JPH0739436B2 - Formestin, a novel physiologically active substance, and method for producing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a novel physiologically active substance formestin (Formestin) having inhibitory activity against N-formylmethionine aminopeptidase and A, B of formestin belonging thereto.
It relates to at least one process of the group consisting of C and D.

[Conventional technology]

Among physiologically active substances produced by microorganisms, it has been reported so far that Ebelactone A and Ebelactone B inhibit N-formylmethionine aminopeptidase [H.
Umezawa (H.UMEZAWA), etc. The Journal of Antibiotics Volume 33, Vol.
1594-1596 (1980)].

[Problems to be solved by the invention]

Ebelactone A and Ebelactone B inhibit N-formylmethionine aminopeptidase and strongly inhibit esterase (the above-mentioned document). Therefore, the specific N-
Inhibitors of formylmethionine aminopeptidase are desired.

An object of the present invention is to provide a physiologically active substance that does not have such problems and a method for producing the same.

[Means for solving problems]

Briefly describing the present invention, the first invention of the present invention relates to a novel physiologically active substance formestin, which has the following general formula: (Wherein R represents a hydroxyl group or hydrogen), an ester thereof, or a salt thereof.

The second invention of the present invention is an invention relating to a method for producing the novel physiologically active substance formestin A, B, C, D, which comprises culturing a formestin-producing bacterium belonging to the genus Actinoma de Yura and physiologically activating the physiological activity from the culture. At least one selected from the group consisting of the substances formestin A, B, C and D
It is characterized by separating and collecting seeds.

One of the formestin-producing bacteria belonging to the genus Actinomadera
An example is the radiant bacterium Actinomadura roseoviolacea MH511-0'F1 isolated from the soil of Hagi City, Yamaguchi Prefecture by the present inventors.

The mycological properties of this strain are as follows.

1. Morphology Under the microscope, the aerial hyphae, which are relatively longer than the branched basal hyphae, are elongated. At the tip of the aerial mycelium, about 10 spore chains are found, forming a hook or helix (sometimes a hard spiral). In addition, pseudospores (diameter about 2μ) are observed. The surface of the spores is smooth.

2. Growth status in various media Regarding the description of color, the standard shown in [] is the Container Harmony Manual (Container Corporation of America).
Color harmony manual).

(1) Sucrose / nitrate agar medium (27 ° C culture) Growth is colorless to pale yellow tea [21e, Mustard], aerial hyphae do not grow, and soluble pigments are not found.

(2) Glucose / asparagine agar medium (27 ° C culture) Growth is extremely slow, and colorless growth is slightly observed around 21 days after culture. No aerial hyphae or soluble pigments are found.

(3) Glycerin / asparagine agar medium (2 with isp-medium 5
(7 ° C culture) Growth is colorless to pale yellow, aerial hyphae are not cured, and soluble pigments are not found.

(4) Starch / inorganic salt agar medium (cultivated at 27 ° C. in isp-medium 4) Growth is colorless to light yellow tea, aerial hyphae do not grow, and soluble pigments are not found.

(5) Tyrosine agar medium (cultivated at 27 ° C in isp-medium 7) Growth is colorless to pale yellow, aerial hyphae do not grow, and soluble pigments are not found.

(6) Nutrient agar medium (27 ° C culture) Growth of light yellow tea [21e, Mustard to 31e, Cinnamon], aerial hyphae did not settle, and soluble pigments were not found.

(7) Yeast-malt agar medium (cultivated in isp-medium 2 at 27 ° C) Nibu red daidai [5pg, Henna] to nibu red [6pg, Barn Re
For the development of d], pink-white aerial mycelia are settled. Soluble dyes are not found. The color of the rear surface of growth changes from nib red to magenta with 1N NaOH, and from nib red to pale with 1N HCl.

(8) Oatmeal agar medium (cultivated in isp-medium 3 at 27 ° C) White to pink aerial hyphae are grown on the development of light pink [5gc, Peach Tan]. Soluble dyes are only slightly pinkish. In addition, the color and the soluble pigment on the backside of development change from gloomy red to magenta with 1N NaOH and from gloomy red to pale with 1N HCl.

(9) Glycerin / nitrate agar medium (27 ° C culture) On the development of colorless to light yellow, white aerial mycelia grow very slightly from about 10 days after culture. Soluble dyes are not found.

