JPS61251693A - Antibiotics: ct-b, ct-c and ct-d - Google Patents

Abstract

NEW MATERIAL:An antibiotic, CT-B, having following properties: colorless crystals; acidic; melting point: 196-200 deg.C; elementary analyses: C 62.67%, H 8.45%, O 26.57%, Na 3.23%; [alpha]<15>D=+12.5 deg. (c=1.0 in chloroform); easily soluble in chloroform, benzene; soluble in ethyl acetate, acetone, isopropanol, ethanol, methanol; hardly soluble in water, hexane; colored with 10% sulfuric acid into yellow, with vaniline-sulfuric acid into red. USE:Antibacterial. It has especially strong bactericidal action on gram-positive bacteria and, in addition, anticoccidium and antiviral actions. PREPARATION:For example, a microorganism in Actinomadura (FERM 5678) is cultured in an appropriate medium, preferably under aerobic agitation at about 30 deg.C for 72-148hr.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to antibiotic CT-B, CT-C and CT-D substances.

The present inventors have tested a large number of microorganisms for the purpose of searching for new antibiotics, and have isolated and searched for the antibiotics produced by the microorganisms.

As a result, an antibiotic different from the antibiotic 76-11 substance originally produced by this bacterium (see Patent Publication No. 1983-33948) was produced in the culture solution and cultured bacterial cells of Kaikoken Bacteria No. 5678. found that it is accumulated.

When these antibiotics were isolated and their physicochemical and biological properties were examined, they were found to be new antibiotics, and were classified as antibiotic CT-B substances, antibiotic CT-C substances, and antibiotic CT-C substances, respectively. It was named antibiotic CT-D substance.

Microorganisms used in the production of antibiotic CT-B substances, antibiotic CT-C substances, and antibiotic CT-D substances include:
All microorganisms can be used as long as they have the ability to produce the antibiotic CT-B substance, the antibiotic CT-C substance, and the antibiotic CT-D substance. Preference is given to Kaikoken Bibori No. 5678.

Incidentally, the mycological properties of Kaikoken Bokuyori No. 5678 are described in Patent Publication No. 1983-33948.

In order to produce the antibiotic CT-B substance, antibiotic CT-C substance and antibiotic CT-D substance of the present invention, the producing bacteria can be cultured using a culture method suitable for each producing bacteria.

For example, in the case of the microorganism strain No. 5678 belonging to the genus Actinomadura used by the present inventors, the culture can be carried out by a known culture method for actinomycetes.

Industrially, aeration and agitation culture is suitable.

In addition, as the production medium, ordinary raw materials used for culturing actinomycetes are used, that is, various carbon sources, nitrogen sources, organic or inorganic salts, and in some cases antifoaming agents may be used in appropriate combinations. can. In general, carbon sources include glucose, starch, glycerin, dextrin,
Sucrose, animal and vegetable oils, etc., and as nitrogen sources, soybean flour, corn steep liquor, wheat germ, ammonia, etc. can be used. In addition, inorganic salts such as calcium carbonate, sodium chloride, potassium chloride, and phosphate are added as necessary. The culture temperature is generally in the range of 25 to 35°C, but preferably around 30°C.The culture time varies depending on various conditions, but is usually 72 to 148 hours.
The production and accumulation amounts of each of the B substance, the antibiotic CT-C substance, and the antibiotic CT-D substance are maximized. Antibiotic CT-B substance, antibiotic CT-C substance and antibiotic C from culture
In order to isolate and purify the T-D substance, the physical and chemical properties of the substance can be utilized, and known means such as means that utilize the difference in solubility with impurities, adsorption power to ion exchange resins or various adsorbents can be used. extraction method using an organic solvent that is immiscible with water.

