JPH03251597A - Versaferrocide-based compound - Google Patents

Abstract

NEW MATERIAL:Compounds of the formula (R is H or methyl) having following physical properties. Visual appearance: colorless oily substance; Molecular weight: 593, 607; Holecular formula: C30H51N5O7, C31O53N5O7; Specific rotatory power: [alpha]=-201.8 deg. (c=0.45, methanol), [alpha] J5=-190.0 deg. (c=0.58, methanol); Solubility: soluble in methanol, ethanol, ethyl acetate and chloroform and insoluble in water, and so on. USE:An anti-pine wood nematode agent. PREPARATION:For example, an asporous imperfect fungi D 1084 (FERM P-11289) is cultured at 20-27 deg.C for 3-10 days preferably under an aerobic condition using the submerged culture method.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Field of Application) The present invention relates to versaferroside compounds having antipine nematode activity.

(Problems to be Solved by the Prior Art and the Invention) Pine blight has become a problem not only in forestry but also in society. However, an effective drug to exterminate pine nematode, which causes pine blight, has not yet been put into practical use.

In view of the current situation, the present inventors isolated a large number of microorganisms, and isolated and purified the active substances produced by the microorganisms, with the aim of searching for novel antipine nematode active substances.

As a result, we found that a new active substance was produced in the culture of a specific fungus, isolated and purified it, and confirmed that this active substance had good antipine nematode activity. This discovery led to the completion of the present invention.

(Means for Solving the Problems) That is, the gist of the present invention resides in a versaferroside compound represented by the following structural formula.

(In the formula, R represents a hydrogen atom or a methyl group.) The versaferroside compound of the present invention is represented by the above structural formula.

Hereinafter, in the present invention, those in the above formula in which R represents a methyl group are referred to as versaferroside A substances, and those in which R represents a hydrogen atom are referred to as versaferroside B substances.

The versaferroside compound represented by the above structural formula is
As will be described later, for example, the nonspore-deficient bacterium D1084 (FERMp-11289)
It can be obtained by culturing.

The microorganism strain DI084 used in the present invention is a type of fungus (Eumycota) newly isolated from a natural dicotyledonous plant, and its mycological properties are as follows.

Mycological properties of strain D1084 Potato dextrose agar (PDA), oatmeal agar (OA), malt agar (MA), and Miura medium (LCA)
), the growth is moderate at 27°C, and after 7 days of culture, the colonies are 5-6 cm in diameter and white fluff-like. When observed under a microscope, no characteristic morphology such as conidia could be observed in any of the media. Furthermore, sterilized rice straw or banana peels were placed on the agar and inoculated, and the cells were observed at 27°C for one month, but no conidia were observed in this case either.

・Growth temperature (cultured on PDA for 1 week): 15-35°C
(Optimum temperature 27°C) Growth pH (LCA, 1 week culture): 2-9, (Optimum growth pH 7-8) Therefore, this strain was designated as the apospore-deficient strain DI084.

In the present invention, the above-mentioned bacteria are cultured in a medium containing nutrients that can be used by ordinary microorganisms. Nutrient sources include glucose, starch syrup, dextrin, sucrose, starch,
Molasses and animal/vegetable oils can be used. Also, soybean flour, wheat germ, cornstape liquor, cottonseed meal, meat extract, peptone, yeast extract, ammonium sulfate, sodium nitrate, urea, etc. can be used as nitrogen sources. In addition, it is effective to add inorganic salts capable of producing sodium, potassium, calcium, magnesium, cobalt, chlorine, phosphoric acid, sulfuric acid, and other ions as necessary. In addition, organic and inorganic substances that aid the growth of bacteria and promote the production of active substance versaferroside compounds can be appropriately added.

The most suitable culture method is a culture method under aerobic conditions, especially a deep culture method. The appropriate temperature for culturing is 10-30°C.
℃, but in most cases, it is cultured at around 20 to 27℃. The production of versaferroside compounds varies depending on the culture medium and culture conditions, but it is usually
Its accumulation reaches its maximum within 10 days. When the accumulation of versaferroside compounds in the culture reaches its maximum, the culture is stopped, and the target substance is isolated and purified from the culture solution.

In order to isolate and purify the versaferroside compound represented by the above structural formula of the present invention from a culture, there are methods that utilize the difference in solubility with impurities, methods that utilize the difference in ionic binding strength, and methods that utilize the difference in adsorption affinity. The means that utilize the difference and the means that utilize the difference in molecular weight may be used alone, in appropriate combinations, or repeatedly.

That is, the culture filtrate is extracted with ethyl acetate and concentrated under reduced pressure.

When the concentrated solution is purified using a combination of silica gel column chromatography, florisil chromatography, etc., a pure versaferroside compound can be obtained.

(Examples) The present invention will be described below in more detail with reference to Examples and Test Examples, but the present invention is not limited by the following Examples unless the gist thereof is exceeded.

Example 1 (1) Cultured starch syrup 4.0%, soybean flour 2.0%, soybean oil 0.3%, Famamedia 1.0%, sungrain 0.5%, calcium monoxide 0.3%, sulfuric acid Iron 0.001%, cobalt chloride 0
．． 0001% of nickel chloride and 0.0001% of nickel chloride (pH 6.0). zu”:)200m1.
Dispense into 20 Erlenmeyer flasks and incubate for 20 minutes at 121°C.
Sterilize under autoclave for minutes. In addition to this, D1084-based substance producing strain, D
1,084 strains were inoculated in one platinum loop, and incubated at 26°C for 6 days.
Culture with shaking at 210 rpm for 1 day.

