JPS5843794A - Antibiotic substance, isohematinic acid - Google Patents

Abstract

PURPOSE:To obtain the titled substance from the cultured product obtained by the cultivation of microorganisms belonging to Actinoplanes genus and capable of producing the titled substance, and to use the titled substance as an antibacterial agent and an agent for promoting the host resistance. CONSTITUTION:Microorganisms belonging to Actinoplanes genus and capable of producing isohematinic acid of formula, e.g. Actinoplanes sp. SANK61681 strain, are cultured in a medium containing known nutrients for actinomycetes, e.g. glucose, glycerin, soybean oil, corn steep liquor, peptone, meat extract, inorganic salts, etc. at 24-30 deg.C and nearly neutral pH condition. After the cultivation, the microbial cells are separated from the medium by filtration, and the objective isohematinic acid is separated from the filtrate with e.g. adsorbent, etc. The obtained isohematinic acid is used as an antibacterial agent or an agent for promoting the host resistance.

Description

[Detailed description of the invention] The invention is a new antibiotic J! ! t isohematic acid (80 ha
m-atinic acld) and its pharmacologically acceptable vine salt, its production method, and an antibacterial agent and host resistance enhancer containing it as an active ingredient. It was discovered that the 5ANKSIlil1 strain belonging to the genus Actinoplanes produced a new antibiotic, and as a result of investigation of this substance, it was found to have the following structural formula9.

C. This substance is hereinafter referred to as inhematic acid.
yloaoccus aureus), Streptococcus faecalis
faec-alig), Escherichia collie (1!
ischerichiacoli), Bacteroides fragilis (Bacter-oldee fragi)
It exhibited antibacterial properties against bacteria such as A. lig), and also exhibited host resistance enhancing effects in experimental mouse infection systems. Therefore, isohematic acid is used for the prevention and treatment of human and animal diseases caused by these.

The present invention also includes pharmaceutically acceptable salts of inhematic acid. Here, pharmacologically acceptable salts include:
For example, alkali metal salts such as sodium and potassium, alkaline earth metal salts such as calcium and magnesium, etc.
-I can give. The mycological properties of the actinomycete strain 8ANK6161H, which produces inhematic acid, are as follows.

1. Morphological characteristics On potato extract/carrot extract agar medium, 28℃, 14
Morphological characteristics of strain 5ANK61681 observed under a microscope after culture for one day t4.・This strain has sporangia, which usually have a diameter of 5 to 16 μm and are spherical to subglobular.

The sporangium contained in the sporangium has an oval shape, and its size is (L8~1.5Xt, 4~LOμ).The sporangium has a flagellum, and the sporangium kpHV, o, /1
．． When placed in M phosphate buffer, sporangiospores become migratory in about 5 minutes.

2 Properties on various culture media The properties after culturing on various culture media at 28°C for 14 days are as shown in Table 1. The color tone display represents the color chip number of the Japan Color Research New Edition Standard Color Chart.

Table 1 8M Not very good, flat, light brown (4 chloride -8-6) agar
Mu M No settlement, 1, 8F No production, 8M Residue not good, flat, brownish white (2-crus/asparagus -9-7) Golden agar Musuma No settlement, sp No production, 8M Too good Flat, light orange glycerin asparagus AM Kishi agar without epiphyte
SP Not produced 8M Abundant, raised, pleated 1 dull yellow orange CS-8-7) Starch inorganic salt agar AM Not attached (from P4) BP Not produced 8M Abundant, raised, pleated, bright brown ( 8-5-7) Tyrosine agar AM No attachment (Eng. 5p7) 8P Light brown (8-5-7)
8M Not very good, flat, pale orange (4-s-r) Nutrient agar AM No settlement (D14ao) 8P H1i no formation 8M
Abundant, raised, pleated, dull yellow-orange (a-a-S) Yeast Malt Agar Mu M Not established (Eng 5p2) ap Not produced 8M Abundant, raised, pleated, yellow-orange (1o -8- 7) Oatmeal agar AM No settlement (Engineering 8P3)
SP No production, 8M Very little and flat, yellow brown (@-7-8) Water Agar AM No epiphyte, BP! ! Neck 8G No formation 8M Not well formed and flat, yellow brown (6-7-8) Potato/carrot extract AM No formation, agar sp No production 8G Well formed 8M Very little and flat, Light yellow brown (4-7-8) Soil extract agar AM No epiphyte sp No production 8G No shape, no formation 8M Bibasic hyphae -El: Soluble pigment 5M = Aerial hyphae
SG: Sporangium 3, Physiological properties 5 The physiological properties of the ANK H681 strain are shown in Table 2.

