JPH0272185A - Novel substance ks-502 and production thereof - Google Patents

Abstract

NEW MATERIAL:A compound expressed by the formula having the following physicochemical properties. Property: white powder.Melting point; 119-120 deg.C, Specific rotatory powder: [alpha]D<23>=-45 deg. (C=0.3, methanol). Solubility; readily soluble in methanol, n-butanol, acetone, THF, dioxane, dimethyl sulfoxide, pyridine, monoethanolamine, acetic acid and alkaline water, soluble in chloroform, ethyl acetate and acetonitrile and insoluble in hexane, carbon tetrachloride, benzene, neutral water and acidic water. Color reaction; positive to iodide, anisaldehyde and ferric chloride reaction, negative to aniline phthalic acid, ninhydrin and Rydon-Smith reaction, etc. USE:Having an action controlling serotonin release of platelets and an antiamnesia action. PREPARATION:A microorganism belonging to the genus Sprothrix such as Sprothrix sp. KAC-1985 (FERM BP-1278) is cultured at 15-25 deg.C and neutral pH for 3-15 days.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to a novel substance produced by a microorganism belonging to the genus Sporothrix, which has an effect of inhibiting serotonin release from platelets and an anti-amnestic effect, and a method for producing the same.

BACKGROUND OF THE INVENTION The following substances have been reported as substances produced by microorganisms that have an effect of inhibiting serotonin release or aggregation of platelets.

・Pyrrothines Chemical and Pharmaceutical Bulletin (Chem,
Pharm, Bull, ) 28.3157-316
2thiolutin: R=cL aureothric+n: R
-CLCH3・'vVF-5239 Journal of Antibiotics (J, ^nti
biot, > 37, 469-474 (1984)-
Staurosporine Agricultural and Biological Chemistry (＾gric, Biol, Chem,) 50°NF (CHO ・W+'-30581 Journal of Antibiotics (J, Anti
Bioshi, ) 37, 1153-1160 (1984
) ■ 1IF-30581A: R=CH, C112C) 13W
F-30581B: R=CH2CH, CHffCH3・
KS-290II i-2 JP-A-61-195689 In addition, no substance produced by microorganisms that has an anti-amnestic effect is known.

Problems to be Solved by the Invention There is a constant need for substances that have an effect of inhibiting platelet serotonin release, an aggregation inhibitory effect, or an anti-amnestic effect.

Means to Solve the Problems With the aim of providing useful new physiologically active substances that can be used as pharmaceuticals or their intermediates, we have conducted research on the products of numerous microorganisms obtained from the natural world. ,
We have discovered that a newly isolated microorganism produces a physiologically active substance that inhibits platelet serotonin release and has an anti-amnestic effect. Isolating and purifying the physiologically active substance,
After examining its physical and chemical properties, it was discovered that it is a new substance. Hereinafter, this substance will be referred to as KS-502.

The present invention will be explained in detail below.

The present invention is a novel substance KS- having the following physical and chemical properties.
502 and a method for producing the same.

Properties: White powder Melting point: 119-120°C Upper optical rotation: [α:l; 3-45° (cO
, 3, methanol) Solubility: Easily soluble: methanol, n-butanol, acetone, tetrahydrofuran, dioxane, dimethyl sulfoxide, pyridine, monoethanolamine, acetic acid, alkaline water Soluble: chloroform, ethyl acetate, acetonitrile Insoluble: hexane, Carbon tetrachloride, benzene, neutral water, acidic water Color reaction: Positive for each reaction of iodine, anisaldehyde, ferric chloride, negative for each reaction of aniline phthalate, ninhydrin, Lydon Smith Ultraviolet absorption spectrum: Acidic and neutral methanol solution: λ□N(ε)252n
m (14,100), 282 nm (6600, sh)
, 300 nm (5400, sh) Alkaline methanol solution: λ1. i(ε)234nm
(18,900,sh), 298nm (24,800
) Infrared absorption spectrum: KBr 3450, 2912.2940.23n, IT24.
1595°1463, 1432.1375.1343.
1245.1161°1135,1062c '' Mass spectrum: S rMs, negative m/z
:647(M-1)-', 392.251NMR spectrum: 'H-NMR (400MHz, CD, OD, δ)
:6.60 (IH, d, J=2.3Hz), 6.
58 (IH, d, J=2.1Hz), 6.51 (
IH, d, J=2.3Hz), 6.38 (I
N, d, J=2.1tlz), 5.54 (
IH, d.

J4.8H2), 4.27(1)1. dd),
ca, 4.1 (2fl).

3, 76 (LH, br t, J=5.9Hz),
3.65 & 3.61 (2H, AB+n ABX,
, bill, 0, JAX=5.6. Jex=7.
0H2).

