JPH0445516B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a new antibiotic Spirocardin A or B and a method for producing the same. The present inventors isolated a specimen belonging to the genus Nocardia from soil collected in Yama-gun, Fukushima Prefecture.
We found that the SANK64282 strain produces spirocardin A or B, a new antibiotic that is effective against Gram-positive and Gram-negative bactericidal bacteria. Therefore, spirocardin A or B can be used for the prevention or treatment of human, animal or plant diseases caused by these bacteria. Here, spirocardine A or B is represented by the following formula. However, in the formula, spirocardine A is

[Formula] is shown, spirocardine B is based on X

[Formula] is shown. Produce spirocardin A or B
The mycological properties of SANK64282 strain are as follows. (1) Morphological characteristics ISP [International Streptomyces projectus]
Streptomyces Project] Prescribed double areas and recommendations of SAWaksman et al. [The Actinomycetes, vol. 2]
Culture on medium. Culture is usually carried out at 28°C.
A morphological feature of the SANK64282 strain is that clear division between aerial and vegetative hyphae is observed several days after inoculation and culture. The segmented hyphae are spherical or rod-shaped. No other special organs were observed. (2) Properties on various culture media Properties when cultured on various culture media at 28°C for 14 days are shown in Table 1. The color tone display represents the color chip number of the Japan Color Research Institute's "Standard Color Chart".

【table】

【table】

[Table] (3) Physiological properties The physiological properties of SANK 64282 strain are shown in Table 2.
Table 3 shows the carbon source utilization.

