JPS61195689A - Novel substance ks-290 ii i-2 and production thereof - Google Patents

Abstract

PURPOSE:To separate the titled novel substance effective to suppress the isolation of thrombocyte serotonin, by culturing a microbial strain belonging to Nocardiopsis genus. CONSTITUTION:A microbial strain belonging to Nocardiopsis genus and capable of producing KS-290 II i-2, e.g. Nocardiopsis sp. K290 (FERM-BP 696) is cultured in a medium under aeration and agitation, using a cultivation method and medium sued in conventional cultivation of actinomycetes. After removing the microbial cells from the medium by filtration, centrifugal separation, chromatography, etc., the objective KS-290 II i-2 is separated from the medium. The novel substance is white powder having a melting point of 240-245 deg.C, soluble easily in methanol and ethanol, insoluble in water, and having the activity to suppress the isolation of thrombocyte serotonin.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a novel substance produced by a microorganism belonging to the genus Nocardiopsis and having an effect of inhibiting platelet serotonin release, and a method for producing the same.

Problems to be Solved by the Prior Art and the Invention Aspirin, prostaglandin E1, etc. are known as substances that have an effect of inhibiting platelet serotonin release. No examples have been found.

With the aim of providing useful new physiologically active substances, we conducted research on the products of numerous microorganisms, both natural and human-made.As a result, we found that cultures of newly isolated microorganisms had an inhibitory effect on platelet serotonin release. We have discovered the fact that physiologically active substances with The substance is then isolated from the culture.

After refining it and examining its physical and chemical properties, it was found to be a new substance. Hereinafter, the substance will be referred to as KS-29011i.
-2.

The physical and chemical properties of KS-290I[i-2, a means for solving the problem, are as shown below.

Properties: White powder Melting point: 240-245°C (decomposed) Specific rotation: [α:]” = +34.9° (C = 0.37
．． CH, 01l) Solubility: Easy to dissolve; Slightly soluble in methanol, ethanol, and dimethyl sulfoxide; Insoluble in chloroform; Water color reaction: Positive for iodine and ferric chloride reactions.

Ultraviolet absorption spectrum (5,5J1g/ml methanol solution); Fig. 1 Infrared absorption spectrum (KBr) 3340, 3050. 2900. 1650. 1
605. 1589. 1493°1454, 1402
．． 1345.1330.1281.1246.1147
゜1129, 1112. 1090. 1045.
1009. 952. 934°922, 906.87
5.838.790.773.744.694゜670
, 648. 620. 579. 527. 490
．． 430 cm-1 high resolution mass spectrum: Actual value; 457.1650 Calculated value; 457.16361 H as CzsLJJ5
-NMR (400MHz, DMSO-da,
δ) 11.68 (1H, br, s), 9.47 (1H
, d, J=7.7Hz), 8.54(11(,br,
s), 8.07 (1H, d, J=7.8Hz), 7
．． 69 (1H, d.

J=8.3Hz), 7.60(1H, +j, J=8
．． 1tlz), ca,? , 5(2)1゜m),
7.3H1H, t, J=7.5flZ), 7.27
(1H, t, J=7.5Hz).

6, 69 (ill, br, s), 6.40 (I
tl, d, J=9.5flz), 5.40(1
H, d, J=3.6Hz), 5.0H3H,br
, s), ca, 4.5 (IH.

m), 4.48 (1H, br, q, J=岨Z), 4.18 (1H, br, s).

4.05 (1H, br, s), 1.70 (3H, d
, J=7.2flz)13C-NMR(100MH
z, DMSO-d, δC) 172.2. 140
．． 1. 138.9. 133.8. 127.5.
125.5°125.1. 125.0. 124.4
．． 122.2. I21.8. 121.1°11
9.7. 119.2. 118.5. 117.4.
114.8. 111.1°109.7. 77.1
．． 76.4. 71.6. 71.4. 66.8.
45.1°15.3 From the above data, KS-290If i-2 was found to be a new compound.