3.09 (2H, br, dd), 2.65 (2H,
br, dd), ca, 1.5 (4N, m>,
1.2-1.4 (16H), 0.88 (3H, t
, J=6.6).

0.87 (3H, t, J=6.7) 3C-NMR (100MHz, CD, 00. δ)
: 176.6. 168.2°164.3. 161
．． 2. 157.3. 154.6. 149.6゜1
44.8. 116.0. 115.7. 115.6
．． 111.1°108.6. 108.4. 102
．． 0. 85.5. 83.5. 78.6°72.2
．． 64.4. 36.5. 34.9. 33.2.
33.1°33.0. 32.6. 31.0. 3
0,? , 30.5. 30.3°23.74. 2
3.70. 14.46. From the physical and chemical properties of 14.43 and above, it was found that KS-502 is a new substance represented by the following formula.

Next, Table 1 shows the Rf values of thin layer chromatography of KS-502 using various developing agents.

Detection was performed by irradiating ultraviolet light at 253.7 nm.

Table 1 Developing solvent acetone Rf value 0.12 KS-502 has platelet serotonin release inhibiting action and anti-amnestic action.

Next, a method for manufacturing KS-502 will be explained.

KS-502 is Sporothrix (Sporothrix)
i) A microorganism belonging to the genus X) and having the ability to produce KS-502 is cultured in a medium, and in the culture, KS-502 is mainly contained in the bacterial cells.
It is produced by producing and accumulating KS-502 and collecting KS-502 from the culture.

The KS-502-producing microorganism is Sporothrix (
Any microorganism may be used as long as it belongs to the genus Sporothrix and has the ability to produce KS-502. As a specific preferred example, Sporothrix sp.
sp,) KAC-1985 strain (hereinafter referred to as KAC-19
85).

The mycological properties of KAC-1985 are as follows.

(1) Growth status on each medium The growth status shown below is shown on both malt extract agar medium and potato glucose agar medium.

Growth is relatively slow, and the diameter of the colony reaches 29 to 33 mm after 30 days of culture at 20°C. The colony is dome-shaped, and both the front and back sides of the colony are white or cream-colored.

Hyphae have septa and extend in and on the medium. The hyphae are colorless, smooth, and well branched, with a diameter of 1 to 6 μm, and hyphal fusions are occasionally observed.

Conidiophores are colorless, smooth, 4.5-20 μm long, 1 wide
．． It is 5 to 2.5 μm and may have partition walls. In some cases, conidia are formed symposially at the tip of the conidiophore, and then the conidiophore is elongated. Conidia are single cells, colorless, smooth, and oblong in shape, with a length of 4 to 7 μm, rarely reaching 10 μm, and a width of 1 to 2 μm. Note that a complete generation of this strain was not observed.

(Support)] Physiological properties Growth temperature: 5-25°C Optimal growth temperature: 15-25°C Growth pH: 3-11 Optimal growth pH: 4-9 Based on the above mycological properties, classification of KAC-1985 The academic position in The General of Fanjai Sporrating in Pure Culture, 2nd edition (The
Genera of Fungi Sporulatin
g in Pure Culture, 2nd ed
Cramer.

Vaduz, J.A., von Arx, 1974). As a result, this strain was found to be Sporothrix sp.
was recognized as belonging to The present inventor named this strain Sporophrynx sp. KAC-1985,
It has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology (August 4, 2017).

When culturing microorganisms, usual culture methods used for culturing fungi are applied. The medium to be used may be either a natural medium or a synthetic medium as long as it contains a suitable amount of carbon sources, nitrogen sources, inorganic substances, etc. that can be assimilated by the bacteria.

Carbon sources include carbohydrates such as glucose, fructose, stabilose, saccharose, lactose, 78m, f-kistrin, mannose, maltose, molasses, and mash potato base, organic acids such as citric acid, malic acid, acetic acid, and fumaric acid, and glutamic acid. Amino acids, glycerol, cottonseed oil, etc. are used.

Ammonium chloride, ammonium sulfate,
Ammonium salts such as ammonium nitrate and ammonium phosphate, amino acids such as aspartic acid, glutamine, cystine, and alanine, urea, malt extract, peptone, meat extract, yeast extract, dried yeast, corn steep liquor, soy flour, and cottonseed meal. , soybean casein, cadiminoic acid, Pharmamedia, soluble benzitapur protein, vegetable and fruit juices, etc. are used.

Inorganic substances include potassium dihydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate, ferrous sulfate, manganese sulfate, cobalt sulfate, zinc sulfate, calcium pantothenate, ammonium molybdate, potassium aluminum sulfate, barium carbonate, calcium carbonate, Cobalt chloride, sodium chloride, magnesium phosphate, etc. are used.

In addition, substances that promote bacterial growth or KS-502 production, such as vitamins and thiamine, can be added to the medium as necessary.