(10) Starch agar medium (27 ° C culture) White aerial mycelium grows lightly on the development of colorless to light yellow sardine. Soluble dyes are not found.

(11) Lime malate agar medium (27 ° C culture) On the development of colorless to light yellow, white aerial hyphae grow slightly from the 21st day after culture.

Soluble dyes are not found.

(12) Cellulose (27 ° C culture) Growth is colorless, aerial hyphae do not grow, and soluble pigments are not found.

(13) Gelatin stab culture Both simple gelatin medium (20 ° C culture) and glucose / peptone / gelatin medium (27 ° C culture) develop colorless to light tea, aerial mycelium does not grow, and soluble dye is not found.

(14) Defatted milk (37 ℃ culture) Growth is bright tea [4ne, Luggage Tan ~ 5ne, Tile Red],
The aerial mycelium does not settle and no soluble pigment is found.

3. Physiological properties (1) Growth temperature range Oatmeal agar medium (Quaker, White Oat
s) oatmeal 1.4%, string agar 3.0%, pH 7.2) and tested at each temperature of 20 ℃, 24 ℃, 27 ℃, 30 ℃, 37 ℃ and 50 ℃, except 50 ℃. It grew at any temperature. The optimum temperature for growth seems to be around 30 ℃ -37 ℃.

(2) Liquefaction of gelatin (15% simple gelatin medium, 20 ° C culture; glucose-peptone-gelatin medium, 27 ° C culture) In the simple gelatin medium, liquefaction was not observed after 29 days of observation. In glucose-peptone-gelatin medium, liquefaction was observed from the first day after culturing, but its action was extremely weak.

(3) Hydrolysis of starch (starch / inorganic salt agar medium and starch agar medium, both at 27 ° C.) Not observed in any medium.

(4) Coagulation / peptonization of skim milk (skim milk, 37 ° C. culture) From the 14th day after cultivation, the milk becomes a solidified state, and the coagulation is completed immediately and the peptone starts. Peptonization is completed in about 2 weeks and its effect is moderate.

(5) Production of melanin-like pigment (tryptone yeast broth, isp-medium 1; peptone yeast iron agar medium, is
p-medium 6; tyrosine agar medium, isp-medium 7, both 27 ° C
Culture) Negative in any medium.

(6) Utilization of carbon source (Pridham-Gotrieve agar medium, isp-medium 9 at 27 ° C.) L-arabinose, D-xylose, D-glucose, D-fructose, L-rhamnose , Raffinose is not used. D-mannitol is likely to be utilized and inositol is probably not. It is not clear whether Syuclaus is used or not.

(7) Dissolution of lime malate (lime malate agar, 27 ° C)
(Culture) Dissolution of lime malate is not observed.

(8) Reduction reaction of nitrate (peptone water containing 0.1% potassium nitrate,
27 ° C. culture in isp-medium 8) Positive.

To summarize the above properties, the MH511-0'F1 strain has a well-extended aerial hyphal helix and a specific pseudosporangium. The surface of spores is smooth.

On yeast-malt agar medium and oatmeal agar medium, white to pink-white aerial hyphae grow on thin pink to dark red growth. Soluble dyes are pink on oatmeal agar. In addition, the color tone of the growth back surface of the above-mentioned medium and the soluble pigment change in color tone depending on pH.

In other cultures, the growth is colorless to pale yellow tea, aerial hyphae and soluble pigments are hardly observed.

The formation of melanin-like pigment is negative. The water degradability of starch was not found, and the proteolytic activity was moderate to weak.

The MH511-0'F1 strain was obtained by Lechevalier et al.
al, International Journal of Systematic Bacteriology (International Journal
of Systematic Bacteriology), 20, 435, 1970)
3 shows Type III B, which is a major component of the cell wall advocated by. That is, it was confirmed that the whole cells contained meso-2.6-diaminopimelic acid and glucose, mannose, ribose, and magiculose as sugar components.

From the above points, the strain MH511-0'F1 is
tinomadura) is considered to be actinomycetes. Based on these properties, as a result of searching for known bacterial species, MH511 was found as a species closely related to the 0'F1 strain, Actinomadura roseoviolacea (Actinomadura roseoviolacea, Reference: Nonomura et al., Journal of Fermentation Engineering, Vol. 49, Vol. 904, 1971).

