JPH0495069A - New bioactive substance ec 40 - Google Patents

Abstract

NEW MATERIAL:EC 40 of the formula having 121-124 deg.C melting point and C15H21NO20 molecular formula. USE:Having a lipoperoxide production inhibition effect and useful as a preventive and a remedy for disease symptoms such as various diseases, inflammations or edemas due to hindrance of circulation in brain, heart, periphery, etc. Useful also as an antioxidant for foods. PREPARATION:The compound of the formula is obtained by culture of an microorganism. As the strain, an EC 40-producing strain belonging to Streptomyces genus, specifically Streptomyces chrestomyceticus EC 40 strain (FERM BP-3032) is used and an aerobic liquid culture is preferably carried out in a suitable culture medium. Culture temperature is suitably 20-37 deg.C, especially preferably 25-30 deg.C. EC 40 is collected from the cultured material, e.g. by extraction using ethyl acetate, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION [Background of the Invention] Technical Field The present invention relates to a novel substance, more specifically, a novel substance EC4 having active oxygen scavenging activity or lipid peroxide production suppressing activity.
Regarding 0.

Prior Art> In the pathological state of living organisms, reactive oxygen species (active
It has been suggested that the locally generated active oxygen species form a complex with endogenous iron to generate strong hydroxyl radicals (OH), resulting in the formation of strong hydroxyl radicals (OH). , large amounts of lipid peroxide are generated in tissues.

This lipid peroxide is known to damage normal organs and tissues including blood vessels and cause secondary lesions such as vascular disorders, inflammation, and edema (Japan Clinical, 46 (I0)
, 64 (I988)). Therefore, substances that scavenge reactive oxygen species and suppress lipid peroxide production are effective as preventive and therapeutic agents for various disorders and diseases such as ischemic heart disease, cerebrovascular disorders, arteriosclerosis, inflammation, kidney disease, and peptic ulcers. It can be said that there is a constant desire for substances that have such an effect.

[Summary of the invention]

Summary> The present invention meets the above needs.

That is, the novel substance according to the present invention is compound EC40 represented by the following formula (I).

Effect> The novel compound EC40 according to the present invention has a lipid peroxide production inhibiting effect as strong as that of vitamin E, as shown in the experimental examples below. It may be said that it was unexpected that a compound with such a relatively simple structure had a high degree of inhibitory effect on lipid peroxide production.

Due to these properties, compound EC40 is useful as a preventive or therapeutic agent for the above-mentioned diseases, or as an antioxidant for various targets such as foods.

[Detailed description of the invention]

[I] New substance EC40 1) Chemical structure The new substance EC40 according to the present invention has a chemical structure represented by the above formula (I).

The chemical structure of EC40 was determined as described above by examining in detail the proton nuclear magnetic resonance spectrum, carbon-13 nuclear magnetic resonance spectrum, ultraviolet absorption spectrum, infrared absorption spectrum, and mass spectrometry spectrum.

2) Physical and chemical properties (I) Appearance Pale yellow powder (2) Melting point 121-124°C (decomposition) (3) Solubility Soluble in methanol, ethanol, benzene, chloroform, acetone, ethyl acetate, n-hexane Slightly soluble in water, insoluble in water (4) Rf value (Merck's "Silica Gel 60F254"
Used) Chloroform-methanol (5:1) 0.42 (
5) FD-MS spectrum (m/z) 247 (M'') (6) Ultraviolet absorption spectrum shown in FIG.

λ (ε) 237 (8500) ■ax
na+ (7) Infrared absorption spectrum is shown in FIG.

(KBr disk method) (8) Proton nuclear magnetic resonance spectrum It is shown in FIG.

(500 MHz, in heavy methanol) (9) Carbon-13
The nuclear magnetic resonance spectrum is shown in FIG.

(I25 MHz, in heavy methanol) (I0) Elemental analysis HN Analysis value α) 72.81 8. 58 5. 66
12. 95 to calculated value') 72.84 8. 56
5.66 12.94 (I1) Molecular formula % Formula % EC40 can currently only be obtained by culturing microorganisms, but it can also be produced by synthetic chemical or microbiological modification of related compounds, or by total synthesis. It could also be produced chemically. It may also be possible to use genetic engineering techniques. That is, it would be possible to integrate a gene involved in the production of compound EC40 into an appropriate microorganism, culture the resulting transformant, and obtain the compound from this culture.