If the microorganism used requires specific substances for growth, it is necessary to add substances necessary for growth.

Culture is carried out by shaking culture method, aeration agitation culture method, etc.
It is carried out at a temperature of 5° C. and a pH around neutrality. After 3 to 15 days of culture, the accumulation of KS-502 reaches its maximum and the culture is completed.

When the accumulated KS-502 is isolated and collected from the bacterial cells, a conventional method for collecting physiologically active substances from the bacterial cells is applied.

That is, obtaining bacterial cells by filtration, centrifugation, etc., extraction from bacterial cells with organic solvents such as methanol or acetone, distribution with water or two or more organic solvents, adsorption resin, silica gel, silanized silica gel, aluminum, cellulose. , K by adsorption/desorption treatment of active substances by column chromatography or thin layer chromatography using diatomaceous earth, magnesium silicate, gel filtration agents, etc.
S-502 can be isolated.

An example of isolating KS-502 from bacterial cells is as follows.

Bacterial cells are obtained by filtering or centrifuging the culture solution. After adding an organic solvent such as methanol to the obtained bacterial cells and stirring them thoroughly, the bacterial cells and the solution or supernatant liquid are separated again by filtration or centrifugation. The solvent is evaporated from the obtained p liquid or supernatant liquid under reduced pressure and concentrated to obtain an aqueous solution. This aqueous solution is then extracted using a suitable water-immiscible solvent such as ethyl acetate.

After concentrating the extract under reduced pressure, silica gel chromatography is performed using a mixed solvent of chloroform and methanol as a developing solvent.

KS-5 by increasing the methanol content stepwise.
Elute 02. Both fractions containing KS-502 are collected, concentrated under reduced pressure, and subjected to column chromatography using Sephadex LH-20 (manufactured by Pharmacia). KS-50
Both fractions containing 2 were collected and concentrated to dryness under reduced pressure to obtain a white powder of KS-502.

Detection of KS-502 during the above purification process was performed using silica gel thin layer chromatography followed by iodine reaction or 253.
This was carried out using a 7 nm ultraviolet irradiation method.

Examples are shown below.

Sporothrix sp.
othrix sp,) KΔC-1985 is used.

Glucose 1.0g/d! , peptone (manufactured by Kyokuto Pharmaceutical Industries) 0.5 g/di, dry yeast Ebios (manufactured by Asahi Beer Co., Ltd.) 0.5 g/dI2, V-8 vegetable juice (manufactured by Campbell >0.2 d1/J, calcium carbonate 0. 3
The seeds were inoculated into 4Qml of a seed medium with a composition of g/di and pH 6.0. The culture was then cultured at 25° C. with shaking until the bacteria grew sufficiently. 10 ml of this seed culture was inoculated into 10 Oml of a fermentation medium having the following composition.

Fermentation medium; glucose 1.0g/J, peptone 0.5g
/a, dry mu mother x bits 0.5 g/a.

V-8 vegetable juice 0.2di/d1. , 'J ncosis (manufactured by Meijiya Co., Ltd. > 0.2 J/J, calcium carbonate 0.5 g/di, pH 6.0) Cultivation was carried out at 25°C for 12 days with shaking. After the cultivation was completed,
Centrifuge 1.32 of the culture solution (RPR-9 manufactured by Hitachi, Ltd.)
-2 type rotor, 7000 rpm). Next, 1.3 for the bacterial body! of methanol was added and thoroughly stirred. Thereafter, bacterial cells were removed by filtration. Methanol was distilled off from the obtained methanol extract under reduced pressure, and an aqueous solution (approximately 10
0m1). After adjusting the pH to 2 with 2N hydrochloric acid, the mixture was extracted three times with 10 Oml portions of ethyl acetate. The ethyl acetate extract was concentrated under reduced pressure and then dissolved in a small amount of chloroform. This was filled using chloroform.
5 Qml silica gel C-200 (manufactured by Wako Pure Chemical Industries)
Using a column, add 5'OOml of chloroform, 5%
Elution was performed sequentially using methanol/chloroform (V/V), 10% methanol/chloroform (V/V), and then 20% methanol/chloroform (V/V) as developing solvents. When the eluted fractions are collected in 18 g portions, KS-502 is eluted in fraction numbers 160 to 201. Both components were collected and concentrated under reduced pressure, and then dissolved in a small amount of methanol. This was filled using methanol.
Elution was performed using a 0 ml Sephadex LH-20 (manufactured by Pharmacia) column using methanol as a developing solvent. When the eluted fractions are separated into 4.4 ml portions, KS-502 is eluted in fraction numbers 19 to 28.

By collecting these two parts and concentrating to dryness under reduced pressure, 92
A white powder of mg of KS-502 was obtained. KS-502 was detected during the purification process by silica gel thin layer chromatography, followed by iodine reaction or 253.7 nm ultraviolet irradiation.

