JPH0466508A - Fungicidal agent - Google Patents

Abstract

PURPOSE:To obtain a new fungicidal agent having high selectivity and safety and exhibiting strong toxicity (growth-inhibiting activity) against microorganisms, especially true fungi, and little toxicity to animal and plant by using anhydrofulvinic acid as an active component. CONSTITUTION:The objective fungicide free from proliferation inhibiting activity to bacteria and animal cells, exhibiting extremely remarkable effect to inhibit the proliferation of dermatic true fungi and plant-pathogenic molds and having high safety can be produced by using anhydrofulvinic acid of formula as an active component. The anhydrofulvinic acid used as the active component can be produced by culturing a microbial strain capable of producing the acid in a nutrient medium.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a novel antibacterial agent, and more specifically to anhydrofulvic acid (Anhydrofulvic Acid).
) Contains a highly safe antibacterial agent.

[Background of the invention]

Fulvic acid having a common core with the anhydrofulpic acid of the present invention is derived from Penicillium griseofulvum (Penicillium griseofulvum).
icillium Uendialihadan) and Sericospora.
It is known that it is produced by veticola (for example, Bioc
hem, J. 29.1102.1935 and Agri
, Biol.

Chea+, 45.1275.1981) However, the characteristics of this compound include yellowing of sugar beet leaves, yellow needle-like appearance in the culture solution, and its use as an acidic metabolite. Evaluation is unknown.

Additionally, anhydrofulpic acid has been synthesized as a confirmation sample for analyzing the structure of fulvic acid (
J,Chem,Soc,Perkin Trans,I
, 3891987), its physiological activity has not been investigated at all.

By the way, various antibiotics have been developed and actually used as antibacterial agents, but when considering the diversity of various pathogenic bacteria, the specificity of the activity of the drugs used against pathogenic bacteria, and the toxicity to living organisms, Development of new antibacterial agents is necessary.

Therefore, an object of the present invention is to provide a less toxic antibacterial agent, particularly an antifungal agent.

In order to achieve this objective, the present inventors focused on using the succinate oxidase system, which has been attracting attention in recent years as a preliminary evaluation system for new antifungal agents. In other words, with regard to the inhibitory activity against succinate oxidase, compounds such as flutolanyl, which have different efficacy against mammals and fungi, are attracting attention (e.g., flutolanyl).
o1. chem, 521445 1981), it is thought that these systems can be used as preliminary evaluation systems for less toxic antifungal agents. Therefore, the present inventors investigated the respiratory function inhibiting activity at the cellular level using bacterial culture fluids having antifungal activity.
Succinate oxidase system activity was measured using mitochondria prepared from S. lis). Thus, among the many cellular respiratory function inhibitors, Penicillium was selected as one that inhibits succinate oxidase system activity.
Penicillium facidum
) (new species), and further purified the fraction having antifungal inhibitory activity, respiratory inhibitory activity, and succinate oxidase system inhibitory activity from the culture solution. The present invention was completed by discovering anhydroflupic acid that has growth-inhibiting activity at extremely low concentrations.

[Structure of the invention]

According to the present invention, a novel antibacterial agent comprising anhydrofulpic acid represented by the following formula is provided.

The anhydrofulpic acid used in the present invention was synthesized in the course of structural analysis of fulvic acid as described above and is known in itself, but it is not known whether it has any physiological activity. However, it does not inhibit the growth activity of grouperia or animal cells, and has an extremely dominant growth inhibitory effect on skin fungi and plant pathogenic fungi.
This invention is disclosed for the first time. The compound can be produced by a microorganism belonging to the genus Penicillium, as described in detail later, but as long as it achieves the effects of the present invention, it can be produced by a chemical synthesis method including the known synthesis methods described above. can also be used.

Antibacterial agents containing this compound as an active ingredient are suitable for use in animal and plant diseases or diseases caused by microorganisms, especially fungi and yeasts, whose respiration at the cellular level is inhibited by this compound, or by environmental pollution (for example, mold on painted walls). outbreak). An example of such a microorganism is S. calomyces (yes)
, Candida, Sh 1zosacchar.

Those belonging to the genera L/L/';L, Hansenula, Rhizopus, Mucor, Penicillium, and Asberghii. Can be mentioned.

In addition, the dosage form of the present invention can be appropriately selected depending on the applied microorganism and application method, but specific examples include preparations for human skin or injections, plant pathogenic mold powders, emulsions, or Examples include additive compositions for hydrating agents and paints. Note that although these dosage forms are not limited by the target of application, typical formulations suitable for various target targets will be explained below.

