JPH07227289A - Microorganism metabolite and antitumor agent comprising the same as active ingredient - Google Patents

Abstract

PURPOSE:To obtain a hydrophobic substance having antitumor activity, as a metabolite of a microorganism of Cordyceps ophioglossoides. CONSTITUTION:Cordyceps ophioglossoides (IFO8, 992) is used and its culture medium is extracted with an organic solvent to give a metabolite of a microorganism as a hydrophobic substance. The substance completely suppresses proliferation of mouse tumor cell YAC-1 in concentration of about 0.1-0.0125mug/ml and is recognized to have antitumor action.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a microbial metabolite and an antitumor agent containing the same as an active ingredient. More specifically, it relates to a microbial metabolite obtained by culturing a microorganism Koji Ceps ophiogrosides and an antitumor agent containing the same.

[0002]

2. Description of the Related Art Microorganisms of the genus Cordyceps contain many mushrooms that parasitize eukaryotic cells, and their metabolites are expected to contain substances that suppress the growth of eukaryotic cells. However, most of them are related to polysaccharides having an immunoreactive function.

As the compounds having a cytotoxic effect, cordycepin found in the culture medium of Kojicep militaryalis and galactosaminoglycan found in the culture medium of Kojicep Ophioglossoides are only known. No, but (J. Chem. Soc. 1951 No. 2299
Page, EP 067 000, Chem. Pharm. Bull. Volume 36 Volume 4505
(Page, 1988), all of these compounds are hydrophilic substances, and it cannot be said that they have strong in vitro cytotoxicity. So far, no hydrophobic substance having strong cytotoxicity has been found. .

[0004]

DISCLOSURE OF THE INVENTION An object of the present invention is to provide a hydrophobic substance which is a microbial metabolite of Cordyceps ophiogrosoides and has antitumor activity.

[0005]

The object of the present invention is as follows.
Microbial Koji Seps Ophioglossoides (IFO 8992)
It is achieved by a microbial metabolite consisting of an extract obtained by extracting the culture medium of 1. with an organic solvent.

[0006] The microorganism Koji Ceps ophioglossoides belongs to the same genus as Cordyceps sinensis, which has been used as an herbal medicine that has been important since ancient times. It is a kind. In the present invention, an extract obtained by extracting the culture medium with an organic solvent is obtained as a microbial metabolite.

Codyceptus ophiogrosides is cultivated by a usual fungal culture method. That is, a static culture method or a shaking culture method is used, and as the medium, any of synthetic medium, semi-synthetic medium and natural medium may be used as long as it contains a nutrient source that can be utilized by this strain. it can.

In the medium, for example, dextrose, maltose, sucrose, dextrin, glycerin, starch, potato decoction, sawdust, corn mill, etc. as carbon sources, malt extract, soybean powder, corn steve liquor as nitrogen sources. , Cottonseed flour, meat extract, yeast extract, peptone, potato decoction, casein hydrolyzate, dry yeast, rice bran, amino acids, ammonium salts, nitrates, etc., and if necessary, inorganic salts such as sodium, potassium and magnesium. , Calcium, zinc, iron and other sulfates, nitrates, carbonates, phosphates, chlorides and the like, respectively, when the medium is used as a solid medium, further agar, gelatin, silica gel, as a gelling agent, Gellan gum and the like are contained.

It is generally advantageous to carry out the culture under aerobic conditions. The culture temperature is about 20 to 30 ° C. and the pH is about 5 to 8.
The respective ranges are preferable, and the culture time varies depending on the composition of the medium, the culture temperature, etc., but is generally about 1 week to 1 month, during which the antitumor substance is produced and accumulated in the medium.

The antitumor substance produced and accumulated in the medium is collected from the medium by appropriately selecting and combining ordinary means for separating the hydrophobic component from the culture medium of the microorganism. It is as follows.

That is, when the antitumor substance is separated from the liquid medium, the mycelium is removed from the medium after culturing and then extracted with ethyl acetate. When the extract is concentrated to dryness, a metabolite having an antitumor effect is obtained as the residue. In the case of separating the antitumor substance from the solid medium, after crushing the solid medium, the solid medium is suspended in an organic solvent capable of dissolving the antitumor substance, for example, alcohol such as methanol or n-butanol, and at room temperature or under heating conditions. After removing the solid matter from the extract, the mixture is concentrated to dryness under reduced pressure at a temperature of 60 ° C or lower, and the water-soluble portion is removed from the residue to obtain a hydrophobic metabolite having an antitumor effect.

[0012]

[Advantages of the Invention] Koji Cepth Ophioglossoids (IF
O 8992) is used to extract this culture medium with an organic solvent to obtain a microbial metabolite as a hydrophobic substance. This substance completely suppressed the growth of mouse tumor cells YAC-1 at a concentration of about 0.1 to 0.0125 μg / ml, and was found to have an antitumor effect.

[0013]

EXAMPLES The present invention will now be described with reference to examples.