Therefore, the MH511-0'F1 strain was compared with Actinomajiyura roseoviolase IMC A-0115 (IFO14098). The outline of the results is shown in Table 1 together with the literature description.

As is clear from Table 1, the MH511-0'F1 strain and Actinomadyura roseoviolase exhibited the same properties.
There is a slight difference in the use of sucrose and inositol, but since it is unclear whether or not they are used,
I can't think of a big difference. Furthermore, the cell wall of this strain exhibits a type III B component, exhibits pseudosporal formation, and develops a reddish tinge, and the physiological properties are also in agreement.

From these facts, the MH511-0'F1 strain was considered to be a species closely related to Actinomadillura roseoviolase. Therefore, the MH511-0'F1 strain was identified as Actinomadura roseoviolacea MH511-0'F1.

The MH511-0'F1 strain was applied for deposit at the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology, and on April 1, 1986, it was accepted as Microindustrial Research Institute of Microbiology No. 8721 (FERM P-8721). As the strain which can be used in the present invention, all of the above-mentioned strains, mutants thereof, and formestin-producing bacteria belonging to the genus Actinoma deyura can be used.

Follow up during the culturing process and purification process of formestin
It was performed based on the measurement of anti-N-formylmethionine aminopeptidase activity by the following method. For its measurement, H.SUDA et al. Biochimica et Biophysica Acta Vol. 616, 60-67
The measurement method described on page (1980) was used. That is, 20 mM N-
Formylmethionine-β-naphthylamide (methionine-β-naphthylamide from Batchem Co. in formic acid-acetic anhydride
N-formylated and crystallized from methanol-water)
Methanol solution [5% (w / v) Tween 20
0.1M Tris-HCl buffer (pH 7.8) in 0.005 ml.
A mixture of 5 ml, 0.1 ml of a solution containing the sample and 0.345 ml of water was heated at 37 ° C for 3 minutes and then purified by DEAE-cellulose column chromatography to obtain 0.05 ml of N-formylmethionine aminopeptidase solution. In addition, 37 ℃ 30
Let react for minutes. After the reaction, 1 ml of 1M sodium acetate buffer (pH 4.2) containing 10% of Tween 20 and 0.1% of Farst Garnett GBC salt was added to stop the reaction, and the absorbance (a) at 525 nm was measured. At the same time, the absorbance (b) of the control when the same reaction was performed except for the sample was measured. In addition,
At this time, the blinded absorbance (a ') corresponding to each
(B ') was also measured. In the blind test, a buffer solution (pH 4.2) was first added, and then an enzyme was added to develop a color without performing an enzyme reaction. The N-formylmethionine aminopeptidase inhibitory activity (%) is calculated by the formula [1- (aa ') / (b-
b ′)] × 100.

Formestin A and formestin B pure by this assay
50% inhibition (IC ₅₀ ) of N-formylmethionine aminopeptidase at concentrations of 0.05 μg / ml and 0.14 μg / ml, respectively. However, neither formestin A nor formestin B inhibited esterase at a concentration of 100 μg / ml.

Formestin C at a concentration of 0.0016 μg / ml and formestin D at a concentration of 1.0 μg / ml inhibited N-formylmethionine aminopeptidase by 50%.

A formestin-containing culture is obtained by inoculating a nutrient source-containing medium with a formestin-producing bacterium belonging to the genus Actinoma deyura and aerobically developing it. As a nutrient source, one that can be used as a nutrient source for actinomycetes is used. For example, commercially available peptone, meat extract, corn staple liquor, cottonseed flour, peanut flour, soybean flour, yeast extract, NZ-amine, hydrolyzate of casein, sodium sulfate, ammonium sulfate, nitrogen sources such as ammonium sulfate, and Commercially available glycerin, sucrose, starch, glucose, galactose, mannose, carbohydrates such as molasses, or carbon sources such as fat, and salt, phosphate,
Inorganic salts such as calcium carbonate and magnesium sulfate can be used. If necessary, a trace amount of metal salt, antifoaming agent, plant, mineral oil and the like can be added. Any of these may be used as long as it can be used by the producing strain and is useful for the production of formestin, and any known culture material for actinomycetes can be used. Liquid culture is preferable for the large-scale production of formestin, and the culture temperature is usually 20 to 40 ° C., preferably 27 to 37 ° C., which can be applied within the range where the producing bacteria grow and formformin is produced. Cultivation can be appropriately selected and performed according to the properties of the formestin-producing bacterium using the conditions described above. Formestin is present in both culture medium and cells. The culture solution can be extracted with a water-immiscible organic solvent such as ethyl acetate or butanol at a pH of 2 or less. The cells can be extracted with an organic solvent such as methanol or acetone, the extract is concentrated under reduced pressure, and solvent extraction can be performed in the same manner as the culture solution. In addition to the above-mentioned extraction method, a known method used for collecting a fat-soluble substance, for example, adsorption chromatography, gel perchromatography, scraping from thin layer chromatography, high performance liquid chromatography, etc. is appropriately combined. Alternatively, it can be collected purely by repeating.