When used as an antifungal agent, this compound is incorporated into the antifungal agent for skin in any proportion, but usually 0.05 to 50% by weight based on the total amount of the antifungal agent, preferably 0.1%. ~
It is used in a range of 30%. Furthermore, the antifungal agent of the present invention can contain various known ingredients to improve performance. These ingredients include additives such as surfactants, excipients, binders, suspending agents, emulsifiers, dispersants, thickeners, stabilizers, wetting agents, etc., as known in the art. can be given. Examples of the surfactant include anionic surfactants such as alkyl sulfate salts, cationic surfactants such as alkyltrimethylammonium halides, nonionic surfactants such as polyoxyethylene sorbitan monooleate, and alkyl sulfonate surfactants. Examples include double-sided surfactants such as betaine.

Next, when using the present compound as an agricultural and horticultural fungicide targeting plant pathogenic fungi, the formulation system includes, for example,
It can be processed into solutions, emulsions, suspensions, fine powders, powders, pastes, and granules, and the dosage form can be changed depending on the target pathogen.

The formulations can be prepared in a known manner, for example by extending the active substance with solvents and/or excipients, optionally using emulsifiers and dispersion aids. On this occasion,
Examples of solvents include water, alcohols (e.g., methyl alcohol, ethylene glycol, etc.), aromatic compounds (e.g., benzene, toluene, etc.), ketones (e.g., acetone, methyl ethyl ketone), amines (e.g., nitinolamine, etc.). is suitable, and one or two of these
More than one species can also be used.

Suitable excipients include viscous substances such as cations and bentonites, talcs such as talc and pyrophyllite, oxides such as diatomaceous earth and white carbon, and vegetable powders such as soybean powder and CMC. You may use 1 type, or 2 or more types.

Emulsifiers include nonionic and anionic emulsifiers (e.g. alkyl sulfonates and alkyl sulfonates) and dispersants (e.g. lignin, methyl cellulose).
can be mentioned.

When applying this compound as an antifungal agent for agriculture and horticulture, when using it in the form of an emulsion, the compound should be used in an appropriate proportion of 10-50%, solvent 10-80%, and surfactant 3-20%. A stock solution is prepared by mixing in a suitable ratio, and at the time of use, it is diluted with water to a predetermined concentration and used by methods such as spraying. When used in the form of a wettable powder, mix 2 to 80% of this compound, 10 to 90% of an excipient, and 1 to 20% of a surfactant in appropriate proportions, and add water as in the case of an emulsion. Use by diluting with etc.

In addition to the present compound, antibacterial agents known per se can also be added to the preparation.

Anhydrofulpic acid used as an active ingredient of the present invention can be produced chemically synthetically as described above, but
It can also be produced by the method described below, in which a microorganism capable of producing the present compound discovered by the present inventors is cultured in a nutrient medium.

Microorganisms with such production ability are obtained by isolating, for example, bacteria belonging to the genus Penicillium from a microbial isolation source such as soil, using a conventional method, and then using these culture solutions to test for antibacterial activity against Candida utilis. It can be obtained by selecting

In the production of the compound of the present invention, the medium used for culturing Penicillium genus may be liquid or solid, but shaking culture or aerated agitation culture using a liquid medium is usually convenient. Any medium may be used as long as it can grow anhydrofulpic acid producing bacteria and accumulate the effective substance of the present invention in the medium. That is, carbon sources include, for example, glucose, lactose, glycerin, starch, sucrose, dextrin, molasses, organic compounds, etc., and nitrogen sources include, for example, peptides, protein hydrolysates such as casamino acids, meat extracts, Yeast extract, soybean meal, cornstarch liquor, amino acids, ammonia salts, nitrates, and various other organic or inorganic nitrogen compounds are used. As inorganic salts, various phosphates, magnesium sulfate, and sodium chloride may be added, and vitamins, nucleic acid-related compounds, etc. may be added for the purpose of promoting bacterial growth. Note that adding antifoaming agents such as silicone, polypropylene glycol derivatives, and soybean oil to the medium may be effective in increasing the amount of the present substance accumulated. In culturing, it is preferable to carry out small-scale pre-culturing in advance and contact the resulting culture with the medium, rather than carrying out main culturing immediately. Conditions such as culture temperature, culture period, culture liquid, etc. are appropriately selected and adjusted to maximize the amount of accumulation of the present invention.
Cultivation may be carried out at 5°C to 35°C for 12 to 3 days, and the pH of the medium is preferably maintained at pH 5.5 to 7.2.

In this way, anhydrofulpic acid is produced and accumulated during the culture. When culturing using a liquid medium, the target substance is mainly accumulated in the liquid part, so it is separated from the filtrate or supernatant after the culture is filtered or centrifuged to remove bacterial cells. Although it is preferable to do so, it is also possible, if necessary, to separate the target product directly from the culture solution without removing the bacterial cells. In order to collect anhydroflupic acid, it is quantified by bioassay using the paper disk method using Candida albicans as the test bacterium.