Next, Table 1 shows the Rf values of thin layer chromatography of KS-290II i-2 using various developing solvents. Detection was performed by irradiating 3,650 people with ultraviolet light.

Table 1 Silica gel thin layer chromatography developing solvent for KS-290 Old-2 Rf value 1, methanol:water-8:2 (v/v) 0.4
92, methanol: water: chloroform = 63:27 + 10 (v/v) 0.42 thin layer: reversed phase silica gel play) RP-18 (Merck & Co., Art
15685) Development: room temperature, ascending method, 1 hour KS-290 old-2 has an inhibitory effect on platelet serotonin release.

Next, the manufacturing method of KS-290 old-2 will be explained.

KS-290II i-2 belongs to the genus Nocardiopsis, and a microorganism capable of producing KS-290II i-2 is cultured in a medium, KS-29011i-2 is produced and accumulated in the culture, and KS-29011i-2 is produced and accumulated in the culture. Manufactured by collecting KS-290 Old-2.

The KS-290II i-2 producing microorganism may be any microorganism that belongs to the genus Nocardiopsis and has the ability to produce KS-290II i-2.

A specific preferred example is Nocardiopsis sp. K-290 strain, which the present inventors isolated from grassland soil in Tama City, Tokyo. The strain was submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology, as part of the Microtechnology Research Institute No. 696 (deposit date: 19B5-1).
It has been deposited as (April 19th).

Observing the morphological characteristics and physiological properties of the K-290 strain,
The test results are as follows.

The fungus exhibits normal or good growth on a variety of natural and synthetic media commonly used for the growth of actinomycetes. Aerial mycelial growth is relatively good on synthetic media, and the color is white or beige. The color tone of the basal hyphae is yellow-gray to beige, but it becomes reddish-brown on tyrosine agar, yeast/malt agar, and peptone/yeast iron agar. No soluble dye formation is observed. There is also no production of melanin-like pigments.

The spores form a chain of 20 or more at the tip, which is simply branched from the aerial hyphae, and are curved. The morphology of the spores is spherical to cylindrical, and the size is 0.6 to 0.8 x O17 to 1.
．． The spore surface is smooth or warty when observed using an electron microscope.
It is. No flagella are observed and no sporangia are found.

Growth status on various media The characteristics of growth and color when cultured on various media at 28°C for 20 days are shown below. Color display is Co1ar
Harmony Manual (Container
Corporation of America)
According to the color classification by

Note that no pigment was produced in any of the media.

(1) Growth on sucrose/nitrate agar medium: Normal aerial hyphae: Medium, natural (2dC) Color of basal hyphae (3ba) (2) Growth on glucose/asparagine agar medium: Good, raised air Medium mycelium: medium to good, pearl (3ba) to beige a
(3gc) Color of basal hyphae: Nyrite Aipori-(2Ca) (3) Glycerol/asparagine agar growth: Normal Aerial hyphae: Good, Veil Blue (14Ca) Color of basal hyphae: Birchmen) (ICb) ~ Dark Cobalt Dale (2ih) (4) Growth on starch agar medium: Normal Aerial hyphae: Moderate to good, Pearl (3ba) Color of basal hyphae: Viscera) (28C) (5) Growth on tyrosine agar medium Growth: Good Aerial hyphae: Good, Bisutsu (38C) Color of basal hyphae: Nude tan (4gc>(6) Growth on nutrient agar medium: Normal Aerial hyphae: Poor, Bisutsu (36C) Color of basal hyphae : Nude tan (4gc) (7) Yeast/malt agar culture Growth: Good, pleated aerial hyphae: Medium, pearl (2ba) Base hyphae color: Amber (3βC) ~ Clove brown <3pρ) (8) Growth on oatmeal agar medium: Poor, granular aerial hyphae: None Color of basal hyphae Nirite Aipori- (2ea) (9) Peptone yeast Growth on iron agar medium: Good, pleated aerial hyphae: None Color of basal hyphae: Camel (3ie) Various physiological properties -The physiological properties of strain -290 are shown below. Regarding the growth temperature range, the results were shown after 2 days, the effects on skim milk, 7' latin and cellulose were observed after 1 month at 28°C, and the others were observed after 3 weeks at 28°C.