Formestin A, B, C and D shape, mass spectrometry, molecular formula, specific rotation, ultraviolet absorption maximum, infrared absorption maximum, solubility, silica gel thin layer chromatography and reversed phase ODS
The thin layer chromatographic Rf values are shown in Table 2 for formestin A and B and in table 3 for formestin C and D.

Further, FIG. 1 shows formestin A, FIG. 2 shows formestin B, FIG. 3 shows formestin C, and FIG. 4 shows formestin D.
3 shows the infrared absorption spectrum of the potassium bromide tablet of.
In each figure, the horizontal axis represents the wave number (cm ^-1 ) and the vertical axis represents the transmittance (%). And in FIG. 5, formestin A and 6
FIG. 7 shows the proton nuclear magnetic resonance spectra of formestin B in FIG. 7, formestin C in FIG. 7 and formestin D in deuterated dimethyl sulfoxide, respectively. In each figure, the horizontal axis represents chemical shift (ppm). The internal standard is tetramethylsilane.

Furthermore, Table ¹³ shows the ¹³ C nuclear magnetic resonance spectrum data of deuterated dimethyl sulfoxide of formestin A, B, C and D.

From the above physicochemical properties, formestin A, B, C and D have the structural formulas of formula II to formula V, respectively.

Examples of salts of formestin are salts with pharmaceutically acceptable cations at the carboxyl group of formestin, for example alkali metal such as sodium, potassium, alkaline earth metal cations such as calcium, magnesium. Examples of the ester include an intramolecular ester, an alkyl ester, an acetoxymethyl ester and a pivaloyloxymethyl ester which are easily metabolized and decomposed in vivo as an ester with a carboxyl group of formestin. Further, as the ester with the hydroxyl group of formestin, there are organic acid esters with formic acid, acetic acid, propionic acid, valeric acid, isovaleric acid and the like.

Formestin is a substance with low toxicity. In an acute toxicity test on mice, formestin A
No deaths were observed even if 0 mg / kg was administered or 500 mg / kg of formestin B was administered.

〔Example〕

Next, the present invention will be described with reference to examples, but the present invention is not limited thereto.

In addition,% in each example is weight%.

Example 1 Glucose 1.0%, glycerol 1.0%, potato starch 1.0 from Actinoma deyura roseoviolase MH511-0'F1 strain (Microtech Lab. No. 8721) cultured in an agar slant medium.
%, Corn staple liquor [Nippon Shokuhin Kako Co., Ltd.]
Made] 1.0%, peptone [Polypeptone manufactured by Daigo Nutrition Chemical Co., Ltd.] 0.5%, yeast extract 0.2%, calcium carbonate 0.32%
One platinum loop was inoculated into four Erlenmeyer flasks equipped with waffles, each of which was dispensed with 110 ml of the liquid medium (pH 7.4), and the mixture was shake-cultured at 30 ° C. for 5 days. Using it as a seed culture, tomato paste 2.4%, dextrin 2.4%, yeast extract 1.2%,
Liquid medium consisting of 0.006% cobalt chloride hexahydrate (pH 7.
(3) was inoculated into 90 waffle Erlenmeyer flasks each containing 110 ml of 0) and cultured at 27 ° C. for 5 days with shaking.