When purifying anhydrofulpic acid from culture solution,
For example, use the culture solution as Diaion HP-20 (manufactured by Ryo Kasei).
It is applied to a column, washed with water, eluted with methanol, dried to dryness, dissolved in chloroform, and extracted with water at pH 9.0. Further, this aqueous layer was adjusted to pH 3.0, extracted with chloroform, concentrated, applied to silica gel, and fractionated with chloroform-methanol. The 99% chloroform fraction is dried, dissolved in DMF, and precipitated by adding water.

By washing this precipitate with methanol and recrystallizing the insoluble matter with DMF-water, anhydrofulpic acid having strong antibacterial activity can be obtained as yellow needle-shaped crystals. This is in NMR agreement with the standard synthesis.

[Effect] The effect of the present invention can be confirmed in part by measuring the physiological activity of the anhydrofulpic acid obtained in this way, for example, the respiratory inhibitory activity against Candida utilis cells. Regarding this inhibitory activity, 12m
It was found that anhydroflupic acid exhibits respiratory inhibition at concentrations below 6.25 μ/ttr1, as measured by oxygen consumption with a C1ark-type oxygen electrode model 100 in the presence of M glucose. The succinate oxygenase system and NADH were detected using mitochondria prepared by a conventional method using Candida utilis cells.
When the activity of the oxidase system was measured, it was found that the succinate oxidase system was anhydroflupic acid 1.56 /
It was completely inhibited at concentrations of d(0,34) or higher. On the other hand, the NADI (oxidase system) is hardly inhibited even by 6.25 g/- or more of anhydrofulpic acid, and this compound
It was revealed that the system up to Q, ie, Complex II containing succinate dehydrogenase, is the point of action. There is information that the Complex H types of animals and fungi are different, and this is presumed to be the cause of the low toxicity to animals.

MJ! L! In addition, this compound exhibits a minimum inhibitory concentration (MIC) against various microorganisms as shown in Table 1 below using the conventional YM liquid medium dilution method. The usefulness of this compound as an antibacterial agent can be understood by comparing it with that of fulvic acid, which has a common core, as a comparison.

l-,,,,! --- Table Antibacterial activity of anhydrofulvin and fulvin (Left below) Note that the above-mentioned respiration inhibitory activity against cells can be specifically measured as follows.

-S Candida j-tilis (C, utilis) AT
CC42402 was used as an indicator bacterium to examine respiratory inhibition against live bacteria. Using YM liquid medium in advance, the bacterial cells were cultured to the mid-logarithmic growth phase, and then treated with 5011Il tartaric acid/NaO.
A bacterial solution was prepared by washing and suspending the bacteria with H buffer (pH 3,0).

Some of them were left for another 22 hours to starve them into cells.
Infiltration was carried out at 25°C. Anhydrofulpic acid at a predetermined concentration was added to these two bacterial solutions, glucose was further added to the starved one to give a final concentration of IIIM, and the respiratory activity was measured after 60 minutes using an enzyme electrode. . As a result, respiratory activity was similarly inhibited in both cases, suggesting that anhydroflupic acid acts directly on mitochondria rather than inhibiting substrate uptake or glycolysis. The results are shown in Table 2.

1 unit 1 table 6.25 5 = 22 4.09 50 C. utilis cells obtained in the same manner as above.
% sucrose, 50mM) Lis HCl2 in
The cells were suspended in +M EDTA (pH 7.5) and disrupted by the glass bead method, and mitochondrial fractions were obtained by sucrose density gradient centrifugation. Using this mitochondrial preparation, a predetermined concentration of anhydroflupic acid (0,39
The oxygen consumption amount derived from NADH and succinate under the conditions where ~25~/d) was added was observed. The results are shown in Table 3.

In this table, the values are shown as relative values, with the oxygen consumption in the absence of anhydroflupic acid added (control) as 100.
Little inhibition of oxidase was observed, whereas succinate oxidase was 0.39 Jig/Id.
Since activity inhibition was observed even at an extremely low concentration of 22 mM, the inhibitory action point of anhydrofulpic acid of the present invention is the pathway from succinic acid to Co, that is, Complex II containing succinate dehydrogenase. Presumed.

12.5 6.25 3.13 1.56 0.78 0.39 5.4 1.3 0.34 0.086 0.022 81.60 B5.20 90.48 , exhibits almost no toxicity to animals and plants (for example, LDS when administered intraperitoneally to mice is 200 ■ or more).

