JPH02231072A - Microbial strain capable of producing fr-900220 substance - Google Patents

Abstract

NEW MATERIAL:Amauroascus sp No.6237 (ATCC No.20595) expressed by formula and its mutant. It has following physical and chemical properties. Elemental analysis (%), C 74.00, H 7.18, N 10.64; molecular weight, 508.2825 (by high- resolution mass spectrometry); molecular formula. C32H36N4O2: melting point, 156-158 deg.C; specific rotation. [alpha] =-583 deg. (c=1, in chloroform); crystal form and color, colorless columnar crystal; etc. USE:A hypotensor, a prophylactic and a gastric juice secretion suppressing agent. PREPARATION:An FR-900220-producing strain belonging to genus Amauroascus is cultured at about 30 deg.C for 50-100hr preferably by submerged culture method using a liquid medium.

Description

DETAILED DESCRIPTION OF THE INVENTION This invention provides a novel pharmacologically active substance, PR-90022.
This is related to bacteria that produce 0 substances. More specifically, the present invention relates to Amaurocus SB 6237 and its mutant strain that produce the novel FR-9 (IQ220 substance), which has antihypertensive effects, anti-infection effects, and suppressive effects on gastric juice secretion. is a member of the genus Amauroascus, which is currently undergoing research to screen for substances produced by microorganisms that have a hypotensive effect.
They discovered that the 6237 bacterium produced the novel FR-900220 substance, and as a result of further intensive research, they completed this invention. Among the bacteria producing the FR-900220 substance, the mycological properties of the bacteria (numbered Nc6237) newly isolated by the present inventors from rotting wood in a tree hollow in Mt. Rokko, Hyogo Prefecture are shown below. (1) Growth status on each medium NQ was placed in the center of a plate made with potato glucose agar, Zwerbeck agar, malt extract agar, YpSs agar, Saiichi Tomil yeast extract agar, and Bennett agar medium.
6237 strain was inoculated in small dots, cultured at 30°C for 10 days, and the bacterial colonies during culture were observed. The results are as follows. <1> Fruiting organs: In general, in old cultures with good growth medium, small poppy-like yellow granules appear here and there near the surface of the colony. Observation of this part with an optical microscope reveals that the grain (size: 600
μ or more, shape: loose net-like oval or spherical, color:
The presence of ascospores (size: 4-6μ, shape: spherical with spiny appendages on the surface, color: yellow to orange) was observed. (Size: 12~14X16~20μ, Shape: Oval, Color:
Colorless and transparent) are arranged irregularly. (i) Asexual spores: All aerial hyphae are torn in old cultures, and asexual spores (
arthrospora). (i) Morphological properties of vegetative hyphae: There are septa and chlamydospores are observed. Table 2 (2) Physiological properties <<1>> Optimal growth conditions (on YpSs agar medium) pH: 6
~8 Temperature: 22-33°C (i) Range of growth pH: 5-10 Temperature = 14-38°C (3) Morphological properties based on slide specimens Fruiting organs, asexual spores, vegetative hyphae, shape on substrate, The color tone is the same as described above. (4) Regarding the mycological properties of the NQ 6237 strain with a negative or higher phenol oxidase reaction, F.
The General of 77 by F.E. C1amants and C.L. Shamr
F Fungi (1964), A Manual by Joseph C. Gilman (1964);
Manual of Soil Fungi) (1
959), H.H.
Kuehn), K. ffuba
ki) and Miphrosia (M
ycologia) Vol. 56, pp. 863-872 (
1964》, J.A.
．． Pers by Bernese (Yon Arx)
oonia) Volume 6 Part 3 Pages 371-380 (1
971), the strain &6237 is a type of Gymnoascus x-(Gym
Amauroascus (Am
It is reasonable to judge that it belongs to the genus (Auroascus). Therefore, the Nc6237 strain was
S.P. Ha 6237 ( Amauroascus
sp. No. 6237). This Amauroascus sp. strain Nll6237 was given Microbial Accession No.
No. 364 and ArCCNc 20595 with American Type Culture ψFlection. FR belonging to the genus Amauroascus used in this invention
- 900220-producing bacteria may be subjected to irradiation treatments such as X-rays and ultraviolet rays, such as nitrogen mustard, azaserine, nitrous acid, 2-aminopurine, N-methyl-N'-
Treatment with mutagenic agents such as nitro N-nitrosoguanidine (NTG), phage contact, transformation, transduction,
By commonly used bacterial strain mutation processing methods such as conjugation, F
The production capacity of R-900220 substance can be increased. The production of the FR-900220 substance by culturing the FR-900220 substance producing bacteria in a medium is in principle similar to the culture method of general microorganisms, but the deep culture method using a liquid medium is usually advantageous. The medium used for culture is FR-, which belongs to the genus Amauroascus.