The liquid and the bacterial cells were separated by filtration, and the liquid was extracted with an equal volume of ethyl acetate at pH 2. The cells were extracted with methanol (2), concentrated under reduced pressure, and extracted with ethyl acetate (2) at pH 2. This was combined with the ethyl acetate extract of the liquid, an equal amount of water was added, and the mixture was redissolved in water at pH 8. The aqueous layer was separated and extracted again with an equal volume of ethyl acetate at pH 2. This is concentrated under reduced pressure 5.
32 g of brown candy was obtained. To this, 320 ml of water and 80 ml of 1N sodium hydroxide solution were added, stirred and dissolved.
After neutralizing this with hydrochloric acid, 5% potassium acetate, 1% citric acid.
Dilute 2 times with a buffer solution containing monohydrate (referred to as buffer solution A) and apply it to a 180 ml reversed-phase silica gel column (silanized silica gel 60 manufactured by Merck) equilibrated with buffer solution A for chromatography. I went. Elution was performed with buffer solution A1 and then with acetonitrile-buffer solution A (1: 5) to fractionate 15 g each.
Fractions No. 89 to 189 having enzyme inhibitory activity were collected, concentrated under reduced pressure, acetonitrile was distilled off, and water was added to adjust to 2. This was applied to 115 ml of Diaion HP-20 [Mitsubishi Kasei Kogyo Co., Ltd.] for desalting, and 10% citric acid monohydrate aqueous solution 1,
After flowing water 1, the mixture was eluted with methanol. The active fractions were collected and concentrated under reduced pressure to obtain 1.36 g (IC ₅₀ = 0.2 μg / ml) of crude powder. This was dissolved in a small amount of methanol, 10 g of cellulose powder (Avicel manufactured by Asahi Kasei) was added, and the mixture was concentrated under reduced pressure to give a dry powder. This was applied to a 200 ml silica gel column filled with butyl acetate and chromatographed.
First, butyl acetate-butanol-acetic acid-water (24: 8: 1: 1) 1
Then, elution was carried out with (6: 4: 1: 1) 1 and then (3: 4: 1: 1) 0.5, and fractions of 15 g were fractionated. The active substance is bimodal, and the active fractions No. 36 to 84 which are eluted first are collected and concentrated under reduced pressure to 0.46 g.
The crude powder I of was obtained. Active fraction to be eluted later No.87-145
Were collected and concentrated under reduced pressure to obtain 0.39 g of crude powder II. Each was separately dissolved in a small amount of methanol and applied to a column (1.6, diameter 4 cm) of Sephadex LH-20 (manufactured by Pharmacia) swollen with methanol in advance, eluted with methanol, and fractionated by 15 g each. When using the coarse powder I,
Active fractions Nos. 58-60 were collected and concentrated under reduced pressure to obtain 0.35 g of crude powder of formestin B. When crude powder II was used, active fractions Nos. 53 to 70 were collected and concentrated under reduced pressure to obtain 0.26 g of crude powder of formestin A.

Next, 0.26 g of this crude powder of formestin A was subjected to high-speed chromatography (manufactured by Senshu Scientific Co., Ltd., Senshu Pack, Nucleosil 5C ₁₈ , 20φ × 300 mm) for purification. As the mobile phase, a mixture of acetonitrile-buffer A (described above) = 1: 3 was used, and the active fraction showing a peak at 67 minutes at a flow rate of 10 ml / min was collected and concentrated under reduced pressure to remove acetonitrile. After that Diaion HP-20 (50m
l), 10% citric acid monohydrate aqueous solution (350 ml) and water (650 ml) were flown, and then the mixture was eluted with methanol. The active fraction was concentrated under reduced pressure to obtain 0.12 g of powder. This was dissolved in a small amount of methanol, applied to a column (1.6, diameter 4 cm) of Sephadex LH-20 (manufactured by Pharmacia) and eluted with methanol to fractionate 15 g each. Active fractions Nos. 58-65 were collected and concentrated under reduced pressure to obtain 113 mg of pure formestin A white powder.

The crude powder of formestin B (0.35 g) was also purified by the same purification method using the same column, and the active fractions were collected to obtain 122 mg of pure formestin B (white powder).