Alternatively, methods such as precipitation, removal of impurities, dialysis, drying, recrystallization, etc. can be appropriately selected and combined. Antibiotic CT-B substance produced in culture, antibiotic CT
The -C substance and the antibiotic CT-D substance are preferably collected, for example, in the following manner.A filter aid 1 such as diatomaceous earth or Radiolite 700 is added to the culture solution and the culture solution is filtered. Each body is extracted with a suitable solvent such as ethyl acetate, butyl acetate, chloroform, butanol, acetone, etc.

The bacterial cells can be extracted with aqueous acetone or aqueous methanol, the organic solvent is distilled off under reduced pressure, and the remaining aqueous solution can be extracted using ethyl acetate or the like.

Next, the bacterial cell extract and the filtrate extract are combined, and the solvent is distilled off from this extract to obtain antibiotic 76-11 substance, antibiotic CT-B substance, antibiotic CT-C substance, and antibiotic C.
Crude crystals of T-D substance are obtained.

In order to isolate each of the antibiotic CT-B substance, antibiotic CT-C substance and antibiotic CT-D substance from these, 5
For example, a silica gel column to be subjected to adsorption chromatography on silica gel, alumina, etc. is prepared with benzene, and a solution of the extract dissolved in a small amount of benzene is poured into it.

Using benzene and a benzene-ethyl acetate mixture, the antibiotic CT-B substance, antibiotic CT
Obtained as the sodium salt of the -C substance and the antibiotic CT-D substance.

The antibiotics CT-B, CT-C and C thus obtained,
The properties of the sodium salt of substance T-D are as follows.

Antibiotic CT-B substance (has the following physical and chemical properties as a sodium salt) (1) Colorless crystal, itself an acidic substance.

(2) Melting point 196-200℃ (in columnar crystals) (3
) Elemental analysis value (actual value %) CB2.67%, 88.45%.

0 25.60%, Na3.23% (4) Specific optical rotation [α)” = +12.5° (C1,
0, chloroform) (5) Ultraviolet absorption spectrum two maximum values methanol and hydrochloric acid in acidic methanol: λmax = 21
7 sjL (E, -260)λmax=264
tap (E 18189) lc轡λmax=301 1llt (HI8 70) IC
#1 In alkaline methanol: ゛λvsax= 263 mtx (E 1t1e+
188) λw+ax=300 tap (HI2
74)/CM (6) Infrared absorption spectrum (measured with potassium bromide tablets)
Characteristic absorption (cm”): 3400.2960, 1710, 1635 = 1605
, 1575. 1450. 1370. 1310°1250, 1205. 1195. 1150. 1
105, 1035. 965. 935. 920(
7) Solubility: Easily soluble in chloroform and benzene, soluble in ethyl acetate, acetone, isopropanol, ethanol, and methanol.
Slightly soluble in water and hexane.

(8) Color reaction: Yellow with 10% sulfuric acid (on silica gel thin layer chromatography, heated at 100°C for 5 minutes after spraying), red with vanillin sulfuric acid.

(9) Thin layer chromatography (using Kieselgel GF254 manufactured by Merck & Co.): Antibiotic CT-C substance (having the following physical and chemical properties as a sodium salt) (1) Colorless crystals, itself an acidic substance.

(2) Melting point: Decomposes at 210°C.

(3) Elemental analysis value (actual value %) C61.20%, H8°27%, 026.57%, Na 3°92% (4) Specific optical rotation [α)” = +19.4° (C1, (5) Ultraviolet absorption spectrum two maximum values methanol and hydrochloric acid in acidic methanol: λmax = 2
17 mp (E,,,,268)λmax
=264 wμ(E::188)λmax=301
rap (E” 6g) lc+m in alkaline methanol: λmax= 2 6 3 mtt (E 1L
, 17g) λmax=300 mfi (E; Yu6
9) (6) Characteristic absorption (Cm-1) in infrared absorption spectrum (measured with potassium bromide tablets): 3400.296
0,1710,1640,1610, 1580. 1
450. 1380. 1300°1255, 121
0. 1200. 1155. 11to, 103
5. 980. 935(7) Solubility: Easily soluble in chloroform and benzene, soluble in ethyl acetate, acetone, isopropanol, ethanol, and methanol.
Slightly soluble in water and hexane.