Table 1 Hydrolysis of starch Positive: (strong) Liquefaction of gelatin Positive (slow), '4 Coagulation of milk 26℃ Negative 37C Positive (strong) Peptonization of milk 26℃ Negative Peptonization of milk 31℃ Positive (weak) ) Melanin-like pigment productivity medium 1 Negative medium 2 Negative medium 3 Degradation of negative casein Degradation of positive (moderate) tyrosine Degradation of negative xanthine Porous growth temperature range (medium 4) & 5-33℃ medium 1 Nidributone yeast extract Broth (Engineering 8Pj) medium 2
: Peptone/yeast extract/iron agar (Technical 5plB) Medium 3: Tyrosine agar (from P7) Medium 4: Yeast/malt agar Also, using Prudham-Godelive agar medium, 2
The carbon source assimilation ability of the Ro8ANK616H strain observed after culturing at 8°C for 14 days is as shown in Table 3.

Table 3 Threat: Frequently used +: Used Person: Use is questionable -2 Not used 1 Niblidhem-Gotlieb agar 2 Niblidhem-Gotlieb agar yeast extract 0
．． 1%4, B. Beaker et al. and M.P. Lechavalier (M.P.) regarding bacterial cell components.
[Applied Microbiology (ppli・fL Microbioxology) method]
), vol. 12, pp. 421-423, 1%64,
Vol. 1S, p. 28 - 243, 1965; H. Prauser, The Actinomycetal.
ea).

311-316, 1970], the acid hydrolyzate of the bacterial cells was analyzed by paper chromatography, and men-2,6-diaminopimelic acid, glycine, and galactose were observed. It was confirmed that it was a 90-year-old actinomycete as described above.
Among the various properties of the 1i81 strain, this strain is particularly characterized by the fact that it forms spherical or subglobular sporangia, that the sporangiospores contained therein are migratory, and that the cell wall type is type L. Among actinomycetes, it was found that it is a strain belonging to the genus Actinoplanes of the family Lanaceae, and Actinoplanes sp.
inoplanes 8p,) 5ANK(illil
Named ll9.

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. 6045. In addition, the identification of this strain 8mu NK111i81 was made using 18P.
(International Streptomyces Project)
es Project)-Standards, Applied Microbial Industry Examination Standards, Bergey sMa
nual of Determinative Bac
teriology) 8th edition, ``The Actinoplanes'' written by N. A. Walcsman and recent literature on actinomycetes. However, it is a well-known fact that the various properties of actinomycetes are not constant and can easily change naturally or artificially. Therefore, all strains are included.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 aerated agitation culture.Medium components include Mori, Known nutritional sources for actinomycetes are used, such as glucose, sucrose, glycerin, maltose, dextrin, starch, soybean oil, and cottonseed oil as carbon sources, and soybean flour, peanut flour, and cottonseed as nitrogen sources. Powder, Farmamine, Fishmeal, Corn Steep Liquor,
Peptone, meat extract, yeast, yeast extract, sodium nitrate, ammonium nitrate, ammonium sulfate, various amino acids, etc., and inorganic salts such as table salt, phosphate, calcium carbonate, trace metal salts, etc. are required! It is added as appropriate depending on the situation. During liquid culture, silicone oil, vegetable oil, surfactant, etc. are appropriately used as antifoaming agents. The pm of the medium is near neutral, and the culture temperature is preferably 24°C to 30°C, particularly around 27U. Changes over time in inhematic acid produced in the culture solution over the course of culture were determined using Bacteroides frgilis as the test bacterium.
Agilis) 8AliK71176 (D-Night culture solution was added to a gum agar medium (manufactured by Hinaga Pharmaceutical Co., Ltd.) to prepare a cellulose, and the culture was measured by the paper disc assay method on a nine-agar plate. Usually after about 140 hours of culture. The production amount of isohematic acid reaches its maximum value.After the cultivation, the inhematic acid present in the liquid part of the culture solution is removed by filtering the bacterial cells and other solid parts using diatomaceous earth as a filter aid, or by centrifugation. and extracted from the filtrate or supernatant.