When culturing a microorganism, a strain belonging to the genus Streptomyces and having an EC40-producing ability is used. Specifically, the present inventors have revealed that the Streptomyces crestomyceticus EC40 strain isolated by the present inventors produces EC40; It is possible to isolate suitable substances from nature by conventional isolation methods. Furthermore, there is still room to increase the production ability of EC40 by subjecting EC40 producing bacteria, including Streptomyces φchrestomyceticus strain EC40, to irradiation or other mutation treatments. As mentioned above, genetic engineering techniques can also be used.

<EC40 strain> The EC40 strain, which the present inventors have discovered as a Streptomyces strain capable of producing EC40, has the following content.

1) Origin and deposit number Strain EC40 was isolated from soil collected in Pedreira, Brazil, and was deposited at the Institute of Microbial Technology, Agency of Industrial Science and Technology on July 27, 1990, and designated as the Third
032 J (FERMBP-3032) number has been obtained.

2) Mycological properties The mycological properties of the EC40 strain are as follows.

(I) Strain EC40 has poor growth on glucose-asparagine agar and nutrient agar, and moderate growth on sucrose-nitrate agar, glycerol-asparagine agar, yeast-malt agar, and oatmeal agar. However, it grows well on starch inorganic salt agar medium and tyrosine agar medium. Aerial mycelium grows well on starch inorganic salt agar medium. Aerial hyphae produced from basal hyphae form simple branches and elongate, and the spore chain has about 30 spores in a spiral shape. The spore shape is cylindrical or oval, and the size is about 0.6 μm x 0.6 μm. 5 μm, and its surface is smooth. Specialized forms such as sporangia, flagellated spores, and sclerotia are not recognized.

(2) Growth status on various media The results of culturing the EC40 strain on various media at 27°C for 3 weeks are shown in Table 1.

(3) Physiological properties The physiological properties of the EC40 strain are shown in Table 2.

(4) Carbon source utilization The carbon source utilization of the EC40 strain (on Pridham-Gotlieb agar medium) is as shown in Table 3.

(5) Analysis of diaminopimelic acid As a result of analyzing diaminopimelic acid, which is one of the amino acids constituting the cell wall, LL-diaminopimelic acid was detected.

The above mycological properties are summarized as follows.

1) The spore chain is spiral and the surface of the spore is smooth.

H) Adhesion of aerial mycelium is good on starch inorganic salt agar medium. The color ranges from white to yellowish white to dark yellow-orange to dark yellowish brown, and the color of the reverse side ranges from yellowish gray to yellowish white to dark yellow to dark yellow.

fft) Tyrosine agar medium, peptone yeast ψ
Neither melanin-like pigments nor soluble pigments were observed in either the iron agar medium or the tryptone yeast liquid medium.

iv) L-arabinose, D-fructose, inositol and sorbitol are not assimilated.

Based on the above properties, I5P (International Streptomyces Project) is described (E, B).

Shirling and D, Gottl
ieb2 Int, J, 5yst,
Bact.

Ill, 139-189.279-392 (I00
g): 19.391-512 (I969); 22.
265-394 (I972)) and Bersey's Manual of Determinants. Bacteriology (Bergey's Manual or Deter
When searching for related known bacterial species using the 8th edition (I974) as a reference, we found Streptomyces crestomyceticus (Streptomyces clestomyceticus).
eptomyceschrestol Wyceticu
s) (Int, J, 5yst, Bact,:
22°2B2 (I972)) appeared to be the most similar.

Therefore, when comparing the EC40 strain and Streptomyces clestomyceticus, although they differ in sugar utilization, L-arabinose and D-fructose, their spore chains are helical and the spores have a smooth surface. , peptone yeast iron agar medium, tryptone yeast liquid medium, and tyrosine agar medium, the fact that melanin-like pigments are not produced, and the color tone of aerial mycelia are in good agreement.

As described above, it is appropriate to identify the EC40 strain as Streptomyces clestomyceticus since it closely matches that of Streptomyces clestomyceticus. Therefore, the EC40 strain was transformed into Streptomyces clestomyceticus (StreptIlyces c.
hrestomycetjcus) E C40 strain.