(8) Color reaction: Pale yellowish brown with 10% sulfuric acid (on silica gel thin layer chromatography, heated at 100°C for 5 minutes after spraying).

(9) Thin layer chromatography (using Kieselgel GF254 manufactured by Merck & Co.): Antibiotic CT-D substance (having the following physical and chemical properties as a sodium salt).

(1) Colorless crystal. itself is an acidic substance.

(2) Melting point 170-174℃ (in columnar crystals) (3
) Elemental analysis value (actual value %) C62.34%, 88.33%, 026.04%, Na3.15% (4) Ultraviolet absorption spectrum two maximum values Methanol and hydrochloric acid in acidic methanol: λmax = 21
7 rap (El″256)l(lII λmax=2 6 4 up (E ″18
0) 1cm λraax= 301 rap (E 64)
C1v1 in alkaline methanol: λmax=263 rap (E''' 164)
ax λmax=300 mp (E” 6g) (5
) Characteristic absorption (Cm-1) in infrared absorption spectrum (measured with potassium bromide tablets): 3400.2970,17
10, 1635, 1605, 1575, 1450, 13
80,1320°1250.1200,1155,11
05.1080, 1060. 1035.935 (6
) Solubility: Easily soluble in chloroform and benzene. Soluble in ethyl acetate, acetone, isopropanol, ethanol, and methanol, slightly soluble in water and hexane.

(7) Color reaction: Light brown with 10% sulfuric acid (on silica gel thin layer chromatography, heated at 100°C for 5 minutes after spraying).

(8) Thin layer chromatography (using Kieselgel GF254 manufactured by Merck & Co.): Table 1 shows the antibacterial spectra of the above antibiotic CT-B, antibiotic CT-C and antibiotic CT-D substances.

Based on the above physicochemical and biological properties, this substance belongs to the polyether group of antibiotics.

However, antibiotics CT-B substances, CT-C substances and CT-
It can be seen that Substance D is a new antibiotic different from known antibiotics based on its properties such as melting point, specific optical rotation single-element analysis value, and infrared absorption spectrum.

Antibiotics CT-B substance, CT-C substance and CT-D substance have a strong antibacterial effect particularly against Durham positive bacteria and are therefore useful as antibacterial agents.

Furthermore, it has anticoccidial, acaricidal, and antiviral effects, and is also useful as a preventive and therapeutic agent for diarrhea, a feed efficiency improver, or a growth promoter for livestock such as cows and pigs.

7, -'/' Example 1 Oatmeal? %, glucose 2%, soybean flour 2%, wheat germ 2% and salt 0.05% (pH
Inoculate 450 mg of a strain of Actinomadura (Accession Number: FAIKEN Bacterium No. 5678) at 33°C for 450 mg.
Incubate for 8 hours. 450 mQ of this culture solution was inoculated into a medium 15Q having the same composition as above, and cultured with aeration and stirring at 33° C. for 2 days using a Jarfer Menta at an aeration rate of 15 fl/min and a rotation speed of 350 rpm.

600 mQ of this culture solution was mixed with 8% glycerin, 1% glucose, 2.5% soybean flour, 0.4% potassium monophosphate, 0.2% dibasic potassium phosphate, 0.1% calcium carbonate, and 0.1% dibasic sulfuric acid. It is inoculated into medium 20Q consisting of 0.1% iron and 0.1% ammonium sulfate, and cultured with aeration and stirring at 33° C. for 210 hours using a Jarfer Menta.

The ventilation amount at this time is 20 Q/min and the zero rotation speed is 350 rpm.