Any medium may be used as long as it contains a nutrient source used by the 900220 substance-producing bacteria. That is, a synthetic medium, a semi-synthetic medium, or a natural medium is used, and the medium composition includes carbon sources such as glucose, sucrose, maltose, glycerin, starch, and liquefied starch, and nitrogen sources such as meat extract, Casein hydrolyzate, veptone, gluten meal, hoon meal, mung bean meal, soybean flour, hoon's liquor, dried yeast, yeast extract, urea, ammonium phosphate, etc. are used. In addition, inorganic salts such as disodium hydrogen phosphate, potassium dihydrogen phosphate, magnesium chloride, magnesium sulfate, and calcium carbonate can also be added to the medium as necessary. In addition, when foaming is significant during culturing, for example, vegetable oils such as soybean oil and linseed oil, saicutadecanol, tetradecanol,
Antifoaming agents such as higher alfurs such as heptanol and silicon compounds may be added as appropriate. The appropriate culture temperature is around 30''C, and good results are often obtained by performing seed culture as the culture volume increases.The appropriate culture time for the main culture is about 50 to 100 hours, and the culture medium As the concentration increases, the culture time may be further extended.The culture conditions described above are applied by selecting the optimal conditions for each according to the characteristics of the production strain used.Next, the culture produced by culture Since the FR-900220 substance is usually accumulated both inside and outside the bacterial cells in a culture, it is generally removed from the bacterial cells and filtrate (supernatant liquid) by means such as centrifugation or filtration. After separation, both are separated, purified, and collected by means used in the production of general antibiotics.That is, the target substance within the bacterial cells is usually separated and extracted from the bacterial cells by extraction means such as solvent extraction.Then, this extraction The product and the above filtrate (supernatant) are subjected to vacuum concentration, solvent extraction, liquid conversion, such as anion exchange resin,
Treatment with resins such as cation exchange resins and nonionic adsorption resins, such as activated carbon, silicic acid, silica gel, alumina,
By combining or repeating treatment with an adsorbent such as cellulose, crystallization, recrystallization, etc. in any order, the target substance, FR-900220 substance, can be separated.
It can be purified. By treating the FR-900220 substance obtained in this way with an acid such as hydrochloric acid, sulfuric acid, maleic acid, or fumaric acid in accordance with a conventional method, an acid addition salt thereof can be obtained. FR-900220 obtained by the above manufacturing method
Substances have the following physical and chemical properties. (a) Elemental analysis value (%) C 74.00 i H 7.18 i N 10.6
4 (portion) Molecular weight: 508.2825 (High resolution mass spectrometry) (c) Molecular formula: C32H36N402 (2) Melting point: 156-158°C <*) Specific optical rotation: [α]: 8=-so゜ (C= 1. In chloroform) (to) Ultraviolet absorption spectrum (Figure 1): λ”': 24
5nm (E=11.000), 300nmffia
X (ε=4.200) (g) Infrared absorption spectrum (Figure 2): vKB”:
3350. 2940. 2g50. 1650.
1600. IIIaX (chi) 14g0, 1464. 1415. 1380
．． 1362.1310, 1300. 128
7. 1270. 1247.1208. 11
80. 1145. 1115. 1075.1
055. 1032. 1015. 1005.
980,967,912,890,830,817
, 795, 780, 752, 738, 690,
655, 595.565. 512, 480.
453 am''H-NMR spectrum: Sppm (CDCI3) 7 1.01 (3H.s
). 1.10 (3}!.s), 2.45 (2
H. d, J=9Hz>. 3.75 (IH.t.J
=9Hz>. 4.9-5.2 (2H.m).
5.10 (IH, disappeared at s.D20). 5.45 (
IH,s). 5.8-6.1 (IH, m). 6
．． 4-7.2 (4H, m) (ri) 13C-NMR spectrum: δppm (CDC1a) ' 22.45 (q),
22.81 (q), 35.19 (t). 40.
71 (s). 60.25 (d). 61.76
(s). 77.11 (d), 109.15
(d). 114.25 (t). 118.61
(d). 124.74 (d), 128.69
(s or d). 128.81 (d or s). 1
43.49 (d). 149.92 (s). 16
6.06 (s) (S) Crystal form and color: Based on the physical and chemical properties of colorless columnar crystals and the results of separate research, FR -
The chemical formula of substance 900220 was determined as follows. Next, the pharmacological effects of the FR-900220 substance will be explained using the following test examples. Test Example 1 [Spontaneously hypertensive rats (Spontane
ousHypartensive Rat. Below is SIR
FR-900220 substance was suspended in physiological saline containing gum arabic, and this was intravenously administered to rats. Administered intraperitoneally and orally. Blood pressure was measured invasively through a cannula inserted into the abdominal aorta of rats. The results are shown in Tables 1, 2 and 3 below. Table I Antihypertensive effect when FR-900220 substance is administered intravenously to 51{H Table 2 Antihypertensive effect when FR-900220 substance is intraperitoneally administered to SIH Table 3 FR-900220 substance is orally administered to SHH Antihypertensive effect test example 2 [hypertensive effect on Donryu rats] Male Donryu rats, 12 weeks old and weighing 350 to 400 g, were used as test animals, and FR was carried out in the same manner as in Test Example 1. - The blood pressure lowering effect of substance 900220 was tested. The results are shown in Table 4 below. Table 4 Antihypertensive effect test example 3 when FR-900220 substance was administered intravenously to Dongli strain rats [Relaxation effect on isolated blood vessels] FR - The relaxing effect of 900220 was measured. The results are shown in Table 5. Table 5 FR-90022 for potassium chloride constricted blood vessels
