KR820001203B1 - Process for producing deoxynarasin antibiotics by fermentation - Google Patents

Abstract

Deoxynarasin(I) and 20-deoxy-17-epinarasin(II) were produced by submerged aerobic fermn. with Streptomyces aureofaciens NRRL 11181. Thus, a preculture of S. aureofaciens was inoculated into 100 medium contg. soybean meal 25, glucose 20, CaCO3 2, Na2SO410 H2O 1, FeSO47 H2O 0.6, MnCl24 H2O 0.3, ascorbic acid 0.018 g/l, soybean oil 20, and Me oleate 20 ml/l and was incubated at 30≰C for 10 hr with aeration and stirred. The pH was lowered to 3 and the broth shaken for 1 hr, filtered, extd. with MeOH contg. NaHCO3. The ext. was concd. and extd. with CHCl3. The CHCl3 ext. was dried and chromatographed on silica gel to sep. 83 mg I and 56 mg II.

Description

Method for producing deoxynaracin antibiotic

The present invention relates to a method for preparing a deoxy narcin antibiotic of the following structural formula (I) or (II), which is useful as an antibacterial agent and an anticoccidial agent and enhances the feed utilization effect of ruminants.

A mixture of deoxynacin antibiotics consisting of 20-deoxynaracin and 20-deoxy-epi-17 naracin is produced by aerobic fermentation of Streptomyces oreo faciens NRRL 11181 in water. 20-deoxynaracin and 20-deoxy-epi-17-naracin are separated by chromatography. Deoxynacin antibiotic mixtures of 20-deoxy-naracin and 20-deoxy-epi-17-naracin are antimicrobial and anticoccidial agents and also enhance the effectiveness of ruminant feed.

There is a continuing need for the development of new veterinary antibiotics, for example, coccidiosis is a major problem in the livestock industry. Coccidiosis is caused by an infection with one or more species of Aimeria or Isolaspora.

See Lund and Farr in "Diseases of Poulty '", 5th ed, Biester and Schwarte, Eds, Iowa State University Press, Ames, Iowa, 1965, pp. 1056-1096). Since the economic loss caused by coccidiosis is large, the development of better anticoccidial agents is required. In the field of veterinary medicine, another economically demanding purpose is to promote the growth of ruminants such as cattle and sheep.

In particular, the purpose is to promote growth by increasing feed efficiency. The mechanism of action of the main component (hydrocarbon) in ruminant feed is well known. That is, the microorganisms in the intestine of the animal decompose the hydrocarbons and convert them into monosaccharides and convert the converted monosaccharides into pyruvate compounds. Pyruvate is metabolized by microbiological methods to produce acetate, butyrate or propionate, known as volatile fatty acids (VFAs). Further details are described in the following references.

[Reference ; Leng in "Physiology of Digestion and Metabolism in the Ruminant", Phillipson etal. Eds., Oriel Press, Newcastle-upon-Tyne, England, 1970, pp. 408-410.]

The comparative effect of using VFA was discussed by Meklov. [See also: "Feedstuffs", June 19, 1971, page 19; Eskeland et al. in J.An. Sci. 33, 282 (1971): and Church et al. in "Digestive Physiology and Nutrition of Ruminants," Vol. 2, 1971 pp. 622 and 625.]

Acetate and butyrate are also used, but propionate is used more efficiently. Moreover, when there is little propionate available, animals develop ketone excess.

Therefore, compounds that promote the production of large amounts of propionate from hydrocarbons to enhance hydrocarbon utilization efficiency and also reduce the occurrence of ketone excess are useful.

20-deoxynaracin and 20-deoxy-epi-17-naracin are novel polyether antibiotics. They are most closely associated with Naracin (antibiotic A-28086 factor A; US Pat. Nos. 4,035,481 and 4,038,384), which are polyether antibiotics. These patents and Biomed et al. The structure of naracin presented by Mass Spectrometry 3, 272-277 (1976) has recently been described in H. It was confirmed by Seto et al. [J. Antibiotics (6) 530-532 (1977)].

