JPS5951998B2 - New physiologically active substance betenone B and its production method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel physiologically active substance bethenone B and a method for producing the same.

For the purpose of searching for new physiologically active substances, the present inventors cultivated microorganisms belonging to the Phomabetae family in an appropriate medium, and found that EA-B-DG, a substance that exhibits plant seed growth inhibitory activity, -14 could accumulate in the culture medium, isolated this physiologically active substance, recognized that it is a new physiologically active substance based on its physicochemical and biological properties, and recognized this physiologically active substance. Physiologically active substance Betaenone B (BetaenOneB)
Hereinafter, it will be referred to as the physiologically active substance bethenone B.

That is, the present invention provides (1) a physiologically active substance bethenone B;
(2) Bethenone B, a physiologically active substance belonging to Homa Bethe
The producing bacteria are cultured in a medium, and physiological activities are detected in the culture. This is a method for producing the physiologically active substance bethenone B, which is characterized by producing and accumulating the physiologically active substance bethenone B, and collecting the same.

Note that hereinafter, the physiologically active substance bethenone B may be simply referred to as "bethenone B."

In producing the physiologically active substance bethenone B of the present invention, the physiologically active substance bethenone B belonging to Homa Bethe
Production bacteria are used.

As an example, Homa Bete Blank PS-13 (Ph
As OmabetaeFrankPS-13), Institute of Industrial Science and Technology Microbiology. Microorganisms deposited with the Institute of Industrial Science and Technology are used (Feikoken Bacterial Submission No. 6556). It goes without saying that the bacteria producing betenone B, a physiologically active substance belonging to the Homa bethe group used in this invention, can be used by increasing its production capacity by, for example, being irradiated with ultraviolet rays or X-rays, or treated with a mutagenic agent. However, the strains that can be used in the present invention include all strains that belong to Homa bethe and produce the physiologically active substance betenone B.

In the method for producing a physiologically active substance of the present invention, the strain is cultured in a medium containing available nutrients.

The medium composition includes, for example, a decoction of yam and potatoes, starch, glycerin, dextrin, carbon sources such as sucrose, maltose, and glucose, peptone, meat-processing scratches, yeast-processing scratches, casein hydrolyzate, corn, stew, etc. Nitrogen sources such as alcohol, gluten meal, corn meal, soybean flour, wheat germ, cottonseed flour, ammonium sulfate, ammonium phosphate, and urea are used. In addition, calcium carbonate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium chloride,
Inorganic salts such as sodium chloride are added. The culture temperature is 2
A temperature of 0 to 30°C is suitable. The seed culture may be a solid culture or a liquid culture. In the case of main culture, static culture, agitation culture, shaking culture, aerated culture, etc. may be performed. The culture medium is cultured for approximately 3 to 15 days with a medium concentration of 4 to 8. During the culture, the amount of the target substance produced is measured to confirm that a substantial amount has been produced in the medium, and then the culture is continued. end. Since the produced Bethenone B mainly exists in the culture liquid, it is purified and collected from the supernatant liquid after removing bacterial cells by centrifugation or filtration.

In order to purify and collect Bethenone B, the means normally used for collecting metabolites of microorganisms can be appropriately used. For example, separation and purification of the target substance from the liquid may be carried out singly, in combination in any order, or repeatedly using methods such as vacuum concentration, freeze drying, solvent extraction, resin treatment, adsorbent treatment, crystallization, and recrystallization.・Collect data. To give a more specific example, after completion of the culture, the culture solution is filtered using a filter aid to remove bacterial cells.

The resulting filtrate is concentrated under reduced pressure, and the concentrated solution is adjusted to IIfI of about 5.5 to 6.5 with glacial acetic acid, and then extracted with an organic solvent capable of dissolving bethenone B, such as ethyl acetate. The extract (ethyl acetate layer) obtained here is concentrated under reduced pressure and subjected to chromatography using a suction agent such as silica gel. When silica gel is used as an adsorbent, a mixed solvent system of chloroform and methanol is used as the elution solvent for development and elution. The eluate is appropriately fractionated, fractions containing the target substance are collected, and the fractions are concentrated to dryness under reduced pressure at low temperature to obtain a crude substance. In order to further purify the obtained crude material, it is again subjected to silica gel chromatography to remove impurities. The physicochemical properties of Bethenone B obtained in Example 1 described below are as follows. (1) Shape: White powder (2) Specific rotation: [α] LO = 0° (C = 1.0, ethanol) (3) Molecular formula: C2lH36O5 This molecular formula is derived from the acetylated product of bethenone B as described below. Estimated (4) Ultraviolet absorption spectrum: Bikoshi11^? VV- - \-v-● v
(5) Infrared absorption spectrum (main peak): (6)
Thin layer talomatography (using silica gel (manufactured by Melta)) Solvent system Rf value taloloformrimethanol (9:1, VN) 0.37 (7)
Solubility: Soluble; Insoluble in methanol, ethanol, taloloform, ethyl acetate; N-hexane, water (8) Color reaction: Obtains blue-blue green color with anisaldehyde reagent on silica gel thin layer chromatography.