Relaxation effect test example 4 of 0 substance [Infection prevention effect] 5-week-old DDY male mice (5 mice per group) were treated with FR-900220 substance at the following predetermined concentration suspended in physiological saline containing gum arabic. Administer intraperitoneally and orally once a day for 4 days. The day after the final administration of R, Candida albicans (Candida albicans)
Dilution of overnight culture (4 x 106 viable bacteria/body) 0, 2
Inject 5 liters into mice intravenously to infect them. The results of determining whether the mice were alive or dead on the 5th day after infection are shown in Table 6 below. Control mice were administered only physiological saline containing gum arabic before infection. Table 6 FR against Candida albicans -
900220 substance against Escherichia coli in the same manner as above.
The infection prevention effect of substance R-900220 was investigated. The results are shown in Table 7 below. (The inoculum amount of Escherichia furi was 105 viable bacteria/mouse.) Table 7 FR-900220 against Escherichia furi
Infection prevention effect test example 5 of substances [acute toxicity FR-900220 substance with 0.5
This suspension was intraperitoneally administered to eight 7-week-old DDY male mice (dose: 200 m
g/kg) and was observed for 1 week after administration. As a result, there were no deaths. As mentioned above, the FR-900220 substance of the present invention has a blood pressure lowering effect and is useful as a blood pressure lowering agent, and also has an infection prevention effect against pathogenic bacteria and is also useful as an agent for preventing and treating infectious diseases. Although the FR-900220 substance and its pharmaceutically acceptable acid addition salts can be administered as such to mammals including humans, they are generally administered as a preparation in combination with various pharmaceutically acceptable carriers. ．． Examples of such formulations include capsules, tablets, granules, powders, solutions, and the like. Further, acid addition salts that may be acceptable as pharmaceuticals include hydrochloride, sulfate, and the like. Further, pharmaceutically acceptable carriers include, for example, sucrose, starch, mannitol, solpit, lactose, glufus, cellulose, talc, calcium phosphate, calcium carbonate, methylcellulose, hydroxypropyl cellulose, gelatin, gum arabic, canoleboxymethino In addition to Rece J Rerose, magnesium stearate, surfactants, water, and the like, carriers commonly used in the above formulations may be used. F
The dosage of R-900220 substance and its pharmaceutically acceptable salts depends on the type of disease, patient's weight, age,
Although it varies depending on the administration method, it is usually 10{~100aag
The optimal dosage is often selected from within the range of /kg/day. Next, an example of producing the R-900220 substance using the FR-900220 substance-producing bacteria of the present invention will be explained. Production example Corn starch 2. tl%, soy flour 1.0%, dry yeast 1.0%, corn steep liquor 1.0% and glufus 0.5% (pH 7.0).
2 shaker flasks each containing 100
Dispense into 5 tubes, sterilize at 120°C for 20 minutes, inoculate each with one platinum loop of Amauroascus SB Na 6237 strain, and culture at 30°C for 72 hours. Separately, Gurufus 2.0%, corn stew, 10%
．． 25%, dry yeast 0.25%, cottonseed meal 0.5% and wheat germ 0.5% (pH 7.0)
Dispense 202 pieces of each into five 30-liter jar fermenters and sterilize at 120°C for 20 minutes. Each jar fermenter was inoculated with 40011111 copies of the preculture obtained above and incubated at 30°C for 72 hours. Obtained culture 75
Add an equal volume of acetone to the solution, stir, and filter. Concentrate the filtrate under reduced pressure to 3 liters. This concentrate was adjusted to pH 2 with 6N hydrochloric acid.
．． After adjusting to 0, extract twice with 3K ethyl acetate. After combining the extracts and neutralizing with 6N aqueous sodium hydroxide solution,
Concentrate to dryness under reduced pressure. This concentrate was mixed with silica gel (1.
2i column chromatography. This column was washed with benzene 32, and the target substance was mixed with 800 ml of a mixture of benzene and ethyl acetate (10:1).
It elutes with The eluate was concentrated to dryness under reduced pressure to obtain an oily substance 2.
Obtain 08 g. This oil was dissolved in a mixture of chloroform and methanol (9a:2), and silica gel (
1.0Il). When the column is developed with the same solvent, the target substance is 1.2 to 1.4 from the start of elution! The elution is carried out in the fraction of . Concentrate this fraction under reduced pressure to obtain 1.5 g of crude crystals of the target substance. The crude crystals were recrystallized from hot methanol to obtain FR-900220.
Obtain 1.35 g of colorless prismatic crystals of the substance.

[Brief explanation of the drawing]

Figure 1 shows the ultraviolet absorption spectrum of the FR-900220 material, and Figure 2 shows the infrared absorption spectrum of the FR-900220 material. Figure 1

Claims (1)

[Claims] (1) Amauroascus sp.
cus・sp) No. 6237 and its mutants.