Table 1 +: Utilized -2 Not utilized ±: Slightly utilized Cultivation/Production of EC40> The compound EC40 is an EC4 belonging to the genus Streptomyces
It can be produced by culturing zero-producing bacteria aerobically in an appropriate medium and collecting the target product from the culture.

The medium can contain any nutrient source that can be utilized by the EC40-producing bacteria. Specifically, for example, glucose, mannitol, galactose, glyceptol, and fats and oils can be used as carbon sources, and soybean meal, fish meal, cottonseed meal, dried yeast, yeast extract, and cornstarch liquor can be used as nitrogen sources. Organic substances such as ammonium salts or nitrates such as ammonium sulfate, sodium nitrate and ammonium chloride are available. Moreover, inorganic salts such as sodium chloride, potassium chloride, calcium carbonate, phosphates, and heavy metal salts can be added as necessary. In order to suppress foaming during fermentation, suitable antifoaming agents such as silicone oil can also be added according to conventional methods.

The most suitable culture method is an aerobic liquid culture method, which is the same as the commonly used method for producing physiologically active substances using Streptomyces. The culture temperature is suitably 20-37°C, preferably 25-30°C. In this way, EC
The production amount of 40 is 6 for both shaking culture and aeration agitation culture.
It reaches a maximum between 0 and 0.

In this way a culture enriched with EC40 is obtained. In culture, a portion of EC40 exists in the culture fluid, or most of it exists in the bacterial cells.

Any convenient method can be used to harvest EC40 from such cultures. One method is based on the principle of extraction, and specifically, for EC40 in culture solution, it is extracted from water-immiscible EC40.
0 solvent (see above) For example, the bacterial cell aggregate obtained by extraction with ethyl acetate, filtration, centrifugation, etc. for EC40 in the bacterial cells is extracted with methanol, ethanol,
There are methods to recover it by treating it with acetone etc. The culture itself can also be subjected to the above extraction operation without separating the bacterial cells. Countercurrent distribution methods using suitable solvents can also be included in the scope of extraction.

Another method for collecting EC40 from cultures is based on the principle of adsorption, in which E is already in liquid form.
For C40a substances, such as culture furnace solution or extract obtained by performing the extraction operation as described above, use a suitable adsorbent, such as silica gel, activated carbon, "Diaion HP20" (manufactured by Mitsubishi Chemical Corporation), etc. EC40 can be obtained by adsorbing the desired EC40 using a solvent and then eluting it with an appropriate solvent.

The EC40 solution thus obtained is concentrated to dryness under reduced pressure to obtain a crude EC40 sample.

In order to further purify the crude sample of EC40 obtained in this way, the extraction method and adsorption method described above may be combined with a gel method, high performance liquid chromatography, etc. as necessary, and the procedure may be performed as many times as necessary. For example, high-performance liquid chromatography using an adsorbent such as silica gel, column chromatography rYMC pack using a gel or filter agent such as Sephadex LH-2DJ (manufactured by Pharmacia) (manufactured by Yamamura Kagaku Co., Ltd.), A suitable combination of countercurrent distribution methods may be used. Specifically, for example, a crude EC40 sample is dissolved in a small amount of methanol,
``Put it on a Sephadex LH-2OJ column, elute the active fraction with methanol, and concentrate to dryness to obtain a pure product with an EC40 rating.

<Applications of EC40> The compound EC40 according to the present invention is useful in that it has an excellent inhibitory effect on lipid peroxide production by scavenging active oxygen species as described below, and is useful for, for example, circulatory disorders in the brain, heart, and peripheral areas. It is expected to be a preventive/therapeutic agent for various diseases and pathological conditions such as inflammation, edema, etc.

The formulation and administration method for use as a medicine is as follows:
Various conventionally known methods can be applied. That is, possible administration methods include injection, oral administration, and rectal administration.

The formulation may be in the form of injections, granules, tablets, powders, suppositories, or the like.

Furthermore, in the case of oral or rectal administration, it may be used as a sustained release preparation.

During formulation, various adjuvants used for pharmaceutical purposes, such as carriers and other auxiliary agents, such as stabilizers, preservatives, soothing agents, emulsifiers, etc., may be used as long as they do not adversely affect the EC40. can be used as needed.

In the formulation, the content of EC40 can vary widely depending on the formulation form.

The dosage of EC40 is 0.1-10 per adult per day.
Although the amount is approximately 0 g, it is more preferable to increase or decrease the amount as appropriate depending on age, pathological condition, and symptoms.