Isohematic acid can be collected by utilizing its physicochemical properties, for example, using an adsorbent. Examples of the adsorbent include activated carbon or adsorption resins such as THEAL ANHALITE XAD-2 and MAD-4.

XAD-7 etc. (Rohm & Haas JR) and Diaion UP 10, HP 20, UP 5
0 grade (manufactured by Mitsubishi Chemical Industries, Ltd.) is used, and the solution containing inhematic acid is passed through the above adsorbent to adsorb and remove the impurities contained in it. It is also possible to collect the sample using a 0 or 1 ion exchange resin which is eluted with n-butanol water, n-butanol water, -acetone water, or the like. Examples of ion exchange resins include Amberlite RO-50 and CG-
50.

Duolite A-2, Dowex 50W
However, after isohematic acid is adsorbed, it is eluted using hydrochloric acid water, various buffer solutions, saline, etc. λ isohematic acid can be used in water-immiscible organic solvents such as ethyl acetate, n-
It is also possible to extract and purify the cultured solution or aqueous solution using butanol, methyl isobutyl ketone, etc. alone or in combination under neutral or acidic conditions.

In order to further purify inhematic acid, nine-partition column chromatography using Sephadex LH-20 (manufactured by Pharmacia), reverse phase partition chromatography using Pretubpack SOD/C (manufactured by Waters, USA) 8, etc. Effective methods include adsorption column chromatography using a carrier such as silica gel or Florisil, an extraction method that utilizes the difference in the distribution ratio of isohematic acid and mixed impurities to the solvent, or a countercurrent distribution method. Diisohematic acid can be purified by repeatedly using the above purification means alone or in appropriate combinations.

Also, like general fat-soluble antibiotics, isohematic acid exists in the bacterial cell part of the culture solution depending on the culture conditions, and after extraction with a hydrophilic organic solvent such as alcohol or acetone, the solvent is removed to form an aqueous solution. Later, cultivate! It can be extracted and purified in the same manner as from #p solution. ,.

If desired, the isohematinic acid thus obtained can be converted into a pharmacologically acceptable salt according to a conventional method.

Inhematic acid thus obtained has the following physicochemical and biological properties.

1) Properties of substance: colorless microcrystalline acidic substance 2) Elemental analysis value (a): C,! 5.22 ;I! , 4.81i:
N.

7.51 3) Melting point = 138℃ 4) Molecular formula: C!　HN.

5) Ultraviolet absorption spectrum: 24:ζ'nff1 ('') The ultraviolet absorption spectrum measured in methanol is the first
This is the passage shown in the figure.

6) Infrared absorption spectrum KBrc11-1"KB
The infrared absorption spectrum measured with the R tablet is as shown in FIG.

7) Nuclear magnetic resonance spectrum (δ: pp engineering) l measured in heavy pyridine using tetramethylsilane as an internal standard.
QQ MH2 nuclear magnetic resonance absorption spectrum is shown in Figure 3. Positive for cresol green reaction, negative for ninhydrin reaction.

1 Thin layer chromatography: xt value A5 Adsorbent: Merck silica gel plate 5715 Developing solvent: Benzene: Ethyl acetate: Acetic acid (10:10:
1) 11) High-pressure V-paper - Qi-eishoku: Q, IM. High-pressure V-paper electric eishoku (55/,) using Toyoji paper A5 1 mm in Tris-HCl buffer (PH7,5).

