JPH07227294A - Production of pseurotin a - Google Patents

Abstract

PURPOSE:To produce pseurotin A having antagonism on apomorphine, etc., by culturing Cordyceps ophioglossoides (IFO-8992) of a fungus, forming and accumulating pseurotin A and collecting the product. CONSTITUTION:One platinum loop of Cordyceps ophioglossoides (IFO-8992) of a fungus is inoculated into a medium and subjected to static culture at 25 deg.C for 20 days. The culture solution is uniformly inoculated into the surface of a potato dextrose agar medium and subjected to static culture at 25 deg.C for 20 days at a dark place. The cultured medium is heated, melted, mixed with n- butanol, stirred at 100 deg.C for 3 hours and cooled to room temperature. An n- butanol layer separated from the solidified agar medium is concentrated and the prepared residue is purified by silica gel chromatography to give the objective pseurotin A of the formula, having antagonism on apomorphine and incitory action on monoamine oxidase.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing shurotin A. More specifically, it relates to a method for culturing microorganisms to produce shurotin A.

[0002]

2. Description of the Related Art Pseurotin A is a compound having a unique structure containing a heterospiro ring,
2- [1 '(S), 2' (S) -dihydroxyhex-3'-n-yl]-
3-Methyl-8 (S) -methoxy-8-benzoyl-9 (R) -hydroxy- (5S) -1-oxa-7-azaspiro [4,4] non-2-ene-4,6-
Zeon

[Chemical 1] Is. This compound was isolated for the first time from the culture fluid of Pseudorotium ovalisstork (Helv. Chim.
Acta Vol. 59, No. 1, page 133, 1976, Vol. 64, No. 1, No.
(P. 304, 1981), and then isolated as a cultured metabolite of Pseudoles celia vage and Aspergillus fumigatus (Proc. Japan Assoc. Mycotoxyco.
l. Volume 22, p. 33, 1985, EP 546 475).

Various studies have been conducted on biological activity, and apomorphine antagonism and inhibition of monoamine oxidase are known (see Proc. Japan Assoc. Mycotoxyc above).
ol. And EP), and effective use of it is expected. However, its production method is unsatisfactory, and for example, from the culture medium of Pseudorotium ovalisstork, shurotin A was obtained in a yield of 30 to 35 mg per liter of the medium, and from the culture medium of Aspergillus fumigatus, the medium of 1 L was used. Only a mixture of Schurotin A and Schurotin D is obtained with a yield of 10 mg per.

[0004]

SUMMARY OF THE INVENTION An object of the present invention is to provide a method having a high production capacity when culturing a microorganism to produce shurotin A.

[0005]

The object of the present invention is as follows.
Microbial Koji Seps Ophioglossoides (IFO 8992)
It is achieved by a method of culturing cultivated sucrose, producing and accumulating shurotin A, and collecting it to produce shurotin A.

[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. However, there are few studies on its biologically active metabolites, and as culture metabolites, ophiocordine having antifungal activity and galactosaminoglycan having antitumor activity are known (Arch. Microbiol. 113). First
21, p. 1977, Agric. Biol. Chem. Vol. 51, No. 10, 280
5 pages, 1987).

[0007] In the method of the present invention, the microorganism Koji Ceps ophiogrosides is used for culturing.
The culture thereof is carried out by a usual fungal culture method. That is, a stationary 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. You 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 culturing time varies depending on the composition of the medium, the culturing temperature, etc., but is generally about 1 week to 1 month, during which shurotin A is produced and accumulated in the medium.

Collection of shurotin A produced and accumulated in the medium from the medium is carried out by appropriately selecting and combining ordinary means for separating and purifying metabolites from the culture medium of the microorganism, and the outline thereof is as follows. Is like.

That is, in the case of separating shurotin A from the liquid medium, the mycelium is removed from the medium after culturing and then extracted with ethyl acetate. After the extract is concentrated to dryness, the obtained residue is purified by column chromatography and further by HPLC. In the case of separating shurotin A from the solid medium, after crushing the solid medium, the solid medium is suspended in an organic solvent capable of dissolving shurotin A, for example, alcohol such as methanol or n-butanol, and extracted under room temperature or heating conditions. After removing solids from the extract, the mixture is concentrated to dryness under reduced pressure at a temperature of 60 ° C or lower, and the residue is purified by column chromatography and HPLC.