Separately, prepare a main fermentation medium with the same composition as above, and
mI! , into 100 500mf Erlenmeyer flasks,
Autoclave at 121°C for 20 minutes. Add 4 m7 of the above seed culture solution to this main fermentation medium! The cells were inoculated and cultured at 26°C for 5 days with shaking at 210 rpm. The resulting culture was centrifuged to obtain a culture supernatant.

(2) Purification of culture 7.32 g of the cultured synovial fluid obtained in (1) above was extracted with an equal volume of ethyl acetate.

The extract was dehydrated with anhydrous sodium sulfate, concentrated under reduced pressure,
3.3 g of oil was obtained. The obtained oily substance was mixed with Avicel (manufactured by Asahi Kasei Industries, Ltd.) and dry-filled into Wakogel C-200 (manufactured by Wako Pure Chemical Industries, Ltd.) for 100mj! of benzene-ethyl acetate mixture (1:1) to benzene-ethyl acetate-butanol mixture (1:1).
: Chromatography developed in 2) was performed.

The active fractions were collected and concentrated to dryness under reduced pressure to obtain an oily substance. This oily substance was mixed with Avicel, placed on a 50 ml column of Florisil (manufactured by Florisil) filled with benzene, and developed with a benzene-acetone mixture.

The active fraction eluted with a benzene-acetone mixture (8:2) was concentrated under reduced pressure. The obtained oily substance was equilibrated with an acetonitrile-water mixture (8:2) on Wakogel LC:
It was placed on 0DS-30K (manufactured by Wako Pure Chemical Industries, Ltd.) and developed with an acetonitrile-water mixture. Collect the eluted active fraction,
Concentration under reduced pressure gave Versaferroside A [52] and Versaferroside B (153).

The structural formula is shown below.

Further, the physical properties of the obtained versaferroside A substance and versaferroside B substance are shown below.

■) Appearance: Colorless oil 2) Molecular weight: Substance A: 593 (HR-MS, 593.3785)
Substance B: 607 (HR-MS, 607.3942)
3) Molecular formula: % formula % ) : ) ) 5) Ultraviolet absorption spectrum: Substance A: λwax (methanol) nm (ε): 21
0 (16,300) Substance B: λ@11+1 (methanol) nm (C):
210 (18,200) 6) Infrared absorption spectrum: The spectra measured in potassium bromide tablets are as shown in Figure 1 (versaferroside A substance) and Figure 2 (versaferroside B substance).

7) Hydrogen nuclear magnetic resonance spectrum: The 500 MHz hydrogen nuclear magnetic resonance spectrum measured in a deuterated chloroform solution is as shown in FIG. 3 (versaferroside A substance) and FIG. 4 (versaferroside B substance).

8) Carbon nuclear magnetic resonance spectrum: The 125M)(z carbon nuclear magnetic resonance spectrum measured in deuterated chloroform solution is as shown in Table 1.

Table 1 Substance A Substance B δ value δ value 11.3 11.3 11.8 11.8 14.4 14.3 15.2 15.3 15.3 15.4 Table 1 Continued δ value 19.5 20. 0 24.1 24.4 25.6 27.2 28.0 28.9 30.8 33.1 34.4 36.0 37.4 46.5 δ value 18.6 19.5 20.0 24. 6 26.2 27.2 28.1 30.8 32.8 33.2 34.5 35.8 36.4 37.6 53.5 Table 1 continued δ value 53.6 55.4 57.9 60. 7 76.1 168.8 169.7 171.0 171.0 173.5 173.7 δ value 53.9 55.5 57.9 60.4 76.0 168.4 169.7 171.0 172. 0 173.6 173.7 9) Solubility: Soluble in methanol, ethanol, ethyl acetate and chloroform, insoluble in water.

Test Example 1 (Anti-pine nematode test) Sample material Versaferroside A obtained in the above example
A methanol solution in which 100 μg of each of the substance and Versaferroside B substance were dissolved was permeated into a cotton ball shaped into a sphere with a diameter of about 5 m11, and then dried. 15,0 on the cotton ball
100μ of a suspension of pine tree nematode prepared at 00 heads/ml. It was then placed on a petri dish medium with a diameter of 4C1 on which Botr construction soil ± s cinera as food for nematodes had been grown in advance. 5 days
Botr tis ci on a petri dish attacked by pine nematodes after being kept at 26°C
Nera was observed.

As a result, no nematode attack was observed on the petri dish on which the cotton balls containing the active substance were placed.

In addition, when each living nematode was collected and counted, and the proliferation rate was calculated in comparison with the control group, it was 3.3% for versaferroside A substance and 1.2% for versaferroside B substance. , each substance inhibited nematode proliferation.

(Effects of the Invention) The novel antipine nematode active substance versaferroside compound of the present invention has the activity as shown in the examples above, and is expected to be useful as a pine needle nematode exterminating drug. Ru.

[Brief explanation of drawings]

FIGS. 1 and 2 are diagrams showing infrared absorption spectra of Versaferroside A and B substances. FIGS. 3 and 4 are diagrams showing hydrogen nuclear magnetic resonance spectra of versaferroside A and B substances.

Claims (1)

[Claims] (1) Versaferroside compound represented by the following structural formula. ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (In the formula, R represents a hydrogen atom or a methyl group.)