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【table】

[Table] +: Assimilate, ±: Weakly assimilate, -: Not assimilate
(4) Regarding intracellular components M.P.
Lechevalier et al.'s method [A.
Dietz et al., Actinomycete classification
As a result of paper chromatography analysis of the acid hydrolyzate of the bacterial cells, mesodiamino pimelic acid, arabinose, and galactose were observed, and the cell wall type was different from the cell wall type. It was confirmed that there is. The glycotype of all bacterial cells was type A. Furthermore, the method of H. Mordarska et al. [Journal of general microbiology]
Microbiology), vol. 71, p. 77, 1972], the lipids in the bacterial cells were analyzed and it was found that LCN-A
(Lipids characteristic of Nocardia) existed. Among the above-mentioned characteristics of the SANK 64282 strain, the hyphae are divided, the cell wall is type A, and the glycoform in the whole cell is type A, and there is LCN in the cell.
The presence of -A revealed that this strain belongs to the genus Nocardia in the family Nocardiaceae among actinomycetes, and was named Nocardia sp. SANK 64282. did. This strain has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, and its microbial accession number is No. 6986.
This is the number. Above, we have explained spirocardin A or B producing bacteria, but it is well known that the properties of actinomycetes, especially Nocardia, are not constant and can easily change naturally or artificially. Bacteria that can be used in the present invention include all strains that produce spirocardin A or B belonging to the genus Nocardia. The culture in the present invention is carried out according to the culture method for general actinomycetes, and is preferably carried out by shaking culture in a liquid medium or by aeration stirring culture. Medium components include, for example, glucose as a carbon source,
maltose, sucrose, mannitrate, molasses,
Glycerin, dextrin, starch, soybean oil, cottonseed oil, etc. as nitrogen sources, soybean flour, peanut flour, cottonseed flour, firmamine, fishmeal, corn steep liquor, peptone, meat extract, yeast, yeast extract, sodium nitrate, Ammonium nitrate, ammonium sulfate, etc., as well as inorganic salts such as table salt,
Phosphates, calcium carbonate, trace metal salts, etc. are added as appropriate. For liquid culture,
Silicone oil, vegetable oil, surfactant, etc. are appropriately used as antifoaming agents. The pH of the medium is near neutral, and the culture temperature is 24℃ to 30℃.
℃, especially around 28℃ is preferred. Changes over time in the titer of spirocardin A or B produced over the course of the culture are shown in Table 1.
aureus) FDA 209P JC-1 as a test bacterium and a paper disk (manufactured by Toyo Kagaku Sangyo Co., Ltd., diameter 8 mm,
Thick) is measured by the assay method. Usually after 72 to 120 hours of culture, spirocardin A
Or B's production reaches its maximum value. Spirocardin A mainly present in the liquid part of the culture solution
Or B: After the completion of culture, bacterial cells and other solid parts are removed by over-operation using diatomaceous earth or the like as an aid, or by centrifugation, and extracted and purified from the liquid or supernatant. obtained by. Spirocardin A or B can be collected by utilizing its physicochemical properties, for example, using an adsorbent. Examples of adsorbents include activated carbon, adsorption resins such as Amberlite XAD-2, XAD-4, and XAD-7 (manufactured by Rohm and Haas), or Diaion HP10, HP20, HP20AG, HP50, etc. (manufactured by Mitsubishi Chemical). (manufactured by Kogyo Co., Ltd.) is used. Spirocardein A or B A liquid containing Spirocardein A or B is passed through a layer of an adsorbent as described above to adsorb and remove impurities contained in the liquid containing Spirocardein A or B, or It can be obtained by adsorbing spirocardine A or B and then eluting it using methanol water, acetone water, n-butanol water, or the like.
Also, a method for collecting neutral fat-soluble substances from the culture solution,
For example, it is also possible to extract from the culture broth or aqueous solution using an organic solvent that is immiscible with water (for example, chloroform, ethyl acetate, n-butanol, etc. alone or in combination). Furthermore,