0.8/, I) moves toward the anode, and when the mobile reaction of Bj', B (bromo phenol blue) used as a standard substance is 1.0, that of inhematic acid is 1.3. It is.

12) Antibacterial crab each fiJ#ill)! The anti-Japanese ability against M.
)t- was measured by the cylinder plate method.

The results are shown in Table 4.

1: Nitsusui sensitive disk medium 2: Gum agar medium 13) Host resistance enhancing effect: The ability of the host to defend against infection was measured by the method described below.

■ Etssielichia coli (]!facherichi
a c611) Ntech11JJO-2 was cultured overnight at 81°C using trypto soy broth liquid medium (Eiken), and then suspended in physiological saline to give 2Xl' cells/ml. This bacterial solution a2- was intraperitoneally inoculated into mice (ICR male, 10 mice per group, 5 weeks old). On the other hand, a predetermined inhematic acid solution prepared with /15M phosphate buffer,
The mice were administered intraperitoneally, subcutaneously, and orally 24 hours before inoculation of the bacterial solution. '/15M phosphate buffer (L2m/
was similarly administered to mice.

Table 5 shows the results of measuring the number of dead mice 2 days after infection.

Control 10/.

vIHlo　　　　　　’/t.

1oes　　　　　　/1゜ Control $/.

Oral 10・4
/1゜■ E. coli 627 was cultured overnight at 37°C using trypto soy broth liquid medium (Eiken), and then suspended in physiological saline and incubated with l' I
o'JI cell/WLltoL*@Cnoli fluid a2- was intravenously inoculated to Ceus (XOR male, 5 weeks old, 1 animal per group). On the other hand, an aqueous isohematic acid solution adjusted to pif 7.6 with caustic soda was subcutaneously administered at a predetermined concentration of 2077 g/mouse 24 hours before inoculation of the bacterial solution. As a control, 12 d of physiological saline was administered to mice in the same manner, and the number of dead mice was measured 2 and 4 days after the infection.

The results are shown in Table 6.

Chapter 6 Table Number of dead mice 107.

Control group 8/.

■ Candida albicans
cana) 5ANK 50157 T-Sabouraud liquid medium was used to culture at 37°C overnight, and then suspended in physiological saline to give 5X1G7Im cells/d.
IL211Ll was introduced into mice (ICR male, 5 weeks old, group 1).
(0 mice) were inoculated intravenously.

On the other hand, an aqueous isohematic acid solution prepared in the same manner as in (1) above was subcutaneously administered at a predetermined water temperature of 20 μg/max 24 hours before the inoculation of the bacterial solution. As a control, physiological saline fi, 211E/ was similarly administered to mice. The number of dead mice was measured 1 day and 10 days after infection.

The results are shown in Table 1〇 Chapter 1 Table Number of dead mice T-eye removal 10 days later Administration group 2/6/1・ 0 Control group　’/　　　　’/.

0 1 Acute toxicity LD against Max by intravenous administration. The value is so
It was o iv/#i.

From the above, inhematic acid is used as an antibacterial agent and host resistance enhancer for various bacterial infectious diseases. The administration forms include subcutaneous injection, intravenous injection, intramuscular injection, parenteral administration such as layered preparations, and oral administration such as tablets, capsules, powders, and granules.

The dosage varies depending on the target disease, route of administration, frequency of administration, etc., but for adults, it is usually preferable to administer 1 mg to 500 μl per day, either once or in divided doses.

Next, the present invention will be explained in more detail with reference to Examples and Formulation Examples.

Example 1 Actinoplanes sp. 81NK61681 strain,
Soor containing medium @Oat shown in medium composition-1
One platinum loop was inoculated into a D volume Erlenmeyer flask, and cultured for 168 hours in a J21C rotary shaking culture machine at 220 rpm.