Experimental example: Inhibition of lipid peroxide production
Homogenize with M phosphate buffer (pH 7,4),
Brain homogenates were prepared. Immediately, 100 μM ascorbic acid was added to this rat brain homogenate and incubated at 37°C.
By incubating the brain homogenate with shaking for 1 hour, reactive oxygen species are generated in the brain homogenate, resulting in the formation of lipid peroxides. In this reaction, the test drug was added to the reaction solution at the same time as ascorbic acid was added, and the incubation was performed in the same way, and the malondialdehyde (MDA) produced in the incubation mixture was
l, Biochea+, 95.351. (I9
It was measured by the thiobarbic acid method according to 79). This MDAjl (a) and control value (b)
(MDAjl when no test drug was added), the lipid peroxide production inhibition rate was determined by the following formula.

Since the test drug is poorly soluble in water, it was dissolved and diluted with methanol before being subjected to the test.

In addition, vitamin E was used as a control drug.

(Results) From the above results, EC40 was found to have the same strong lipid peroxide production inhibiting effect as vitamin E.

Production> 1) Preparation of Seed Mother The medium used was prepared by dissolving the following components in 1 liter of water and adjusting the pH to 7.4.

Glycerol 30.0゜Fishmeal 20. og calcium carbonate 2.0g 100ml of the above medium was dispensed into a 500ml Erlenmeyer flask with warts, and after sterilization, Streptomyces thalestomai superiorticus strain EC40 (FERM
BP-3032) from the slant to each flask.
Platinum loopfuls were inoculated and cultured with shaking at 27°C for 4 days, which was used as a preculture solution.

Next, an antifoam agent (CA 1
23) 30 liters of culture solution with 02% added
100 ml of the above preculture solution was added to the sterilized liter jar fermenter, and the mixture was heated at 27° C. for 3 days at 150 rpm.

The seed mother was cultured with aeration with stirring at 0.5 VVM.

2) Add 30 liters of the above seed mother to 800 liters of production medium having the same composition as that used in preparing the cultured seed mother and sterilize it into a 1.6 ton jar fermenter. Aeration stirring culture was performed at 80 rpm and 0.6 VVM for 6 days at °C.

3) Collection of EC40 After culturing under the above conditions, 23 kg of Celite is added to the culture solution (800 liters) and filtered through a furnace cloth to obtain bacterial cells. This bacterial body was extracted twice with 150 liters of acetone,
After concentrating the extract to 12 liters, the pH was adjusted to 2.
Extract twice with twice the amount of ethyl acetate. After the extract is dehydrated by adding anhydrous sodium sulfate and filtered, the filtrate is concentrated to obtain 300 ml of concentrate. After dissolving this in 300ml of chloroform, 5 liters of hexane was added,
The precipitate is filtered and the filtrate is concentrated. Silica gel made by equilibrating the concentrate with hexane (Wako Pure Chemical's "Wacoal Gel")
C-200J) column (I0 cmφ x 50 marks), and after flowing 5 liters of hexane, it was eluted with hexane-acetone (5:1).

The active fraction is concentrated to dryness to obtain 4 grams of crude EC40.

This crude sample was reused in a silica gel column (5 cmφ x 5
0 cm) and elute under the same conditions as above. The active fraction is concentrated to dryness to obtain 3.5 grams of crude EC40.

This crude sample is dissolved in a small amount of methanol, subjected to gel filtration using a Sephadex LH-20 column (5 cmφ x 100c+n), and developed with methanol. This gel filtration was performed three times, and both EC40 portions were concentrated to dryness to obtain 1.7 g of a purified EC40 product.

[Brief explanation of drawings]

FIG. 1 is a reproduction of the ultraviolet absorption spectrum of EC40 in methanol. FIG. 2 is a reproduction of the infrared absorption spectrum of EC40 obtained by the KBr disk method. FIG. 3 is a reproduction of the 500 MHz proton nuclear magnetic resonance spectrum of EC40 in heavy methanol. FIG. 4 is a reproduction of the 125 MHz carbon-13 nuclear magnetic resonance spectrum of EC40 in heavy methanol. Applicant's agent Mr. Sato

Claims (1)

[Claims] Compound EC40 represented by the following formula (I). ▲There are mathematical formulas, chemical formulas, tables, etc.▼(I)