[0012]

EFFECT OF THE INVENTION By culturing the microorganism Koji Cepth Ophioglossoides (IFO 8992), shurotin A
Can be produced with high production capacity.

[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 it was sterilized in an autoclave at 120 ° C. for 15 minutes) and inoculated to 20 ml, followed by stationary culture at 25 ° C. for 20 days. 20 ml of this culture broth was suspended in 1 L of distilled water (39 g of Eiken Chemicals potato dextrose agar medium) for main culture, and sterilized in an autoclave at 120 ° C for 15 minutes, and then 30 cm in length, 22 cm in width, (Prepared by pouring into a 6 cm-deep stainless steel lid vat) was uniformly inoculated on the surface, and then cut into about 2 cm square dice with a sterilized knife. In order to improve the contact between each dice-shaped medium and air, they were 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.

The medium after culturing 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. The obtained residue was subjected to silica gel column chromatography (Wako Gel C-300 30 g,
The eluent: benzene / ethyl acetate = 1/1) was used for purification to obtain 60 mg of shurotin A as a pale yellow crude powder. This was further purified by HPLC (column inert sylprep ODS, eluent: methanol / water = 55/45) to obtain 45 mg of shurotin A as colorless crystals. The melting point, ¹ H NMR spectrum, ¹³ C NMR spectrum, and IR spectrum of this crystal were in good agreement with the data described in the above literature, and were identified as shurotin A.

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).
Using 1 g of peptone, 40 g of glucose and 20 g of agar), 37 mg of shurotin A was obtained.

EXAMPLE 3 One platinum loop of a tilted culture of Koji Ceps ophiogrosides (IFO 8992) was suspended in a seed culture medium (Eiken Chemicals potato dextrose agar 39 g in distilled water 1 L).
After sterilization at 120 ° C for 15 minutes in an autoclave, 10 ml of each was inoculated into the center of a petri dish having a diameter of 9 cm and a depth of 20 mm when heated, and inoculated into a center of one plate, and statically cultured at 25 ° C for 7 days .
The circular colonies thus obtained were divided into 20 equal parts with a sterilized scalpel, and all of them were contained in a volume of 5 L containing 1 L of a main culture medium (medium having the same composition as the seed culture medium of Example 1). The cells were inoculated into an Erlenmeyer flask and cultivated in the dark at 25 ° C for 2 weeks.

After completion of the culture, the mycelium was removed 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. The residue was purified by silica gel column chromatography (Wakogel C-300 30g, eluent: benzene / ethyl acetate = 1/1) and further HPLC (column inert silt prep ODS, eluent: methanol / water = 55/45). Purified with
52 mg of Schurotin A was obtained as colorless crystals.

Example 4 In Example 3, a malt extract-containing culture medium (DIFCO product malt extract 10 g, DIFCO product BACTO- was used as a main culture medium.
Dissolve 1 g of peptone and 40 g of glucose in 1 L of distilled water, then
Sterilized at 20 ° C. for 15 minutes) was used to obtain 31 mg of shurotin A.

Example 5 A 5 L Erlenmeyer flask containing 1 L of the main culture medium of Example 4 was inoculated with 20 equally divided circular colonies of Example 3 as an inoculum and shaken at 25 ° C. for 21 days. Cultured. 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 concentrated to dryness. The resulting residue is
Silica gel column chromatography (Wako gel C-30
0 35 g, eluent: ethyl acetate / benzene = 1/1),
Furthermore, preparative thin-layer chromatography (Merck Company Art.
13895, developing solvent: ethyl acetate / benzene = 3/2) to obtain 57 mg of shurotin A as colorless powder.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Office reference number FI technical display location (C12P 17/18 C12R 1: 645)

Claims (1)

[Claims] 1. A method for producing shurotin A, which comprises culturing a microorganism Koji sepus Ophioglossoides (IFO 8992), producing and accumulating shurotin A, and collecting the shurotin A.