Next, the platelet serotonin release inhibiting effect and anti-amnestic effect of KS-502 will be explained using experimental examples.

Experimental example 1゜Effect on serotonin release from platelets (1) Method 9.25 VO1 of white rabbit blood was taken from the carotid artery at 77 mM.
Ethylenediaminetetraacetic acid (hereinafter abbreviated as EDTA)
It was collected in 0.75 vol and centrifuged at 200×g for 15 minutes to obtain platelet rich plasma (PRP).

This PRP was converted into [14C]-serotonin (2μCi/1
00ml PRP) at 37° C. for 1 hour to release [I40]-serotonin. After that, 6
Platelet sediment was obtained by centrifugation at 50×g for 15 minutes. This platelet sediment was dissolved in Tris-buffered saline (pH 7, 4, 1mME).
Finally, after washing with Ca”free T
yrode solution to a concentration of 109 cells/ml.

Add 5ρ of the test drug solution to 0.475n+1 of the [14C]-serotonin-activated platelet suspension prepared in this way and incubate at 37°C for 3 minutes, then add the stimulant thrombin (final concentration 0.250/ml) or A23187
(#concentration 2μM) + Add CaCL (0.5mM),
Incubate for an additional 3 minutes. Immediately, reaction solution Q,
Take 3 ml and add ice-cold 0.1M formaldehyde-5.
The reaction was stopped at 0 d in 30 mM EDTA solution. 3
．． After centrifugation at 0 °C for 10 minutes at 00 rpm, remove the supernatant at 250 °C.
g was taken and radioactivity was measured using a liquid scintillation counter.

(2) Table 2 of experimental results Concentration (47ml) Seroton release inhibition $ (%) 10
．． 02.7 30, 0 28.9 50, 0 51.4 As shown in Table 2, KS-502 inhibited platelet serotonin release in a concentration-dependent manner.

Experimental Example 2 Scopolamine Effect on Amnesia (1) A light/dark box apparatus was used as the experimental apparatus. That is, a bright room of 15 x 9 x 1 cm illuminated with a 4W white fluorescent lamp and 1
It consists of a dark room of 5 x 14 x 18 cm, and the two rooms are 3
It is separated by a guillotine door at 3 am. Each room has a stainless steel grid floor.
A weak electric current can be passed through the darkroom floor.

First, the test drug (10mg/
0.1 ml) was intraperitoneally administered to male DD mice weighing 24 to 28 g, and immediately thereafter, 0.1 ml of scopolamine was administered intraperitoneally to male DD mice weighing 24 to 28 g.
Amnesia treatment was performed by intraperitoneally injecting 5 mg/kg. Thirty minutes after the amnesia treatment, an acquisition trial was conducted using the following method.

That is, the mouse was placed in a bright room, the guillotine door was opened 10 seconds later, and immediately after the mouse's limbs had completely entered the dark room, the floor grid was energized and the foot 5hock (0,1
8 mA for 2 seconds) and immediately removed. Note that mice that took 60 seconds or more to enter the dark room from the light room were excluded from this test.

A test trial was conducted 24 hours after the acquisition trial. That is,
The mouse was placed in the light room again, and the latency from opening the door 10 seconds later until the mouse completely entered the dark room was measured.

The maximum measurement time was 600 seconds, and the latency time of mice that showed avoidance for 600 seconds or more was 600 seconds. The number of animals in one group was 30 or more.

Experimental results table Normal control Amnestic control KS-502 administration 4815 ± 48.2 87.6 ± 21.2' 213.5 ± 33.3c 212.6 ± 38.2C 249, 7 ± 35.5' 202, 6 ±39.6 @ + 0, 156 + 0, 625 + 2.5 + 10+ Standard error above the mean Comparison with normal control group, p<Q, comparison with 0GOL amnestic control group, p<0.01: Amnestic control group p<0.001 = Comparison with amnestic control group, p<0.05 As shown in Table 3, the K for scopolamine-induced amnesia
S-502 showed significant anti-amnestic effects over a wide range of O, 156-10 mg/kg.

Departure Akira of Effect Fruit The present invention Inhibition of serotonin release by platelets KS, a new substance with anti-amnestic and anti-amnestic effects 502 characteristic

Claims (3)

[Claims] (1) Formula ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ A new substance KS-502 represented by KS-502. (2) Cultivating a microorganism belonging to the genus Sporothrix and capable of producing KS-502 in a medium, producing and accumulating KS-502 in the culture, and collecting KS-502 from the culture. KS-5 featuring
02 manufacturing method. (3) The microorganism is Sporothrix sp.
porothrix sp. ) The manufacturing method according to claim (2), which is KAC-1985 Kaikoken Joyori No. 1278.