(1) Assimilation of carbon sources D-glucose, 1-inositol, D-fructose, and L-rhamnose are assimilated, but L-arabinose and D
- Xylose, D-mannitol, sucrose and raffinose are not assimilated.

(2) Liquefaction of gelatin: Not liquefied.

(3) Coagulation and peptonization of skim milk Although peptonized, there was only slight coagulation.

(4) Cellulose decomposition effect: None 5 Starch hydrolysis effect: Yes (6) Growth temperature range: 10-42℃ (7) Melanin-like pigment production: None -290 strain is a mesophilic bacterium. Yes, and forms aerial mycelia on agar media. The spores are formed as a curved chain of 20 or more from the sporophyte, which is simply branched from the aerial hyphae, and no motility is observed. In the basal hyphae, branching was frequently observed, but no clear division was observed.

In hydrolytic analysis of whole bacterial cells, men-type diaminopimelic acid was detected, and characteristic sugars such as magicolose were not detected. Therefore, the strain was designated as Nocardiopsis sp.
sis sp,) K-290.

When culturing the microorganism, the usual culture method used for culturing actinomycetes is 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.

As carbon sources, carbohydrates such as glucose, fructose, stahyrose, precipitated L-dextrin, mannose, maltose, and molasses, organic acids such as citric acid, malic acid, acetic acid, and fumaric acid, amino acids such as glutamic acid, or glycerol 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, peptone, meat extract, yeast extract, dried yeast, corn steep liquor, soybean flour, cottonseed meal, soybean casein , casamino acids, Pharmamedia, 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, common salt, etc. are used.

Other substances, such as vitamins and thiamine, which promote the growth of eugenics or the production of KS-290 old-2 can be added to the medium as necessary.

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

Culture is carried out using shaking culture method, aeration agitation culture method, etc.
It is carried out at a temperature of 0°C and a pH around neutrality.

After 3 to 15 days of culture, the accumulation of KS-290II i-2 reaches its maximum and the culture is completed.

When KS-290II i-2 accumulated in a culture is isolated and collected from a culture solution, a conventional method for collecting physiologically active substances from a culture solution is applied.

In other words, removal of bacterial cells by filtration, centrifugation, etc., absorption of active substances by column chromatography or thin layer chromatography using adsorption resins, silica gel, silanized silica genepe aluminum, cellulose, diatomaceous earth, magnesium silicate, gel filtration agents, etc. KS by desorption processing etc.
290 II i-2 is isolated.

An example of isolating KS-290II i-2 from a culture fluid is as follows.

The bacterial cells are removed by filtering or centrifuging the culture solution. The obtained furnace liquid or supernatant liquid is treated with an adsorption resin, Diaion HP-10 (manufactured by Mitsubishi Chemical Industries), to adsorb the active substance to the resin.

Then, it is eluted with a suitable solvent such as methanol, and the eluate is concentrated under reduced pressure to remove the solvent and form an aqueous solution.

Next, a water-immiscible solvent such as ethyl acetate or butyl acetate is added to this aqueous solution for extraction.

The extract is concentrated under reduced pressure and subjected to Diaion HI"20SS (manufactured by Mitsubishi Chemical Industries) column chromatography. 90% (V/V) methanol is used as the developing solvent. Then, the resulting eluate was concentrated under reduced pressure and chromatographed using Sephadex LH=20 (manufactured by Pharmacia) (developing solvent: chloroform: methanol).
1: Perform IV/V).

Then, the obtained eluate was concentrated under reduced pressure, and KS-290
A white powder of IIi-2 is obtained.