5 ml of this culture solution 1K - medium composition 1 2 medium 8
0-S: 1'F was inoculated into a 50OMl Erlenmeyer flask,
The cells were cultured at 28° C. in a rotary shaking incubator at 220 rpm. Centrifuge the culture solution at 30,001"l) ml for 15 minutes,
The progress of the culture was followed while measuring the antibacterial activity of the supernatant liquid against Bacteroides fragilis 8AMK71176 using a PETXll-disk, and the production amount reached SOO μg/g in 96 hours.

Example λ Actinoplanes SP 8AIJ] im61681
The strain was grown in 5OO containing 80 m of medium indicated by medium composition -1.
- One platinum loop was inoculated into a Erlenmeyer flask, and cultured for 1681 iP at 8 cis in a rotary shaking incubator at 220 rpm. The obtained culture solution 5+117i was inoculated into a soy-sized Erlenmeyer flask containing a medium 80d shown in medium composition -3, and cultured at 28° C. in a rotary shaking culture machine at 22 Orpm. The resulting culture solution was then diluted with 30 e Oram.
Centrifuge for 0 minutes, and use a paper disk to collect the supernatant and collect Bacteroides flavoris 8um M11111@
The antibacterial power against kl:・j is measured! If you follow the progress of culture, 12
The production amount reached 250 μg/R1 at 0 hours.

Example: Actinoplanes sp.
A platinum loop was inoculated into a m Erlenmeyer flask and cultured for 120 hours at 28°C in a 22 Orpm rotary shaking incubator.

351!1lt of the obtained culture solution - medium composition - 2 containing 700 m of the medium shown in 1 was inoculated into an Erlenmeyer flask,
Incubate at 28°C in a rotary shaker incubator at 220 rpm.
Cultured for hours. Next, the obtained culture solution was used as a seed culture solution, and 8 containing 300 t of the medium shown in medium composition -4- was prepared.
Main culture II was performed by inoculating 3.00 into a tank. The conditions for main culture were aeration of 1150 t/min and internal pressure of 0.5 kg/min.
CIL % rotation speed 190 rl) m at 28°C 13
It was cultured for 9 hours. After the culture was completed, the mixture was filtered using a filter press using celite as an auxiliary agent, and when combined with the image body washing solution, 350 tons of culture p solution was obtained. The amount of inhematinic acid in the culture p solution was measured by the paper disk method using Bacteroides fragilis 8ANK71 [6 as the test bacterium, and it was found to be 250 μg/−. After adjusting 350 t of this P solution to pII3 with hydrochloric acid water, it was subjected to F treatment in a filter press using Celite as an auxiliary agent, to obtain 350 t of F solution.

The obtained ν solution was adsorbed on a column of Diaion IIP 20,60 t, and after washing the column with water, it was eluted with 3o-hydrated acetone to obtain active fraction 12 (It). This active fraction was absorbed under reduced pressure. 4 tons of concentrated liquid was obtained.

After adding 400 F of common salt to this concentrated solution, 400 F of ethyl acetate was added.
Extracted twice using t. After combining the extracts, a. under reduced pressure.
Concentrate to 3t, then chloroform-ethyl acetate (1
: 1) After sufficient swelling and equilibration with the solvent system of 9 Sephadex LH-20 (Pharmacia Co., Ltd.
Developed with the same solvent system added to I), and the eluate was converted into (Lst
It was fractionated.

The fractions 11 to 15 were collected and concentrated to dryness under reduced pressure to obtain 20.3f of crude crystals of inhematic acid.

The obtained crude crystals of 28.3 F were recrystallized using hot methanol to obtain colorless fine crystals of isohematic acid &1F.

Here, the composition of the medium used in Examples 1 to 3 is shown.