It is also possible to remove mixed acidic or basic substances by washing the organic solvent layer containing spicaldein A or B with dilute alkaline water, dilute acidic water, or the like. In order to purify spirocardin A or B obtained in this way, partition column chromatography using cellulose such as Avicel (manufactured by Asahi Kasei Industries, Ltd.) or Sephadex LH-20 (manufactured by Pharmacia), etc. Graphite; adsorption column chromatography using a carrier such as silica gel, alumina, or florisil; reversed-phase column chromatography using a reversed-phase carrier; or to a solvent with impurities mixed with spirocardine A or B Extraction methods that utilize differences in distribution rates or countercurrent distribution methods can be said to be effective methods. Spirocardin A or B can be purified by repeatedly using the above purification means alone or in appropriate combinations. Or spirocardin A
Or B, like general lipid-soluble antibiotics, exists in the bacterial cell portion of the culture solution depending on the culture conditions.
In this case, it is possible to perform extraction using a hydrophilic organic solvent such as alcohol or acetone, remove the solvent from the extract, prepare an aqueous solution, and then extract and purify it in the same manner as from a culture solution. Spirocardin A thus obtained has the following physicochemical and biological properties. (1) Properties of substance: Neutral pale yellow powder. (2) Specific rotation: [α] ²⁵ _D −52.7° (c, 0.48, CHC
₃ ) (3) Molecular formula: C ₂₀ H ₃₀ O ₆ (measured by high-resolution mass spectrometry) (4) Molecular weight: 366 (FD/MS) (5) Ultraviolet absorption spectrum: λ _nax nm (E1% 1cm) in methanol The ultraviolet absorption spectrum measured at 280 (sh) nm (E1% 1 cm
10) shows maximum absorption. (6) Infrared absorption spectrum: ν _nax cm ^-1 The infrared absorption spectrum measured by the liquid film method is shown in Figure 2. (7) Nuclear magnetic resonance spectrum: δ: ppm The nuclear magnetic resonance spectrum (400MHz) measured in chloroform using TMS (tetramethylsilane) as an internal standard is shown in Figure 3. (8) Solubility: Soluble in chloroform, ethyl acetate, acetone, ethanol and methanol, insoluble in water. (9) Color reaction: Positive for potassium permanganate and sulfuric acid. (10) Thin layer chromatography: Rf value 0.30 Adsorbent: Merck silica gel plate No. 5715 Developing solvent: Ethyl acetate:benzene (1:1) (11) Antibacterial spectrum Spirocal against general Gram-positive and Gram-negative bacteria Minimum inhibitory concentration (MIC) of Dein A
is a 2% glycerin-added sensitive disc agar medium (manufactured by Nissui Pharmaceutical Co., Ltd.), against molds and yeasts.
Sabouraud agar medium supplemented with 0.2% yeast extract,
For anaerobic bacteria, GAM agar medium (Nissui Pharmaceutical
Co., Ltd.); for mycoplasma, fresh yeast extract, DNA, dipotassium phosphate;
It was measured by an agar medium dilution method using a PPLO agar medium supplemented with PC-G. The results are 4th to 6th
As shown in the table. In addition, spirocardin B has the following physicochemical and biological properties. (1) Properties of substance: Neutral pale yellow powder. (2) Specific rotation: [α] ²⁵ _D −35.5° (c, 0.62, CHC
₃ ) (3) Molecular formula: C ₂₀ H ₃₂ O ₆ (measured by high-resolution mass spectrometry) (4) Molecular weight: 366 (FD/MS) (5) Ultraviolet absorption spectrum: λ _nax nm (E1% 1cm) in methanol The ultraviolet absorption spectrum measured at 280 (sh) nm (E1% 1 cm
5) shows maximum absorption. (6) Infrared absorption spectrum: ν _nax cm ^-1 The infrared absorption spectrum measured by the liquid film method is shown in FIG. (7) Nuclear magnetic resonance spectrum: δ: The nuclear magnetic resonance spectrum (400 MHz) measured in _ppn deuterated chloroform using TMS (tetramethylsilane) as an internal standard is shown in FIG. (8) Solubility: Soluble in chloroform, ethyl acetate, acetone, ethanol and methanol, insoluble in water. (9) Color reaction: Positive for potassium permanganate and sulfuric acid. (10) Thin layer chromatography: Rf value 0.15 Adsorbent: Merck silica gel plate No. 5715 Developing solvent: Ethyl acetate:benzene (1:1) (11) Antibacterial spectrum Spirocal against general Gram-positive and Gram-negative bacteria Minimum inhibitory concentration (MIC) of Dein B
is a 2% glycerin-added sensitive disc agar medium (manufactured by Nissui Pharmaceutical Co., Ltd.), against molds and yeasts.
Sabouraud agar medium supplemented with 0.2% yeast extract,
For anaerobic bacteria, GAM agar medium (Nissui Pharmaceutical
Co., Ltd.); for mycoplasma, fresh yeast extract, DNA, dipotassium phosphate;
It was measured by an agar medium dilution method using a PPLO agar medium supplemented with PC-G. The results are 4th to 6th
As shown in the table.

