JPS62123194A - Novel substance no.8345-c having plant growth retarding and antibiotic activity - Google Patents

Abstract

NEW MATERIAL:An antibiotic No.8345-C. External appearance: white needle crystal; elementary analysis (%): C, 63.0; H, 7.5; N, 4.1. molecular weight, 323. molecular formula; C17H25NO5. specific rotatory power: [alpha]D<24>+17.3 deg. (C 0.075, methanol) melting point; 212-214 deg.C. solubility; soluble in water. color reaction; positive to potassium permanganate and negative to ninhydrin. USE:An antimicrobial agent, agricultural chemical, etc., having antimicrobial activity and plant growth retarding activity. PREPARATION:Perenisporia medullaepanis AJ8345 (FERM-P No.8181) strain is cultivated, preferably in a liquid medium at 6.0-8.0pH and 20-35 deg.C under an aerobic condition by a spinner culture method with aeration.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a novel substance &8'345-C. This substance has plant growth inhibitory activity and antibiotic activity,
Used in the agricultural and pharmaceutical fields.

(Prior Art) Conventionally known substances having both antibiotic action and plant growth inhibitory action include cycloheximide (Japanese Patent Publication No. 45-22754) and anisomycin.

Toyocamycin (Agric, Biol, Chem
, 36.2013 (1970)), Herbimycin A, 13 (J, Antibiot,.

29, 863 (1976)), Herbimycin (J. Antibiot.

32, 255 (1979)) Cytovaricin (No. 5
Abstracts of the 5th International Pesticide Conference, I [b-3, 1982), Bialajos (Summary of the 5th International Pesticide Conference, ■a-19
, 1982), among which bialaphos has been put into practical use as a herbicide.

In addition, antibiotics produced by basidiomycetes include polyacetylene compounds, terpenoid compounds, aromatic compounds, and nucleic acid analogs (Fermentation and Industry + Vol-34 + A11P
843) and other non-protein amino acids are known (Chemistry and Biology 14, 205 (1976)), but there are no reports on the plant growth inhibitory effects of these substances.

(Problems to be Solved by the Present Invention) The problems to be solved by the present invention are to provide a novel and useful substance that has both plant growth inhibiting action and antibiotic action.

(Means for Solving the Problems) In order to solve the above-mentioned problems, the present inventors cultivated a large number of basidiomycetes for the purpose of searching for a novel substance that has both plant growth inhibitory action and antibiotic action. As a result of U'e search,
Bacillus subtilis was cultured in a methanolic extract of mycelia of a strain belonging to the genus Pereniporia in Davis's minimal medium.
A3345-C, a substance that has the growth inhibitory effect of AJ1316 (AJ1316) and at the same time inhibits plant growth.
was found to be produced. Then, as a result of pure isolation of this effective substance from the total culture solution, investigation of its physical and chemical properties, and comparative study with known substances, it was found that 8345-C
discovered that it differs from known substances in its physical and chemical properties.

The present invention has been made based on these findings.

Note that the present inventors previously discovered Pereniporium
An application has been filed for new antibiotics A8345-A and B produced by S. sdullaepanls AJ8345 strain (Japanese Patent Application No. 125498-1983).

Patent application No. 163657/1983).

48345-C of the present invention is derived from Pereniporia (Pereniporia).
It is produced by basidiomycetes belonging to the genus I-poria). An example of this is Pereniporta m@dullae.
panis) AJ8345°FERM-P8181
(hereinafter referred to as AJ8345 bacterium) is the 483 bacterium of the present invention.
45-Ck has advantageous production properties.

In order to obtain the whole A 8345-C of the present invention, A 8345-C producing bacteria belonging to the genus Illeniporia can be cultured by a conventional method for producing this substance. For industrially advantageous production, A 8345-C producing bacteria can be cultured under aerobic conditions with aeration in a medium containing various nutrients.

The culture conditions and the composition of the medium may be those that allow general basidiomycetes to grow. In other words, the medium is basically a carbon source,
A nitrogen source, an inorganic salt, and if necessary, vitamins, precursors, etc. may be added.

Examples of carbon sources include glucose, arabinose,
Xylose, starch, dextrin, glycerin, mannitol, organic acids, molasses, potato, etc. are used alone or in mixtures, and nitrogen sources include, for example, peptone, soybean flour, corn stizo liquor, malt extract, amino acids. , rice bran, malt, urea, ammonium salt, etc., or a mixture thereof. Further, if necessary, an antifoaming agent such as silicone oil, soybean oil, or surfactant may be added.