After the culture is completed, the culture solution is heated at 60° C. for 10 minutes, and then the pH is lowered to 4.0. Further, 800 g of filter aid (trade name Niradiolite 700) is added and filtered under pressure to separate the filtrate and bacterial cells.Next, after adjusting the pH of the filtrate and washing solution to 9.0, ethyl acetate of 101 and 5a is added. Extract twice using

On the other hand, after adjusting the pH of the bacterial cells to 9.0, acetone 10Q
Extract twice. The acetone extract of the bacterial cells is concentrated under reduced pressure to remove acetone, and then extracted twice with 2Q ethyl acetate. This extract and the extract from the filtrate were combined, concentrated under reduced pressure, and the residue was dissolved in a small amount of benzene.
00°5cm x 40cm) column, benzene 2Q, benzene-ethyl acetate (1:1) 2Q.

76-
11 substances, CT-B substance, CT-C substance and CT-D substance, can be obtained singly or in a mixed state. A single fraction is collected and concentrated under reduced pressure, and then methanol is added and allowed to stand to obtain crystals.

The fractions of the mixture are subjected to the column chromatography described above repeatedly to obtain a single fraction, and crystals are similarly obtained.

As a result, 7 g of sodium salt of antibiotic 76-11 substance, 6 g of sodium salt of antibiotic CT-B substance
00 mg, 300 mg as the sodium salt of the antibiotic CT-C substance and 100 mg as the sodium salt of the antibiotic CT-C substance, respectively. Each antibiotic thus obtained exhibits the above-mentioned physicochemical and biological properties.

Test Example 1 Anti-coccidiosis test drug and feed; Antibiotics CT-B, CT-C, and CT-D were evenly mixed into a complete compound feed for young chicks manufactured by Oriental Co., Ltd. to the test concentration. Medication was continued from 2 days before oocyst inoculation until the end of the test (8 days after infection). Feed was provided ad libitum.

As a comparative drug, monensin, which is known as an anti-coccidiosis drug, was used.

Test chicks: The test chicks were male chicks of the egg-laying breed Shaver Star Cross, 7 days old (9 days old at the time of infection), raised in a manner that completely prevented coccidia infection, and 5 chicks were used per group.

Inoculum oocysts and inoculum dose: A susceptible strain of Eimeria tenella isolated and identified at the Livestock Hygiene Laboratory of the Ministry of Agriculture, Forestry and Fisheries was used. The inoculation amount was 3 x 10' mature oocysts per chick, which were orally inoculated into each chick using a metal probe.

Determination of effectiveness: The determination of effectiveness is the relative increase rate of chicks.

Anticoccidial index (A, C) based on survival rate, oocyst value by intestinal homogenate, and intestinal lesion value.

1,) was calculated and judged based on the index value -Anticoccidial index (A, C, 1,) = (relative increase in weight survival rate) - (oocyst value + lesion value) A, C, 1,120 The following are inappropriate as anticoccidial agents.

A, C, less than 1,160 is less effective as an anti-coccidial agent.

A, C, 1,160-180 are moderately effective as anticoccidial agents.

A, C, 1,180 or higher is extremely effective as an anti-coccidial agent.

a) Body weight measurement The weight is determined by the amount of salary increase for each group at the end of the experiment (body weight at the end of the experiment -
The weight at the time of infection) was determined, and the relative weight gain rate was determined, setting the uninfected, unmedicated control group as 100.

b) Oocyst value On the 8th day after infection, the number of oocysts in the cecum was counted using intestinal homogenate, and the oocyst value was determined as follows.

[Method using intestinal homogenate]

C) Intestinal tract lesion degree The chicks were sacrificed at the end of the test (8 days after infection), and the intestinal tract was examined macroscopically to determine the degree of lesion. In addition, the lesion degree was determined as follows.

(-)O: The cecum is completely normal, and if there is a bleeding spot, it is rated as 10.

(+) 1: The shape of the cecum is normal, and the contents are slightly fluid and yellowish in color. The cecal mucosa is partially slightly swollen and whitish.