Example 1 One platinum loop of a tilted culture of Koji Ceps ophiogrosides (IFO 8992) was placed in a medium for seed culture in an Erlenmeyer flask with a capacity of 200 ml (39 g of Eiken Chemical's potato dextrose agar medium was distilled). After suspending in 1 L of water and separating the insoluble agar portion by filtration, 20 ml of the product (sterilized at 120 ° C. for 15 minutes) was inoculated in an autoclave and statically cultured at 25 ° C. for 20 days. 20 ml of this culture solution was used as a medium for main culture (Eiken Chemical Co., Ltd., potato dextrose agar medium 39 g, distilled water 1 L).
Suspended in water, sterilized in an autoclave at 120 ° C for 15 minutes, and then injected into a stainless steel lid vat with a length of 30 cm, a width of 22 cm, and a depth of 6 cm) to uniformly inoculate the surface. After that, it was cut into about 2 cm square dice with a sterilized knife. In order to improve the contact with air, each dice-shaped medium was randomly placed in a vat so as not to adhere to each other, and statically cultured at 25 ° C. for 21 days in the dark.

After the culture medium is heated and melted,
500 ml of n-butanol was added, and the mixture was stirred at 100 ° C for 3 hours.
After cooling to room temperature, the n-butanol layer separated from the solidified agar medium was concentrated to dryness. 100 ml of water is added to the obtained residue.
And 200 ml of ethyl acetate were added for liquid separation, and then the ethyl acetate layer was concentrated to dryness at a temperature of 50 ° C or lower to obtain microbial metabolites 13
7 mg was obtained as a brown candy.

Example 2 In Example 1, malt extract-containing agar medium was used as the main culture medium (malt extract 10 g of DIFCO product, BACTO-product of DIFCO product).
Peptone (1 g), glucose (40 g) and agar (20 g) were suspended in distilled water (1 L), sterilized in an autoclave at 120 ° C for 15 minutes, and then poured into a stainless steel lid vat with a length of 30 cm, a width of 22 cm, and a depth of 6 cm. (Prepared by
115 mg of microbial metabolite was obtained as a brown candy.

Example 3 One platinum loop of a tilted culture of Koji Ceps ophiogrosides (IFO 8992) was suspended in a seed culture medium (Eiken Chemical Co., Ltd. potato dextrose agar medium 39 g in distilled water 1 L) in an autoclave. After sterilization at 120 ° C. for 15 minutes, 10 ml of each was inoculated into a center of a petri dish having a diameter of 9 cm and a depth of 20 mm when heated, and the mixture was inoculated at 25 ° C. for 7 days. The circular colonies obtained in this way are
Divide into approximately 20 equal parts with a sterilized scalpel, and all of them are the main culture medium.
(A medium having the same composition as the medium for seed culture in Example 1) Inoculate into a 5 L Erlenmeyer flask containing 1 L, and
The cells were cultivated in the dark at static temperature for 2 weeks.

After completion of the culture, the mycelium was separated by filtration, the filtrate was diluted with water twice, and then extracted twice with 1 L of ethyl acetate. The ethyl acetate layer was washed with water and then concentrated to dryness at a temperature of 50 ° C. or lower to obtain 95 mg of a microbial metabolite as a brown candy-like substance.

Example 4 In Example 3, the medium for main culture (DIFCO product malt extract 10 g, DIFCO product BACTO-peptone 1 g and glucose 40 g)
Was dissolved in 1 L of distilled water and then sterilized at 120 ° C. for 15 minutes) In a 5 L Erlenmeyer flask containing 1 L, the round colony of Example 3 was divided into about 20 equal parts Then, the cells were shake-cultured at 25 ° C for 21 days.

After completion of the culture, 1 L of ethanol was added to the medium to separate insoluble matter by filtration, and the filtrate was concentrated to dryness at a temperature of 60 ° C or lower. After adding 200 ml of water to the residue, the mixture was extracted with 200 ml of ethyl acetate. The ethyl acetate layer was washed with water and then concentrated to dryness at a temperature of 50 ° C. or lower to obtain 95 mg of a microbial metabolite as a brown candy-like substance.

[Tumor cell growth inhibition test] Mouse tumor cell Y
RPMI 1640 medium containing ^{AC-1 10 6 cells / ml} (10% of the FC
100 μl of S, 100 U / ml penicillin and 100 μg / ml streptomycin was added to each well of a 96-well microplate, and the microbial metabolites (5% dimethylsulfoxide solution) obtained in each of the above Examples at various concentrations To the above R
(Diluted with PMI 1640 medium and adjusted to each prescribed concentration)
100 μl was added to each well and incubated at 37 ° C. in a 5% CO ₂ incubator for 72 hours. As a result of determining the presence or absence of proliferation of tumor cells by comparing the change in color of the medium with a control,
The following results were obtained as the concentration at which the growth of tumor cells was completely suppressed. Microbial metabolite concentration (μg / ml) Example 1 0.1 Example 2 0.05 Example 3 0.025 Example 4 0.0125

Claims (2)

[Claims] 1. A microbial metabolite consisting of an extract obtained by extracting a culture medium of microbial cordyceps ophioglosides (IFO 8992) with an organic solvent. 2. An antitumor agent comprising the microbial metabolite according to claim 1 as an active ingredient.