Example 2 Glucose 1.0%, glycerol 1.0%, potato starch 1.0 from Actinoma deyura roseoviolase MH511-0'F1 strain (Microtechnology Research Institute No. 8721) cultured in an agar slant medium.
%, Corn steep liquor [Nippon Shokuhin Kako Co., Ltd.]
A liquid medium (pH 7.4) consisting of 1.0%, peptone [Polypeptone manufactured by Daigo Nutrition Chemicals Co., Ltd.] 0.5%, yeast extract 0.2%, calcium carbonate 0.32% was dispensed in 110 ml aliquots into four waffle Erlenmeyer flasks. Each platinum loop was inoculated and shake cultured at 30 ° C. for 6 days. Glycerol as a seed culture
1.5%, soluble starch 1.5%, soybean powder [Ajinomoto Co., Ltd. Prolithi] 0.5%, fish meal 1.5%, calcium carbonate 0.2%
Inoculate 20 ml of 10 waffle Erlenmeyer flasks (2) into which 500 ml of liquid medium consisting of
The cells were shake-cultured for 3 days. Using this as a seed culture, 4 seeds were inoculated into 200 fermenters containing the same medium (latter) 100.
Culturing was carried out for 3 days under the conditions of aeration rate of 100 rpm and agitation rate of 150 rpm.

After completion of the culture, the culture was heat-treated at 95 ° C for 10 minutes, cooled, passed through a filter press, and liquid 100 was applied to a column of Diaion HP-20 (3.5) and washed with water 15,
Elution was performed with 80% methanol water 8. The eluate was concentrated under reduced pressure to obtain Concentrate 1 (IC ₅₀ = 0.00115 μg / ml). To this, 170 ml of a 10% aqueous solution of citric acid monohydrate, 425 ml of a 20% aqueous solution of potassium acetate, 340 ml of acetonitrile, 100 ml of water, and 500 ml of buffer A (described above) were added. This was applied to a 450 ml reversed-phase silica gel column [silanized silica gel 60 (described above)] equilibrated with buffer A, and eluted with acetonitrile-buffer A (1: 5) 4 to fractionate 18 g each. Then, it was eluted with acetonitrile-buffer A (4:15) 2 and fractionated in 16 g portions. Active fractions No. 46-130 (fraction containing formestin A and B) and No. 171-290 (fraction containing formestin C and D) were collected. Fraction containing formestin C and D
Citric acid monohydrate 50 to 2.1 (IC ₅₀ = 0.0088μ / ml)
0 g was added and dissolved to adjust the pH to 2.8. This was extracted twice with an equal volume of ethyl acetate, the extracts were combined and concentrated under reduced pressure to give 34.74 g.
An extract (IC ₅₀ = 2.33 μg / ml) was obtained. To this, 100 ml of acetonitrile and 500 ml of buffer A were added and dissolved, and the mixture was again applied to a column of silanized silica gel 60 (450 ml) for purification. Acetonitrile-buffer A (1: 5) (600 ml) was eluted, and then (4:15) 3 was eluted, and 16 g fractions were fractionated. Active fraction No.
181-270 were collected to give a solution of 1380 ml (IC ₅₀ = 0.11 μ / ml). Water was added to this to 3.5, and it was applied to a column (100 ml) of Diaion HP-20 that had been equilibrated with 0.5 M potassium chloride solution in advance. 200 ml of 0.5 M potassium chloride solution, 500 m of water
After washing with l, it was eluted with 80% methanol water. The active fractions were collected and concentrated under reduced pressure to obtain 0.26 g (IC ₅₀ = 0.024 μg / ml) of crude powder. To this, 3 ml of acetonitrile and 7 ml of buffer A are added and dissolved, and the supernatant obtained by removing the precipitate by centrifugation is subjected to high performance liquid chromatography (Wattman's Partidyl 10-ODS-3, Magnum 20 column, 22φ x 500 mm) for purification. I went. A mixture of acetonitrile-buffer A = 3: 7 was used as a mobile phase and was eluted at a flow rate of 9 ml / min to fractionate 9 ml.

Active fractions containing formestin C Nos. 101-120 (145 ml, IC
₅₀ = 0.04 μ / ml) and formestin D containing active fraction No.
A solution of 122-150 (214 ml, IC ₅₀ = 0.42 μ / ml) was obtained.
Each was diluted 3 times with water and applied to a column of Diaion HP-20 (50 ml) that had been equilibrated with 0.5 M potassium chloride solution in advance, and 100 ml of 0.5 M potassium chloride solution and 300 ml of water were added.
After washing with ml, each was eluted with 80% methanol water. The active fractions were collected and concentrated under reduced pressure to obtain 36.5 mg of crude powder of formestin C (IC ₅₀ = 0.0065 μg / ml) and 0.11 g of crude powder of formestin D (IC ₅₀ = 0.193 μg / ml). Then, 36.5 mg of crude powder of formestin C was dissolved in 3 ml of 80% methanol water and applied to a column (560 ml) of Sephadex LH-20 preliminarily equilibrated with 80% methanol water, and eluted with 80% methanol water to obtain the active fraction. Collect and concentrate under reduced pressure 10.2 mg
A powder of potassium salt of formestin C (IC ₅₀ = 0.0018 μg / ml) was obtained.