(9) Nuclear magnetic resonance spectrum: Furthermore, the acetylated product of bethenone B will be described.

That is, when Bethenone B is acetylated with acetic anhydride in pyridine by a conventional method, Bethenone B acetate is obtained as crystals (melting point: 103.5 to 108.0°C).
The results of elemental analysis of Bethenone B acetate are as follows: FOundC: 67.25%;
H9.42% Caled C: 67.29%, H: 9.61% Molecular formula is C23H38O6, therefore betenone B
The molecular formula of C2lH36O. It was estimated that

Next, the biological properties of bethenone B will be described. In other words, as a physiological activity test, a filter paper was placed in a shear dish,
Dissolve 1 mg of the sample in ethyl acetate, apply it uniformly to Fuchi paper, air dry, and then dry in a suction desiccator for 5 hours.

Add 4ml of distilled water to this to make a 250p aqueous solution,
Sow 22 lettuce seeds on paper, put them in an airtight container,
After standing in a dark room for 72 hours, the radicle length and hypocotyl length were measured, and the seed growth inhibition rate was calculated using the following formula. As a result, it was found that Bethenone B exhibited a radicle growth inhibition rate of 20% and a hypocotyl growth inhibition rate of 3%.

These properties have also been observed in aphideicorin, a specific inhibitor of DNA polymerase α produced by the same strain of Homa bethe, so it is presumed that bethenone B also has other physiological activities, and it is believed that bethenone B also has other physiological activities. It is expected to be used as a medicine. The physiologically active substance bethenone B was detected and quantified using silica gel thin layer chromatography (silica gel 60F manufactured by Talc).
.

. . Thickness: 0.2 mm, developing solvent: chloroformrimethanol = 9:1, V/V) color former: 2,4-DNP reagent, heating). Next, examples of the present invention will be shown. Example 1 Phomabetae Frank PS-13 strain (Phomabetae FrankPS-13; Microtechnical Research Institute No. 6556) was used as a microorganism, inoculated into the following seed medium, and precultured at 25°C for 10 days. I did it.

Seed medium: Cut 200 g of peeled yams into 1 cm cubes, add about 11 g of tap water, and autoclave (1 kg/
The decoction was filtered through four pieces of gauze to obtain 11.

Add 20 g of sugar and 20 g of powdered agar to this, and heat in an autoclave for an additional 5 minutes to dissolve. Next, the pre-cultured inoculum was added at 15 ml per 500 ml flask.
Dispense 0ml into sterilized main culture medium using a platinum loop,
Static culture was performed at 25°C for 15 days.

Claims (1)

[Claims] 1. Physiologically active substance bethenone B (1) having the following properties:
Shape: White powder (2) Specific rotation: [α]_D^2^0=
0° (c=1.0, ethanol) (3) Molecular formula: Estimated molecular formula: C_2_1H_3_6O_5 (4) Ultraviolet absorption spectrum: ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (5) Infrared absorption spectrum (main peak): ▲Math,
There are chemical formulas, tables, etc. ▼▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (6) Thin film chromatography [using silica gel (manufactured by Merck & Co.)]: Solvent system Rf value Chloroform: Methanol (9:1 V/V) 0.37 (
7) Solubility: Soluble; Insoluble in methanol, ethanol, chloroform, ethyl acetate; N-hexane, water (8) Color reaction: On silica gel thin layer chromatography, exhibits blue-blue green color with anisaldehyde reagent (9) Nuclei Magnetic resonance spectrum: ▲ Contains mathematical formulas, chemical formulas, tables, etc. ▼ 0.669 (3H, d, J = 6.35 Hz), 0.863 (3H, t, J = 7Hz) 1.106 (1H, s) , 1.151 (1H, d, J = 6.84 Hz), 1.261 (3H, s), 1.319 (1H, t, J = 13.19 Hz), 1.404 (3H, s) , 1.4-1.5 (2H, m), 1.554 (1H, s), 1.567 (3H, s), 1.605 (1H, s), 1.837 (2H, m), 2.307 (1H, dt, J=14Hz, 3Hz), 2.493 (1H, t), 2.66-2.69 (2H, m), 2.817 (1H, dt), 3.110 ( 1H, dq), 3.658 (1H, s), 3.76-3.85 (1H, m), 3.87-3.97 (1H, m), 2 Belongs to Phoma betae Cultivating bacteria producing the physiologically active substance bethenone B in a nutrient medium,
1. A method for producing the physiologically active substance bethenone B, which comprises producing and accumulating the physiologically active substance bethenone B in a culture and collecting the same.