【table】

【table】

[Table] Ifisil

【table】

[Table] As a result of comparing the properties of spirocardin A or B as described above with those of known antibiotics, there was no match and it was determined that it is a new antibiotic. From the above, spirocardin A or B is used as an antibacterial agent to control various bacterial infectious diseases. The administration form includes parenteral administration such as subcutaneous injection, intravenous injection, intramuscular injection, and suppository, and oral administration using tablets, capsules, acid preparations, granules, and the like. The dosage varies depending on the target disease, route of administration, frequency of administration, etc., but for adults, it is usually preferable to administer 500 mg to 2000 mg per day, either once or in divided doses. Next, the present invention will be explained in more detail with reference to Examples. Example 1 Culture medium composition-1 for Nocardia SP.SANK64282 strain
One platinum loop was inoculated into a 500 ml Erlenmeyer flask containing 80 ml of the medium shown in , and cultured at 28°C for 96 hours in a rotary shaking incubator at 220 rpm. 25 ml of this culture solution was inoculated into four 2-volume Erlenmeyer flasks containing 500 ml of the medium shown in medium composition-2, and cultured at 28° C. for 24 hours in a rotary shaking incubator at 220 rpm. Inoculate 750 ml of the obtained culture solution into two 30-volume jar fermenters each containing 15 of the same medium, and stir at 28°C.
Aeration and agitation culture was carried out for 67 hours at 150 revolutions/min and aeration rate of 10/min. When 1.5 kg of Celite 545 (manufactured by John's Manville Products Corporation, USA) was added as a supernatant to the obtained culture solution 30 and filtered, solution 30 (PH 7.1) was obtained. This culture solution was extracted twice with 15 portions of ethyl acetate, washed with 30 portions of saturated brine, dehydrated with anhydrous sodium sulfate, and concentrated to dryness under source pressure to obtain 1.2 oily substance. This oil was dissolved in benzene and adsorbed on a silica gel column (manufactured by Mallinckrodt, USA, 10 g of silica gel) prepared in advance with benzene. After washing with benzene, benzene:ethyl acetate was applied in sequence at 3:1, 1:1, and 1. :20ml of mixed solvent of 2
It was fractionated, developed and eluted. Spirocardine A
was eluted in fractions No. 6 to 9, and spirocardine B was eluted in fractions No. 10 to 13.
Each was concentrated to dryness under source pressure to obtain 260 mg of spirocardein A as an oil and 235 mg of spirocardein B as an oil. The obtained crude spirocardin A was again passed through a silica gel column (10
ml) and developed with 60 ml of a mixed solvent of benzene, chloroform, and 5% acetone-chloroform, fractionated into 5 ml portions, and eluted up to fraction No. 36. Spirocaldein A is fraction No.15
eluted at ~24. By combining the active fractions and concentrating to dryness under reduced pressure, 27 mg of spirocardin A was obtained.
was gotten. On the other hand, the obtained crude spirocardine B was adsorbed onto a silica gel column (10 g) again and developed with 50 ml of each benzene and a mixed solvent of benzene:ethyl acetate of 3:1, 2:1, 1:1, and 1:2. The mixture was fractionated into 7 ml portions and fraction No. 43 was eluted. Spirocaldein B is fraction No.19
eluted at ~31. The active fractions were combined and concentrated to dryness under reduced pressure to obtain 80 mg of spirocardin B. Medium composition-1 Glucose 1% Glycerin 1% Sucrose 1% Fresh yeast 1% Soybean flour 2% Oatmeal 0.5% Casamino acids 0.5% Calcium carbonate 0.1% Nitsusan Daisu Home CB-442 (antifoaming agent) PH7.0
0.01% Medium composition-2 Glucose 2% Lastagen FK 1% Fresh yeast 0.9% Meat extract 0.5% Polypeptone 0.5% Calcium carbonate 0.3% Sodium chloride 0.5% Nitsusan Days Home CB-442 (antifoaming agent) PH7.0
0.01% Example 2 Nocardia SP.SANK64282 strain in medium composition-1
One platinum loop was inoculated into a 500 ml Erlenmeyer flask containing 80 ml of the medium shown in , and cultured at 28°C for 96 hours in a rotary shaking incubator at 200 rpm. 25 ml of this culture solution was inoculated into two 2-volume Erlenmeyer flasks containing 500 ml of the medium shown in Medium Composition-2, and cultured at 28°C for 48 hours in a rotary shaking incubator at 220 rpm. 750 ml of the obtained culture solution was inoculated into a 30-volume jar fermenter containing medium 15 shown in medium composition-2, and incubated at 28°C.
Cultured for 24 hours. The obtained culture solution 15 was added to the same medium.