(++) 2: The shape of the cecum is almost normal, and swelling of the mucous membrane is seen all over. There is no bleeding in the contents, and the mucus is yellowish and discolored. A small number of white dots of necrosis and hemorrhagic spots are seen within the mucosa.

(+++) 3: The atrophy and deformation of the cecum are clear and it becomes slightly longer than the rectum. The cecal wall is markedly thickened and becomes brittle, without any normal contents, often filled with blood clots or off-white cheese-like degeneration.

Petechiae may still remain.

The lesions extend to the base of the intestine, but not to the rectum.

(++++) 4: The atrophy and deformation of the cecum are significant, generally sausage-shaped, and its length is the same as or shorter than the rectum. The lesion extends to the 173rd to 174th positions of the rectum. The rest is the same as (+++)3.

Groups 1 to 3 were fed with feed containing antibiotics CT-B, CT-C, and CT-D at predetermined concentrations from 2 days before oocyst inoculation, and group 4 contained the antibiotic monensin as a comparison drug. Raised on the same feed.

The fifth group is an infected, non-medicated control group in which animals were inoculated with oocysts and fed a basal diet that does not contain the test drug, and the sixth group is an uninfected, non-medicated control group.

Table 2 Anticoccidial effect Based on the results of this test, antibiotics CT-B, CT-C and CT
It can be seen that each of the -D substances has an excellent anti-coccidiosis effect.

[Brief explanation of drawings]

Fig. 1 shows the ultraviolet absorption spectrum of the sodium salt of the CT-B substance of the present invention, Fig. 2 shows the ultraviolet absorption spectrum of the sodium salt of the CT-C substance of the invention, and Fig. 3 shows the ultraviolet absorption spectrum of the sodium salt of the CT-B substance of the present invention.
FIG. 4 shows the infrared absorption spectrum of the sodium salt of the CT-B substance of the present invention, and FIG. 5 shows the infrared absorption spectrum of the sodium salt of the CT-C substance of the present invention. , FIG. 6 shows the infrared absorption spectrum of the sodium salt of the CT-D substance of the present invention.

Claims (1)