KS-290II i-2 was detected during the purification process by silica gel thin layer chromatography, followed by iodine reaction or 3650-person ultraviolet irradiation method.

Examples are shown below.

-290 (
(Feikoken Article No. 696) is used. Glucose LOg/dE, soluble starch 1.0g using the bacteria as a first type medium
/a, meat extract 0.3g/a, yeast extract 0.5g/a
de, Bactodrypton (manufactured by Difco) 0.5 g/d
e, calcium carbonate 0.2 g/dL pH 7°2-7
．． 40 m in a 300 m Erlenmeyer flask with a composition of 4
Inoculate the seed medium of 1. Then, the culture is carried out at 30° C. with shaking until the bacteria grow sufficiently. 4 Qml of this seed culture solution is inoculated into 40 Qml of seed J3 soil of the same composition in a 21 Erlenmeyer flask. Then, the cells are cultured with shaking at 28°C for 3 days. This seed culture solution 1.21 is inoculated into the following fermentation medium 111 in a 30μ jar fermenter.

Fermentation medium Nigelcose 0.5g/dll, soluble starch 3.0g/a, soybean flour 3.0g/dL cornstarch liquor 0.5g/J, yeast extract 0.5g/de
.

Calcium carbonate O: 3 g/a, pH 7.2~
7.4°Culture is carried out at 30°C for 9 days with aeration and agitation. After culturing, the culture solution was centrifuged continuously (15,00 rpm>
do.

Add 1M of supernatant liquid to Diaion HP-10 (Mitsubishi Chemical Corporation ■
21 water, 2
After washing with 20% (V/V) methanol in 1, elute with methanol in 21. Collect all eluates and concentrate for 20 minutes.
Qml and extracted with 600ml of ethyl acetate. After dehydrating the ethyl acetate layer with anhydrous sodium sulfate, it was washed with Celite-54.
Add 50 g of No. 5 (manufactured by Hanui Chemical Co., Ltd.) and concentrate to dryness. 182ml of Diaio 78P filled with water
-20SS (manufactured by Mitsubishi Chemical Industries, Ltd.) column is supplied to the top end.

40 Qml of 70% (V/V) methanol, plus 5
After washing with 00ml of 80% (V/V) methanol, 90ml
Elute with % (V/V) methanol. KS-290 is added to fraction numbers 6 to 21 when each eluted fraction is separated by 15 g.
Old-2 is eluted. Both fractions are combined and concentrated under reduced pressure to obtain approximately 260 mg of oil. 5ml of this
Chloroform:methanol-1:1 (V/V)
The cells are dissolved in a solvent of the same composition and supplied to the upper end of a 55 ml Sephadex LH-20 (manufactured by Pharmacia) column filled with a solvent of the same composition, and eluted as a developing culture using a solvent of the same composition as that of the packed culture. When the eluted fraction was separated into 5g portions, fraction numbers 42 to 47 were obtained as KS-290U i-2.
is eluted. When these two parts are collected and concentrated to dryness, it becomes approximately 28.6
mg of white powder is obtained.

In addition, the detection of KS-290 old-2 during the above purification process
Silica gel thin layer chromatography was followed by iodine reaction or 3650-person ultraviolet irradiation method.

Next, the inhibitory effect of KS-290 Old-2 on platelet serotonin release will be explained using experimental examples.

Experimental example Effect on serotonin release from platelets 1) Method 9.25 vol of white rabbit blood was taken from the carotid artery at 77 mM8.
Collected in DTA 0.75vol, 200Xg, 15
Platelet rich plasma (PRP) was obtained by differential centrifugation.

This PRP is (+4C)-serotonin (2 μCi/10
(14C']-serotonin was incorporated by lhr incubation at 37°C with 0ml PRP).Then, it was centrifuged at 650Xg for 15 minutes to obtain a platelet sediment.The platelet sediment was incubated with Tris-buffered saline (pH 7,4゜1
Finally, Ca +
+-free Tyrode solution 1Q9cells/
ml.