According to Seto et al, naracin is closely related to salinomycin (salinomycin = 4-demethyl-naracin). Jay more recently. W. Wesley et al reported on the C-17 epimer of deoxy- (0-8) -salinomycin.

See J. Antibotics 30 (7) 610-612 (1977). Although the epimers of deoxysalinomycin are similar to the epimers of deoxynacinine of the present invention, the chemical process of making deoxynacinine epimers from deoxysalinomycin epimers is still unknown.

The present invention relates to a mixture of deoxy naracin antibiotics consisting of at least two individual components 20-deoxy naracin and 20-deoxy-epi-17-naracin. This mixture is produced by culturing a strain of microorganism Streptomyces oreofesin NRRL 1181.

The term "antibiotic mixture" as used in the field of fermentation and in the present invention refers to a mixture of individual antibiotic components produced simultaneously, not chemical complexes. As with the production of antibiotics by fermentation, the proportion of individual components produced in the antibiotic mixture will vary depending on the fermentation conditions.

Pharmaceutically toxic salts of 20deoxynaracin and 20-deoxy-epi-17-naracin are also part of the present invention. For the sake of simplicity, the term "deoxynaracin antibiotic" is used to refer to deoxynaracin antibiotic mixtures, 20-deoxy naracin, 20-deoxy-epi-17-naracin and 20-deoxynacin and 20-de It is used against one of the pharmaceutically harmless salts of oxy-epi-17-nacin.

The deoxynaracin antibiotics of the invention inhibit the growth of microorganisms that cause disease in animals and plants. One aspect of this activity is deoxynaracin antibiotics. Deoxynacin antibiotics also enhance the efficiency of ruminant feed.

The deoxynaracin antibiotic mixture consists of 20-deoxy naracin and 20-deoxy-epi-17-naracin, which are obtained as a mixture by fermentation. 20-deoxynaracin and 20-deoxy-epi-17-naracin are separated into individual compounds from the deoxy naracin mixture as described below. The deoxynaracin mixture also contains small amounts of components removed by the described process. Deoxynacin antibiotic mixtures are soluble in most organic solvents but insoluble in water.

[20-deoxynaracin]

One of the components of the present invention, 20-deoxynaracin, has the same absolute configuration as naracin. The structure of 20-deoxynacin is represented by formula (I).

[20-deoxy-epi-17-narcin]

The structure of 20-deoxy-epi-17-naracin, which is another component of the present invention, is represented by the following structural formula (II).

The best way to distinguish between naracin (A-28086 factor A), 20-deoxy naracin and 20-deoxy-epi-17-naracin is by the 13C nucleus using the resonance spectrum (nmr) spectrum. The following Table ¹³ summarizes the ¹³ Cnmr spectrum of the compounds, where each compound is in free acid form and tested in CDCl ₃ (in ppm).

TABLE 1

Other good ways to distinguish 20-deoxy-naracin from 20-deoxy-epi-17-nacin and known A-28086 factors are paper chromatography and thin layer chromatography. For example, the Rf values of Naracin (A-28086 Factor A) A-28086 Factor D, 20-deoxynaracin and 20-deoxy-epi-17-naracin are measured microorganisms in various paper-chromatography. As measured using the Bacillus subrelease ATCC 6633 as shown in Table 3.

TABLE 2

Rf values of these antibiotics in thin layer chromatography using silica gel are shown in Table II.

TABLE 3

Deoxynaracin antibiotics (20-deoxy-naracin and 20-deoxy-epi-17-naracin) are available in several types, such as methanol, ethanol, dimethylformamide, dimethyl sulfoxide, ethyl acetate, chloroform, acetone and benzene. Soluble in organic solvents, only small amounts are soluble in non-polar organic solvents such as hexane and insoluble in water.