Inoculate a 600 volume tank containing 300, stir at 28℃
Aeration and agitation culture was carried out for 115 hours at 100 revolutions/min and aeration rate of 300/min. When 15kg of Celite 545 was added as a supernatant to the obtained culture solution 300 and filtered, the solution
290 was obtained. This culture solution was extracted twice with 160 ml of ethyl acetate and concentrated to dryness under reduced pressure, yielding 43 g.
of oil was obtained. This oil was dissolved in n-hexane and adsorbed on a silica gel column (manufactured by Mallinckrodt Co., USA, 250 g of silica gel) prepared in advance with n-hexane.
The mixture was fractionated into 20 ml portions using a mixed solvent of n-hexane solutions containing 20, 30, 40, and 50% acetone, and developed and eluted.
Spirocardein A is eluted in fractions No. 54 to 85, and spirocardein B is eluted in fraction No. 86.
eluted at ~100. When each was concentrated to dryness under reduced pressure, 7.6 g of spirocardein A and 4.5 g of spirocardein B were obtained as oils. The obtained crude spicaldein A (7.6 g) was again adsorbed onto a silica gel column (50 g) and mixed with 300 ml of benzene:ethyl acetate (4:1, 3:1, 2:3).
The mixture was developed with a mixed solvent of 1:1 and eluted in fractions of 20 ml. Fractions No. 14 to 44 were collected and concentrated to dryness under reduced pressure to produce spirocardine A2.4.
g of oily substance was removed. On the other hand, 4.1 g of the obtained crude spirocardin B was similarly subjected to chromatography again using a silica gel column (50 g). 300ml each of benzene:ethyl acetate 3:1, 2:1, 1:1,
It was developed with a 1:2 mixed solvent, fractionated into 20 ml portions, and eluted. Fractions Nos. 45 to 60 were collected and concentrated to dryness under reduced pressure to obtain 471 mg of spirocardine B as an oil. The obtained spirocardin A or B was subjected to reverse phase chromatography (manufactured by Merck & Co., Ltd.,
Further purification was performed using Rover Column Ricroprep RP-8 (size B). spirocardine
Dissolve 2.4 g of A in 4 ml of methanol, adsorb 1 ml of it onto Rover Column Ricroprep RP-8 (size B) prepared in advance with 60% methanol water, and then measure the refraction of the eluate using a refractometer. It was developed and eluted with 60% methanol water while checking the ratio.
Spirocardin A starts from 30 minutes after sample adsorption36
The active fraction was eluted in 60 ml. Repeating the same procedure three times for the remaining 3 ml of sample yields a total of 180 ml of active fraction.
was gotten. Methanol was distilled off from the obtained active fraction under reduced pressure, extracted with 100 ml of ethyl acetate,
The extract was washed with saturated brine, dehydrated with sodium sulfate, and concentrated to dryness to obtain 170 mg of spirocardin A as a yellow powder. Similarly, spirocardine
470 mg of B was dissolved in 2 ml of methanol and the same operation as for spirocardin A was carried out under the same conditions. Spirocardin B was eluted between 33 and 43 minutes after sample adsorption, and 110 ml of active fraction was obtained.
When the same operation was repeated for the remaining 1 ml of sample, 110 ml of active fraction was obtained. From the obtained active fraction, methanol was distilled off under reduced pressure, extracted with 100 ml of ethyl acetate, the extract was washed with saturated saline, dehydrated with Glauber's salt, and concentrated to dryness to obtain spirocal as a yellow powder. 190 mg of Dein B was obtained. Medium compositions-1 and 2 used in this example are the same as those used in Example 1.

[Brief explanation of drawings]

FIG. 1 shows the ultraviolet absorption spectrum of spirocardine A, FIG. 2 shows the infrared absorption spectrum of the same substance, and FIG. 3 shows the nuclear magnetic resonance spectrum of the same substance. FIG. 4 shows the ultraviolet absorption spectrum of spirocardine B, FIG. 5 shows the infrared absorption spectrum of the same substance, and FIG. 6 shows the nuclear magnetic resonance spectrum of the same substance.

Claims (1)

[Claims] 1 formula Spirocardin A or B with However, in the formula, in spirocardine A, X represents a group [formula], and in spirocardine B, X represents a group [formula]. 2. A method for producing spirocardin A or B, which comprises culturing spirocardin A or B-producing bacteria belonging to the genus Nocardia and collecting spirocardin A or B from the culture. 3 The spirocardin A or B producing bacterium belonging to the genus Nocardia is Nocardia sp.SANK 64282
2. The production method according to claim 2, which is a strain (KAIKOKEN HYUKYO NO. 6986).