The medium is preferably a liquid medium, and the ... of the medium is about 6.0 to about 8.
．． The culture temperature is preferably adjusted to about 20 to about 35°C.

After completion of the culture, A 8345-C may be separated and collected from the culture in accordance with a conventional method for separating and collecting fermentation products from the culture. That is, Wash 8345-C is collected by appropriately combining extraction methods using various organic solvents, chromatography using various active adsorbents, and the like.

Next, a manufacturing example of A 8345-C will be shown as an example, but this example is not intended to limit the scope of the present invention in any way.

Example I Production and structural analysis of A 8345-C AJ8345 bacteria grown on potato dextrose agar slopes were treated with 1% soluble starch, 2% glucose, and 0 ammonium sulfate.
．． 5%, potassium phosphate 0.05%, magnesium sulfate 0.05%, sodium chloride 0.05%, potato extract 70 El/l, trace metals 1 m, l/l fermentation medium ( 100 to 500d Yosakaro flask
The cells were inoculated into a microorganism (dispensed in mJ aliquots) and cultured at 27° C. for 20 to 30 days with shaking or static culture.

Medium trace metal CuSO4'5H200,64,9 FaSO4'7H200,11g MnC12'4H200,79g ZnSO4'7H200,151 Distilled water 100ml From the obtained culture 061, collect bacterial cells by filtration,
1 was extracted twice with methanol, and the methanol solution was concentrated under reduced pressure to dryness, and then dissolved in a small amount of methanol. Next, it was purified by silica dull column chromatography (Roper column Si 60, size B, manufactured by Merck & Co.). Methanol-ethyl acetate (5:95) was used as the developing solvent. The active fractions were collected, concentrated to dryness under reduced pressure, dissolved in a small amount of methanol, and purified by Dal chromatography using 5ephadex LH-20k (column volume: 30M).

Methanol was used for development. The active fraction was collected and concentrated under reduced pressure, and then the active component was purified and isolated by HPLC using a semi-preparative μBondapak Cl8 (registered trademark 1 manufactured by Nippon Waters) column. The mobile phase contains 3
A 0% CH,CN aqueous solution was used. At a retention time of approximately 40 minutes, A 8345-C was collected. By the final method, approximately 5 mq of A 8345-C was finally obtained from the culture solution of 61.
isolated.

The antibiotic A 8345-C obtained in this way is
It has the physical and chemical properties described below.

(1) Appearance: White needle-like crystals (2) Elemental analysis values: Carbon 63.0%, Hydrogen 7.5%, Nitrogen 4.1% (weight ratio) (3) Molecular composition: High resolution EI-MS m/ z: 32
3.17327 (M+) (4) Molecular formula: C47H25NO5 (5) Specific optical rotation: [α], +17.3° (C0.
075, methanol) (6) Melting point: 212-214°C (Solubility: Soluble in methanol and ethyl acetate.

Dissolves slightly when washed with water.

(8) Ultraviolet absorption spectrum (Uv), Me0Hnm
=211 (9) Infrared absorption spectrum: The spectrum measured with potassium bromide tablets is as shown in FIG.

QO) Hydrogen nuclear magnetic resonance spectrum: The coverage measured in heavy methanol is as shown in Figure 2.

Uυ carbon nuclear magnetic resonance spectrum: The spectrum measured in heavy methanol is as shown in Figure 3.

α power Color reaction: Potassium permanganate positive, ninhydrin negative High resolution EI-MS m/z: 323.1732
7, the molecular formula C47H25NO5 was derived. Furthermore, ultraviolet absorption spectrum (UVλ2 211 nm), infrared absorption spectrum (IRV, '::rc1730, 1
720, 1680 cm-'), the number of α, β-unsaturated r-lactones in the molecule is 22 (
Table 1). Therefore, the number of remaining exchangeable protons is three. these are.

It is thought that it is bonded to an oxygen atom or a nitrogen atom -oc
The existence of cH3 (δc173.4) is known, and by adding the ninhydrin negativity and the partial structure of 1C-NH= derived from the above infrared absorption spectrum data, the three exchangeable protons are It is thought that one bond is bonded to the nitrogen atom and the remaining two bond to the oxygen atom as -OH.