As described above, anhydrofulpic acid, which is the active ingredient of the present invention, exhibits a very specific mechanism of action by itself.
Since it also has strong antibacterial activity, it is clear that any composition or dosage form will have the desired effect as long as it does not adversely affect these activities. especially,
The various dosage forms described above can be applied to target animals or plants.

The appropriate amount of anhydrofulpic acid, which is the active ingredient of the present invention, is not critical as it largely depends on the treated microorganism in the subject (eg, human or plant), the age and individual symptoms in humans, and the mode of application. 1 used in the therapy of humans with a body weight of approximately 65 kg
The daily dose (oral or parenteral) is approximately 50 to 500 μ. Note that those skilled in the art will be able to easily determine the optimal dose by conducting simple preliminary tests for each efficacy.

EXAMPLES The following sections are provided to more specifically illustrate the present invention.

M1 Neebeishu Glucose 1.8%, Polypeptone 0.5%, Yeast Extract 0.3%, KH2PO40, 17%, KH2PO40
, ], 4% Mg, 50.7 FI 200.005% was dispensed into 3 sterilized flasks at 100 RiV and then inoculated with the SAM 2570 strain of the present invention at 25°C. Permeabilization culture was performed for weeks. The entire volume of this culture was mixed with 10 L of a medium containing the above medium composition with the addition of 0.75% tartaric acid and adjusted to a pH of 2.0.
After sterilization, the mixture was added to a L fermenter, and cultured with aeration and stirring at 25°C for one week. After culturing, by removing the bacterial cells by filtration, the culture filtrate 9.2
I got L.

9.2L of the culture filtrate obtained in the above culture was injected into Diaion H.
It was applied to a P-20 (manufactured by Mitsubishi Kasei) column (10 x 38 cm), washed with water, eluted with methanol, and the dried product under reduced pressure was eluted with 500j+1 alkaline water (pH 9,0).

This extracted aqueous layer was lowered to pH 3, extracted with 500 ml of chloroform, and concentrated under reduced pressure. This concentrate was applied to a silica gel column (4×30), eluted with a chloroform:methanol (99:1) mixture, and dried under reduced pressure. After dissolving the dried product in dimethylformamide, water was added to obtain a precipitate. The precipitate was washed with methanol and treated again with dimethylformamide to further increase its purity, yielding 250 square yellow needle-like crystals. The NMR spectrum of this compound was consistent with synthetic anhydroflupic acid.

Formulation example 1 An ointment was prepared in a conventional manner according to the following formulation.

White petrolatum 25.0% stearin alcohol 22.0 Propylene glycol 12.0 Sodium lauryl sulfate 2.0 Ethyl paraoxybenzoate 0.3 Propyl paraoxybenzoate 0.2 Anhydrofulpic acid
5.0 salicylic acid
0.1 purified water
Remaining total IT: 100.0% Formulation Example 2 A liquid preparation for external use was prepared according to the following recipe in a conventional manner.

Remaining days below diphenhydramine hydrochloride Dipotassium glycylate anhydrofulpic acid lauro macrogol citric acid water 1.0% 0.5 5.0 0.5 0.1 remainder 100.0% Prepare hydrating powder 2o　Agricultural surplus bladder-bacterial wound Formulation example 1 Follow the recipe below and use the conventional method. did.

Diatomaceous earth alkylhenzenesulfonic acid anhydroflupic acid 75.0% 5.0 20.0 100.0% *When using, the main rule is to use phosphoric acid to pH = 3.0 to 4.
．． Dilute to 0.

Formulation example 2 A wettable powder was prepared in a conventional manner according to the following formulation.

White carbon 1000% diatom ± 47.0 Tsurupol 5039 (*) 3.0
Anhydroflupic acid 40.010
0.0% Hontoho Chemical Industry Co., Ltd. trademark, surfactant whose main component is polyoxyethylene alkylaryl ether sulfonate *When using, add powder to phosphoric acid to pH = 3.0 to 4.
．． Dilute to 0.

Formulation example 3 An emulsion was prepared in a conventional manner according to the following recipe.

Xylene 25.0% Dimethylformamide 30.0 Tsurpol 3005X (*) 15.
0 Anhydrofulpic acid 30.01
00.0% Trademark of Toho Chemical Industry Co., Ltd., mixture of nonionic surfactant and anionic surfactant [Effects of the invention] According to the present invention, it is highly toxic to microorganisms, especially fungi (growth inhibition It is a highly selective and highly safe antibacterial agent that exhibits a high degree of activity) but has little toxicity to animals and plants. Such an antibacterial agent can exhibit the desired effect by using anhydrofulpic acid as its active ingredient.

Margin below

Claims (1)

[Claims] 1. Anhydrofulvic acid represented by the following formula ▲ Numerical formulas, chemical formulas, tables, etc. ▼
An antibacterial agent comprising lvicAcid).