It should be noted, however, that 20-deoxynacin free acid is unstable in alcohol and converted to 20-deoxy-epi-17-narcine free acid. For example, 20-deoxy naracinic acid (435.1 mg) is dissolved in methanol (40 ml), left for 4 hours at room temperature, the solution is evaporated to dryness under reduced pressure, and the residue is redissolved in dioxane to give Drying yields 417.8 mg of 20-deoxy-epi-17-narcin acid.

Other materials that are chromatographically matched to A-28086-I described in US Pat. No. 4,038,384 are produced with the deoxynarcin mixture. The material is initially co-precipitated with deoxynacin antibiotics, but it is easily separated by silica gel chromatography. Using ethyl acetate as the developing solvent on silica gel thin layer chromatography, this material is less polar than 20-deoxynaracin or 20-deoxy-epi-17-naracin. Vanillin spray reagent (0.5 ml concentrated sulfuric acid + 3% vanillin per 100 ml solution) is suitable for detection. When scattered and heated with vanillin, the substance shows blue spots, as in A-28086-I, while deoxynacin antibiotics show yellow spots that become dark black.

20-deoxynaracin and 20-deoxy-epi-17-naracin form salts. Alkali-metal, alkali-earth metal salts and amine salts of pharmaceutically innocuous 20-deoxynarasine and 20-deoxy-epi-17-narasine are part of the present invention. "Pharmaceutically nontoxic" salts are those salts which do not increase the toxicity of the compound compared to the rhinitis form in warm blooded animals. Representative and suitable alkali metal and alkaline earth metal salts of 20-deoxynaracin and 20-deoxy-epi-17-naracin include sodium, potassium, cesium, rubirium, barium, calcium and magnesium salts. Suitable amine salts of 20-deoxy-naracin and 20-deoxy-epi-17-naracin are ammonium and primary, secondary, tertiary, C ₁ -C ₄ -alkyl ammonium and hydroxy-C ₂ -C ₄ -Alkyl ammonium salts.

Examples of amine salts include 20-deoxynaracin and 20-deoxy-epi-17-naracin as ammonium hydroxide, ethylamine, secondary-butylamine, isopropyl amine, diethylamine, diisopropylamine, ethanolamine And those formed by reacting with triethylamine, 3-amino-1-propanol and the like.

Cationic salts of alkali metals and alkaline earth metals of 20-deoxynaracin and 20-deoxy-epi-17-naracin are prepared according to commonly used processes. For example, antibiotics in free acid form are dissolved in a suitable solvent such as acetone or dioxane-water, and a solution containing a stoichiometric amount of inorganic base is added to the solution. The salt thus formed is separated by filtration or evaporation of the solvent.

Salts with organic arms are prepared in a similar manner. For example, gaseous or liquid amines are added to a solution of the antibiotic component in a suitable solvent such as acetone and the solvent and excess amine are evaporated off.

The preparation of a salt of one of the deoxynaracin antibiotics should be appropriately selected early in the separation process.

For example, the pH of the broth is adjusted as a suitable base or a suitable cationic salt is added to the extractant.

It is well known in the veterinary art that the form of antibiotics is not important when treating animals as antibiotics. This is because in most cases, the drug changes in the animal's body in a form other than the administered form. Therefore, the salt form administered is not critical to the method of treatment. The salt form, however, is chosen with regard to economy, convenience and toxicity.

The novel antibiotics of the present invention are prepared by culturing the deoxy naracin-producing strain Streptomyces oreofesine under aerobic conditions in a suitable culture medium until a substantial antibiotic action occurs. Antibiotics are recovered using various separation and purification procedures commonly used in the art.

Microorganisms useful for the production of deoxynaracin antibiotics are obtained by inducing mutations of Streptomyces oreofesine NRRL-8092 into N-methyl-N'-nitro-N-nitrosoguanidine. The taxonomic basis for which S. oreofesin NRRL-9092 is classified as a strain of Streptomyces oreofesin is described in US Pat. No. 4,038,384.

Streptomyces oreofessin cultures effective for the production of deoxynaracin antibiotics have been deposited with the US Department of Agriculture's Northern Research Center as part of a stock collection of bacteria (Peoria, Illinois), which was proclaimed NRRL-11181. s. The microbiological properties of Oreopaciens NRRL-11181 are as follows.