[Scope of Claims] 1) Antibiotic CT-B substance and its salt, which are characterized by having the following physical and chemical properties as a sodium salt. (1) Colorless crystal, itself an acidic substance. (2) Melting point 196-200°C (in columnar crystals) (3) Elemental analysis values (actual value %) C 62.67%, H 8.45%, O 26.57%, Na 3.23% (4 ) Specific optical rotation [α] ^1^5_D = +12.5° (c
1.0, chloroform) (5) Ultraviolet absorption spectrum: Maximum value in methanol and hydrochloric acid acidic methanol: λmax = 217 mμ (E^1^%_1_c_m260
) λmax=264mμ(E^1^%_1_c_m189
) λmax = 301 mμ (E^1^%_1_c_m70) In alkaline methanol: λmax = 263 mμ (E^1^%_1_c_m188
) λmax=300mμ (E^1^%_1_c_m74) (6) Infrared absorption spectrum (measured with potassium bromide tablets)
Characteristic absorption (cm^-^1): 3400, 2960, 1710, 1635, 1605,
1575, 1450, 1370, 1310, 1250,
1205, 1195, 1150, 1105, 1035,
965, 935, 920 (7) Solubility: Easily soluble in chloroform and benzene, soluble in ethyl acetate, acetone, isopropanol, ethanol, and methanol.
Slightly soluble in water and hexane. (8) Color reaction: Yellow with 10% sulfuric acid (on silica gel thin layer chromatography, heated at 100°C for 5 minutes after spraying), red with vanillin sulfuric acid. (9) Thin layer chromatography (using Merck Kieselgel GF_2_5_4): Solvent system Rf value Ethyl acetate 0.22 Ethyl acetate-methanol (20:1) 0.50 〃-〃(50:1) 0.25 Ethyl acetate -Benzene (1:1) 0.00 〃-〃(5:2) 0.05 Chloroform-methanol (20:1) 0.65 〃-〃(50:1) 0.15 2) As the sodium salt, the following Antibiotic CT-C substance and its salt characterized by having physicochemical properties. (1) Colorless crystal, itself an acidic substance. (2) Decomposes at a melting point of 210°C. (3) Elemental analysis values (actual value %) C 61.20%, H 8.27%, O 26.57%, Na 3.92% (4) Specific optical rotation [α] ^1^5_D = +19. 4°(c
1.0, chloroform) (5) Ultraviolet absorption spectrum: maximum value methanol and hydrochloric acid in acidic methanol: λmax = 217 mμ (E^1^%_1_c_m268
) λmax=264mμ(E^1^%_1_c_m188
) λmax = 301 mμ (E^1^%_1_c_m68) In alkaline methanol: λmax = 263 mμ (E^1^%_1_c_m178
) λmax=300mμ (E^1^%_1_c_m69) (6) Infrared absorption spectrum (measured with potassium bromide tablets)
Characteristic absorption (cm^-^1): 3400, 2960, 1710, 1640, 1610,
1580, 1450, 1380, 1300, 1255,
1210, 1200, 1155, 1110, 1035,
980, 935 (7) Solubility: Easily soluble in chloroform and benzene, soluble in ethyl acetate, acetone, isopropanol, ethanol, and methanol.
Slightly soluble in water and hexane. (8) Color reaction: Pale yellowish brown with 10% sulfuric acid (on silica gel thin layer chromatography, heated at 100°C for 5 minutes after spraying). (9) Thin layer chromatography (using Kieselgel GF_2_5_4 manufactured by Merck & Co.): Solvent system Rf value Ethyl acetate 0.65 Ethyl acetate-methanol (20:1) 0.78 〃-〃(50:1) 0.72 Ethyl acetate -Benzene (1:1) 0.20 〃-〃(5:2) 0.40 Chloroform-methanol (20:1) 0.76 〃-〃(50:1) 0.40 3) As the sodium salt, the following Antibiotic CT-D substance and its salt characterized by having physicochemical properties. (1) Colorless crystal, itself an acidic substance. (2) Melting point 170-174°C (in columnar crystals) (3) Elemental analysis values (actual value %) C 62.34%, H 8.33%, O 26.04%, Na 3.15% (4 ) Ultraviolet absorption spectrum: Maximum value in methanol and hydrochloric acid acidic methanol: λmax = 217 mμ (E^1^%_1_c_m256
) λmax=264mμ(E^1^%_1_c_m180
) λmax = 301 mμ (E^1^%_1_c_m64) In alkaline methanol: λmax = 263 mμ (E^1^%_1_c_m164
) λmax=300mμ (E^1^%_1_c_m68) (5) Infrared absorption spectrum (measured with potassium bromide tablets)
Characteristic absorption (cm^-^1): 3400, 2970, 1710, 1640, 1610,
1580, 1450, 1380, 1320, 1250,
1200, 1155, 1105, 1080, 1060,
1035, 935 (6) Solubility: Easily soluble in chloroform and benzene, soluble in ethyl acetate, acetone, isopropanol, ethanol, and methanol.
Slightly soluble in water and hexane. (7) Color reaction: Light brown with 10% sulfuric acid (on silica gel thin layer chromatography, heated at 100°C for 5 minutes after spraying). (8) Thin layer chromatography (using Kieselgel GF_2_5_4 manufactured by Merck & Co.): Solvent system Rf value Ethyl acetate 0.50 Ethyl acetate-methanol (20:1) 0.75 〃-〃(50:1) 0.65 Ethyl acetate -Benzene (1:1) 0.10 〃-〃(5:2) 0.25 Chloroform-methanol (20:1) 0.70 〃-〃(50:1) 0.25