 2 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.
  • Example 4 Obtaining funds with antitumor activity and antioxidant action.
  • Example 5 Obtaining funds with antitumor activity and antioxidant action.
  • Example 2 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.
  • 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.
  • 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.
  • TPO Tumor growth inhibition
  • TPO tumor growth inhibition coefficient
  • Example 5 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.

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Abstract

L'invention concerne la médecine. Une souche du champignon Hypsizygus ultramarius (Bull.) Redhead S-ulm 1-01060 (collection de l'Institut de recherche de nouveaux antibiotiques KKM GU NII im. Gauze de l'Académie russe des sciences médicales) utilisée pour fabriquer un produit possédant une activité antitumorale et une action antioxydante. Le procédé de fabrication du produit comprend la culture du champignon Hypsizygus ultramarius (Bull.) Redhead S-ulm 1 en position immergée, accompagnée d'une aération sur un milieu de culture comprenant des sources de carbone, d'azote et des sels minéraux ainsi que l'extraction du produit.
PCT/RU2008/000563 2007-12-11 2008-08-20 Souche du champignon hypsizygus ultramarius (bull.) redhead s-ulm 1 utilisée pour fabriquer un produit possédant une activité antitumorale et une action antioxydante et procédé pour fabriquer un produit possédant une activité antitumorale et une action antioxydante sur la base de cette souche Ceased WO2009075605A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
RU2007145748 2007-12-11
RU2007145748/13A RU2007145748A (ru) 2007-12-11 2007-12-11 Штамм гриба hypsizygus ulmarius (bull.) redhead s-ulm1, используемый для получения средства, обладающего противоопухолевой активностью и антиоксидантным действием и способ получения средства, обладающего противоопухолевой активностью и антиоксидантным действием, на основе этого штамма

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WO2009075605A1 true WO2009075605A1 (fr) 2009-06-18
WO2009075605A8 WO2009075605A8 (fr) 2009-12-10

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PCT/RU2008/000563 Ceased WO2009075605A1 (fr) 2007-12-11 2008-08-20 Souche du champignon hypsizygus ultramarius (bull.) redhead s-ulm 1 utilisée pour fabriquer un produit possédant une activité antitumorale et une action antioxydante et procédé pour fabriquer un produit possédant une activité antitumorale et une action antioxydante sur la base de cette souche

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WO (1) WO2009075605A1 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2416418C1 (ru) * 2009-12-10 2011-04-20 Открытое Акционерное Общество Завод Экологической Техники И Экопитания "Диод" Способ получения средства, обладающего противоопухолевой активностью, и средство, полученное этим способом (варианты)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS57206618A (en) * 1981-06-12 1982-12-18 Noda Shiyokukin Kogyo Kk Preparation of antitumor substance
SU1291027A3 (ru) * 1981-02-10 1987-02-15 Куреха Кагаку Когио Кабусики Кайся (Фирма) Способ получени гликопротеина,обладающего противоопухолевой и лектиноподобной активностью
RU2082755C1 (ru) * 1990-12-04 1997-06-27 Ил-Янг Фармасьютикал Ко., Лтд. Штамм ganoderma lucidum - продуцент протеогликана gоо9, обладающий противоопухолевой и иммуностимулирующей активностью
RU2192873C1 (ru) * 2001-05-08 2002-11-20 Герасименя Валерий Павлович Препарат, влияющий на тканевой обмен, и применение штамма гриба pleurotus 1137 для его получения
US20060057157A1 (en) * 2004-08-25 2006-03-16 Gavish-Galilee Bio Applications Ltd. Mushroom extracts having anticancer activity

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SU1291027A3 (ru) * 1981-02-10 1987-02-15 Куреха Кагаку Когио Кабусики Кайся (Фирма) Способ получени гликопротеина,обладающего противоопухолевой и лектиноподобной активностью
JPS57206618A (en) * 1981-06-12 1982-12-18 Noda Shiyokukin Kogyo Kk Preparation of antitumor substance
RU2082755C1 (ru) * 1990-12-04 1997-06-27 Ил-Янг Фармасьютикал Ко., Лтд. Штамм ganoderma lucidum - продуцент протеогликана gоо9, обладающий противоопухолевой и иммуностимулирующей активностью
RU2192873C1 (ru) * 2001-05-08 2002-11-20 Герасименя Валерий Павлович Препарат, влияющий на тканевой обмен, и применение штамма гриба pleurotus 1137 для его получения
US20060057157A1 (en) * 2004-08-25 2006-03-16 Gavish-Galilee Bio Applications Ltd. Mushroom extracts having anticancer activity

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WO2009075605A8 (fr) 2009-12-10

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