Medium composition-1 Glucose 1.0 t Oatmeal 0
5 Glycerol t, (l Casamino acid a5 Sucrose 10g 0aOO
, Q, i yeast 1.0 Antifoaming agent OΩ1 dog bean flour 20 Glucose s, o 裳 Meat extract α4(-
Su1! #S 0. I MaCt a25 soybean flour 1.0 0aCO (15 polypeptone 0.4 antifoaming agent 05 (p before sterilization)
Hr, O) Medium composition-3 Chrycerol 0.5%, l', ·, Learn Steep Liquor 15 Sucrose Z,
g Co0t2・6H200,001 Soy flour 1.0 Antifoaming agent 0.01
Medium composition-4 Glucose 20% Polypeptone 0.
5 Soluble starch 1.0 Maot
α5 raw yeast o, s caco,
B3 meat extract o, s antifoaming agent
0.1N bottle: The antifoaming agent used was “Disfoam 0B”.
-442J manufactured by Nippon Oil & Fats Co., Ltd.

Examples of formulations are shown below.

Formulation example 1. Capsules for oral administration Isohematic acid 100~lactose
100 Corn starch 14LS Magnesium stearate 1.5, ::, 3
5oWI9 Powder of the above formulation was mixed and passed through a 30-mesh sieve, and the powder 3501115J was placed in a No. 2 gelatin capsule to form a capsule.

Formulation Example λ Injection Isohematin rR50M9'tN, N-dimethylacetamide Q, dissolved in 311Ll, then 4.7'Ill of 716M phosphate buffer (pTlB, 9) was added, sealed in a 5-ampule, and sterilized according to the usual method. It was made into an injection.

[Brief explanation of drawings]

Figure 1 shows the ultraviolet absorption spectrum of inhematic acid, Figure 2 shows the infrared absorption spectrum of the same substance, and Figure 3 shows the infrared absorption spectrum of the same substance.
The figure shows the nuclear magnetic resonance spectrum of the same material. Patent applicant Sankyo Co., Ltd. agent Patent attorney Shoji Kashi Figure 1 procedural amendment (voluntary) September δ, 1980 Commissioner of the Japan Patent Office Kazuo Wakasugi■ Case indication Patent application No. 141519 of 1982 2. Name of the invention: Antibiotic inhematic acid 3. Relationship with the person making the amendment Patent applicant address: 6 Name, 3-1 Nihonbashi Honmachi, Chuo-ku, Tokyo 103 (185) Sankyo Co., Ltd. Representative Director and President Yoshinori Kawamura 4. Residence of the agent: Sankyo Co., Ltd., 1-2-58 Hiromachi, Honmonagawa-ku, Tokyo 140 5. Number of inventions to be increased due to amendment: None 6. Subject of amendment: Detailed description of the invention in the specification 7. Contents of the amendment As shown in the attached sheet 1. "SM Not very good, flat, pale orange glycerin agar" on page 5 of the specification, Suno Ragin Agar SP Not produced, 8G Not formed ” 1'-SM Not very good, flat, light orange (4-8-7)
SG not formed,” he corrected. 2. Correct "Potato/Carrot Extract Agar" from the second line from the bottom to the last line of page 6 to read "Potato Extract/Carrot Extract Agar." 3. Correct "Medium 4: Yeast/Malt Agar" in the 5th line from the bottom of page 8 to "Medium 4: Yeast/malt agar (ISP 2) J." 4. 2 lines from the bottom of page 9. Correct ``±- use is questionable'' to ``± 2 weak use.'' 1 Correct "glucose" in line 7 of page 12 to "glucose". 6. On page 12, line 8, "glycerin" is corrected to "glycerol." 7. On page 28, line 3, "soluble starch" is corrected to "soluble starch."that's all

Claims (1)

[Scope of Claims] Inhematic acid represented by 1.2〇00H and a pharmacologically acceptable salt of 7. 2. A method for producing isohematic acid, which comprises culturing isohematic acid-producing bacteria belonging to the genus Actinoplanes and collecting isohematic acid from the culture. The inhematic acid-producing bacteria belonging to the genus Actinoplanes is Actinoplanes sp. BA! 1K61681
(Feikoken Bibori No. 6045)
Manufacturing method described in section. 4. An antibacterial agent containing inhematic acid and its pharmacologically acceptable salt as an active ingredient. 4. A host resistance enhancer containing isohematic acid and its pharmacologically acceptable salt as an active ingredient.