NRRL 11181 consists of short, straight or curved spores and belongs to the rectus-flexibilis (RF) classification. The spontaneous chains consist of up to 10 spores per chain, and rarely produce aerobic mycelium and are very poor even when present. The best substrate of aerobic mycelium is ISP No. 4. The color of aerobic mycelium or secondary proliferator is gray to white. Primary proliferators or substrate myceliums are well formed and grow best on medium such as ISP No. 7 and Bennetts agar. The color of the substrate mycelium is generally grayish and often the conidia coremia is observed. Spores have an elongate or cylinder shape and have a smooth surface, the size of which is 0.8-0.9 μ × 1.1-1.4 μ, with an average size of 0.85 × 1.25 μ. The above results are observed and measured from an electron micrograph.

From soil samples obtained from Mount Ararat, Turkey, the culture of originally isolated sinin streptomyces oreofesine is mutated and the culture identified as NRRL 11181 is isolated. The first soil isolated is incubated on agar plates and subcultures are obtained by removing individual colonies from the surface of the agar plates. One single isolate obtained from the culture of the original soil isolate is the culture identified here as NRRL 5758. The cultures selected in nature are again plated and a single colony is selected from it. Repeat the natural selection process eight times and select the ninth generation of single-colon isolates again.

The ninth generation of isolates are matured in chemical agar media containing 1% galactose as a single carbon source. The spore suspension was prepared by moving the spores by shaking the agar medium with ultrasonic waves, and then Whatman No. The suspension is filtered through one to minimize mycelial fraction.

The spore suspension is grown in a liquid medium mixture (pH adjusted to 8.8) at 30 ° C. for 72 hours, which contains 2 mg / ml N-methyl-N′-nitro-N-nitrosoguanidine. The culture growing on the medium is harvested and homogenized with a tissue mill.

The homogenized culture is diluted and spread flat on Hanton's medium and incubated at 30 ° C. until individual colonies can be distinguished. One of the individual selected colonies is used as starting material for the new culture, indicated as NRRL 8092.

Cultures designated as NRRL 8092 are grown on chemical agar media containing glucose as a single carbon source. A suspension consisting of spores and mycelium fraction is prepared by shaking the agar medium with ultrasound. The mixed spores and mycelium suspension were treated with 2 mg / ml of N-methyl-N'-nitro-N-nitrosoguanidine in a liquid medium mixture at pH 8.8 for 96 hours, shaken and harvested. Plate as described above. One of the selected individual colonies on an agar plate is grown to prepare a culture labeled NRRL 11181.

As opposed to given in the present invention. Oreopaciens NRRL 5758, NRRL 8092, or NRRL 11181 by mutation. It is common knowledge in the art to be able to produce other strains with the same biosynthesis (ie, the ability to produce 20-deoxynaracin and 20-deoxy-epi-17-naracin) as oreofaciens NRRL 11181. Anyone with a will know. s. N-methyl-N'-nitro-N-nitroso guanidine is used to obtain oreofaciens NRRL 1181, but other known mutagens such as ultraviolet, X-ray, radiofrequency, radiation and other chemical reagents induce similar mutations. Can be used to Part of the invention is also a method of producing a deoxynacin antibiotic mixture by culturing Streptomyces oreofaciens having the same biosynthesis as NRRL 11181.

The culture medium used to grow Streptomyces oreofaciens NRRL 11181 is one of several media. Any culture medium is preferred which is economical, optimum yield and facilitates separation of the product. For example, the preferred hydrocarbon sources in large scale fermentation methods are tepioca dextrin and sucrose, and glucose, corn starch, furactose, mannose, maltose, lactose and the like are also used. Corn oil, peanut oil, soybean oil and fish oil are sources of carbon. Preferred nitrogen sources are enzyme-gassed casein, with peptones, soy flour, cottonseed flour, amino acids such as glutamic acid and the like. Nutrient inorganic salts added to nutrient media include soluble salts that produce ions such as sodium, magnesium, calcium, ammonium, chloride, carbonate, sulfate, and nitrate.