On the other hand, the number of unsaturations calculated from the molecular formula is 5, and the double bond is 1.
Since it contains one C=O, it is a bicyclic compound. Furthermore, 'H,'C-NMR spectrum-CI-1
In addition to containing 2-CH2-CH2-, this compound was estimated to be a sesquiterpene compound having the skeleton e of drimane fl. A compound having the above physical and chemical properties is a new substance if there are no other applicable substances.

Table A 13C-NMR spectrum data of 8345-C Chemical shift 13Cpeak (ppm) K Group'HA
19.6 -CH2-g, IB
21.4-CH3" (12) 1,7-CH5f D 23.6-CH5b E 33.8-CH3aF
34.4 -C-G
37.1 TaC-H37,9-CH2-c
, k l 44.3 -CH2-d,
eJ 50.2':/cH-h
K 64.8; CH-0-IL
74.5') SH-0-mM
78.2 ;CH-0-nN
144.2 ;(:=0
151.1'):, c=P 17
2.1 N=OQ 173.4
c=.

This was done using dimensional NMR spectrum data.

Example 2 Biological activity of A 8345-C Antibiotic A 8345-C exhibits growth inhibiting effect on Bacillus bacteria in Davis's minimal medium having the composition shown in Table 2. The added medium did not show any growth inhibiting effect. The minimum inhibitory concentrations (MIC) for typical bacterial strains are shown in Table 3.

Table 2 PH7,0 Furthermore, A 8345-C has the effect of suppressing the growth of plants, especially during the germination of lettuce and edible seeds.
Inhibited root elongation. Details are shown in Table 4.

Table 3 Biological activity of Ya8345-C Antibacterial activity Escherichia colt JC-2AJ20
05 >50 >50 Sarcina 1utea
AJ1217 12.5) 50Xant
homonas oryzae AJ2783
)50 >50Bacillus 5ubtilis
AJ1316 1.56) 50Bacill
us ceraus AJ11252 1.56
) 50 liters of medium, [) Avis minimal medium 2, 1) Avis minimal medium + 0.2 polypeptone, 0.1% yeast extract Table 4 Plant growth inhibitory effect Growth (compared to untreated plot)

[Brief explanation of drawings]

FIG. 1 is an optical external absorption spectrum measured in a potassium bromide tablet of &8345-C. FIG. 2 is a hydrogen nuclear outgas resonance spectrum of A 8345-C measured in heavy methanol. FIG. 3 is a carbon nuclear magnetic resonance spectrum of A 8345-C measured in heavy methanol. FIG. 4 is an H-C Nft correlation two-dimensional nuclear magnetic resonance spectrum of A 8345-C measured in heavy methanol. 41,1, Out,,・ノ(Bow 9.・':,','!4.
82, ゛i procedure amendment! ! (Method) Revision Office Liangmiya 2, Name of the invention Plant 1 A new product with inhibitory activity and antibiotic activity! No. 8345-C Address 5, Kyo (Nee-chome), Chuo-ku, Tokyo 8 bow (shipment date nqwa61i [2025th) 5, correction J,
No. 8 due to an increase in the number of inventions, attached 1 copy of application for correction of inventory of stores etc.

Claims (1)

[Claims] 1. New substance No. 1 characterized by having the following physical and chemical properties. 8345-C (1) Appearance: White needle-like crystals (2) Elemental analysis values: Carbon 63.0%, Hydrogen 7.5%, Nitrogen 4.1% (weight ratio) (3) Molecular weight: EI-MSm/z :323(M^+)(
4) Molecular formula: C_1_7H_2_5NO_5 (from high resolution EI-MSm/z: 323.17327) (5) Specific optical rotation: [α]^2^4_D+17.3° (C
0.075, methanol) (6) Melting point: 212-214°C (7) Solubility: Soluble in methanol and ethyl acetate, slightly soluble in water. (8) Ultraviolet absorption spectrum: UVλ^M^e^O^H
_m_a_xnm: 211 (9) Infrared absorption spectrum: The spectrum measured with potassium bromide tablets is as shown in FIG. (10) Hydrogen nuclear magnetic resonance spectrum: The spectrum measured in heavy methanol is as shown in FIG. (11) Carbon nuclear magnetic resonance spectrum: The spectrum measured in heavy methanol is as shown in FIG. (12) Color reaction: Potassium permanganate positive, ninhydrin negative.