To 0.475 ml of the [14C]-serotonin-activated platelet suspension prepared in this manner, the test drug solution 50% was added, and after incubation at 37°C for 3 minutes, the stimulant thrombin (
#Concentration 0.25 V/ml) or A23187 (final concentration 2 μM) + CaC12 (0.5 mM) was added and further incubated for 3 minutes. Immediately add reaction solution 9.
Take 3ml and add 5ml of ice-cold 0.1M formaldehyde.
M BDTA solution 30111 was added to stop the reaction.

3. Supernatant 250I after centrifugation at 0°C for 10 min at OOOrpm
IIl was taken and radioactivity was measured using a liquid scintillation counter.

2) Experimental Results As shown in Table 2, KS-29011i-2 inhibited the release of nicerotonin in a concentration-dependent manner.

Effects of the Invention KS-290II i-2 has a serotonin release suppressing effect.

[Brief explanation of the drawing]

FIG. 1 is an ultraviolet absorption spectrum of KS-290 Old-2. Procedural amendment written μ month S date 1985

Claims (3)

[Claims] (1) KS-290, a new substance with the following physical and chemical properties
IIi-2 Properties: White powder Melting point: 240-245℃ (decomposed) Specific rotation: [α]^2^0_D=+34.9° (c=0
．． 37, CH_3OH) Solubility: Easy to dissolve; Slightly soluble in methanol, ethanol, and dimethyl sulfoxide; Insoluble in chloroform; Water color reaction: Positive for iodine and ferric chloride reactions. Ultraviolet absorption spectrum (methanol solution): Fig. 1 Infrared absorption spectrum (KBr) 3340, 3050, 2900, 1650, 1605,
1589, 1493, 1454, 1402, 1345,
1330, 1281, 1246, 1147, 1129,
1112.1090, 1045, 1009, 952, 9
34, 922, 906, 875, 838, 790, 77
3, 744, 694, 670, 648, 620, 579
, 527, 490, 430cm^-^1 High resolution mass spectrum: Actual value; 457.1650 Calculated value; 45 as C_2_6H_2_3N_3O_5
7.1636^1H-NMR (400MHz, DMSO
-d_6, δ) 11.68 (1H, br.s), 9.4
7 (1H, d, J = 7.7Hz), 8.54 (1H, b
r. s), 8.07 (1H, d, J=7.8Hz), 7
．． 69 (1Hd, J=8.3Hz), 7.60 (1H,
d, J=8.1Hz), ca. 7.5 (2H, m), 7
．． 31 (1H, t, J = 7.5Hz), 7.27 (1H
, t, J=7.5Hz), 6.69 (1H, br.s)
, 6.40 (1H, d, J = 9.5Hz), 5.40 (
1H, d, J=3.6Hz), 5.01 (3H, br.
s), ca. 4.5 (1H, m), 4.48 (1H, b
r. q, J≒7Hz), 4.18 (1H, br.s),
4.05 (1H, br.s), 1.70 (3H, d, J
=7.2Hz)^1^3C-NMR (100MHz, D
MSO-d_6, δc) 172.2, 140.1, 13
8.9, 133.8, 127.5, 125.5, 125
．． 1, 125.0, 124.4, 122.2, 121.
8, 121.1, 119.7, 119.2, 118.5
, 117.4, 114.8, 111.1, 109.7,
77.1, 76.4, 71.6, 71.4, 66.8,
45.1, 15.3 (2) Belongs to the genus Nocardiopsis, KS-290IIi
-2 production ability is cultured in a medium, KS-290IIi-2 is produced and accumulated in the culture, and K-290IIi-2 is produced and accumulated in the culture.
A method for producing a novel substance KS-290IIi-2, which comprises collecting S-290IIi-2. (3) The microorganism is Nocardiopsis sp. K-2
The manufacturing method according to claim 2, characterized in that it is manufactured by 90 Keikoken Joyori No. 696.