Dissolve this in a very small amount of methanol and add 20 ml of ethyl acetate.
And 10% citric acid in water (20 ml) were added and solvent extraction was performed.
The mixture was extracted with ml of ethyl acetate, the extracts were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. This was dissolved in a small amount of methanol and equilibrated with methanol Sephadex LH-20
It was applied to a column of (50 ml) and eluted with methanol. The active fractions were collected and concentrated under reduced pressure to give 6.9 mg of pure formestin C.
Of white powder was obtained. This formestin C is 0.0016μg / ml
N-formylmethionine aminopeptidase at a concentration of 50
% Inhibition.

0.11 g of crude powder of formestin D obtained by high performance liquid chromatography was added to 0.6 ml of acetonitonil and buffer solution A1.4.
High-performance liquid chromatography (manufactured by Wattman Co., Pertidyl 10-ODS-2, Magnum 9, 9.4φ x 250)
mm) and divided into two times for purification. Acetonitrile-buffer A = 3: 7 was used as the mobile phase. Elute at a flow rate of 2 ml per minute to 2 m
Fractionated by l. Fractions of formestin D No. 21-27 were collected and diluted with water to 200 ml. Reversed phase silica gel (YMC-GEL-ODS,
Yamamura Chemical Laboratory) 50 ml column, washed with 100 ml of 10% citric acid monohydrate in water and 300 ml of water, then eluted with 80% methanol and concentrated under reduced pressure to 93 mg (IC ₅₀ = 1.1 μg / ml)
To obtain crude powder of formestin D. This is methanol 3m
It was dissolved in l and applied to a column of Sephadex LH-20 (560 ml) and eluted with methanol. Collect the active fractions and concentrate under reduced pressure.
4.3 mg of pure formestin D white powder was obtained. This formestin D inhibited N-formylmethionine aminopeptidase by 50% at a concentration of 1.0 μg / ml.

〔The invention's effect〕

As described in detail above, the present invention provides a novel physiologically active substance formestin that specifically inhibits N-formylmethionine aminopeptidase.

It was recognized that formestin has a humoral antibody production-enhancing effect in mice and is a substance that enhances immunocompetence. Therefore, it is expected to enhance the antibody-dependent cancer cell-damaging activity, and has utility as an antitumor immunostimulator. Furthermore, it is expected to have an effective action against bacterial infections.

[Brief description of drawings]

1 to 4 are infrared absorption spectra of potassium bromide tablets of formestin A, B, C and D, and FIGS. 5 to 8 are formestin A, B, C and D, respectively.
FIG. 3 is a proton nuclear magnetic resonance spectrum of deuterated dimethylsulfoxide of.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Office reference number FI technical display location (C12P 21/02 C12R 1:03)

Claims (6)

[Claims] 1. The following general formula I: A physiologically active substance formestin, an ester thereof, or a salt thereof, which is a compound represented by the formula (wherein R represents a hydroxyl group or hydrogen). 2. A compound represented by the general formula I is represented by the following formula I:
I: The physiologically active substance formestin A according to claim 1, which is a compound represented by: 3. A compound represented by the general formula I is represented by the following formula II:
I: The physiologically active substance formestin B according to claim 1, which is a compound represented by the formula (1). 4. A compound represented by the general formula I is represented by the following formula I:
V: The physiologically active substance formestin C according to claim 1, which is a compound represented by: 5. A compound represented by the general formula I is represented by the following formula V: The physiologically active substance formestin D according to claim 1, which is a compound represented by the formula (1). 6. A method for producing a formestin-producing bacterium belonging to the genus Actinoma de Yura, wherein at least one selected from the group consisting of physiologically active substance formestin A, B, C and D is separated and collected from the culture. A method for producing A, B, C and D of the physiologically active substance formestin.