Trace elements essential for the growth and development of microorganisms should also be contained in the culture medium. Such trace elements are generally present as impurities in the barley components in a sufficient amount of medium that satisfies the growth needs of the microorganisms.

If foaming is a problem, it is necessary to add a small amount of antifoaming agent such as polypropylene glycol (ie 0.2 ml / l) to a large fermentation broth.

Although not essential, adding a small amount of oil, such as soybean oil, increases the production of antibiotics.

It is preferred to aerobic fermentation in water in the tank throughout the deoxynacin antibiotics until a net mass is produced. Small amounts of antibiotics can be obtained by shaking-flask culture. It is preferable to use vegetable inoculum because the time lag in antibiotic production is related to the contamination of large tanks with spores of microorganisms. Plant inoculum can be prepared by inoculating a small amount of culture medium with a mycelium fragment or spore form of a microorganism to obtain a new culture of vigorously growing microorganisms. This plant inoculum is then transferred to a large tank. The medium used to grow the plant culture is the same as that used in large scale fermentation methods, but other mediums may be used.

Deoxynacin-producing microorganisms are grown between 20 ° and 40 ° C. The optimum temperature for deoxynacin production is between 27 ° and 30 ° C.

Sterile air is passed through the culture medium as is commonly used in aerobic culture processes. The volume of air used in the tank production method to grow microorganisms sufficiently is at least 0.1 volume of air per volume of culture medium for 1 minute. For sufficient antibiotic production, the volume of air used in the tank process is preferably 0.25 volume of air per volume of culture medium for 1 minute. The high solubility of oxygen does not inhibit the antibiotic process. Samples of mycelium extract or gravy are tested during fermentation of antibiotic effects against microorganisms known to be sensitive to this antibiotic.

Antibiotic production is fermented by extracts from mycelial soil or juicy test samples with antibiotic action against organisms sensitive to antibiotics. An analytical microorganism useful for testing the antibiotics of the present invention is Bacillus subtilis ATCC 6633. This bioassay is conveniently performed by paper-disc analysis on a donor plate.

Other convenient measurement methods are semi-automatic systems (Autoturb ^R microbiological assay ststem, Elanco).

Turbidity analysis in the phase.

[See N.R. Kuzel and F.W. Kavanaugh in J. Pharmaceut. Sic. 60 (5), 764and 767 (1971)]

carrousel)에 넣는다.The following test parameters are used when testing deoxynaracin antibiotics. Staphylococcus aureus (H-Heatley) NRRL B-314 is incubated in nutrient broth (ph 7) for 4 hours at 37 ° C. Test samples and standards are dissolved in methanol-water (1: 1). A-28086 Factor A, a standard, was used in autoturb carousel at concentrations of 1,2,4 and 5 mog / ml.

carrousel).

The initial pH of the inoculated culture medium will vary depending on the medium used. In general, the initial pH is in the range of 6.0 to 7.5. The pH at the end of the fermentation is slightly higher, ranging from 6.5 to 8.0.

In general, the antibiotic effect is measured on the second day of fermentation. Antibiotic effects are generally highest between the fourth and tenth days.

The above-described deoxynarcin antibiotics produced under aerobic fermentation conditions are then recovered from the fermentation broth by conventional methods used in fermentation methods. Antibiotics produced during fermentation are present in mycelia and filtered broth.

Deoxynarcin antibiotics are best recovered when performed in combination with filtration, extraction and adsorption chromatography. The preferred solvent used to separate deoxynaracin antibiotics from the whole or filtered fermented broth is ethyl acetate, although other conventional solvents may also be used.

A particularly advantageous method of isolating deoxynaracin antibiotics is to lower the pH of the fermented broth to 3.0. Filtration at this pH allows the deoxynacin antibiotic to easily separate as a mycelium. This method is described in the recovery of similar antibiotics A-28086 factors A, B and D and salinomycin. [Reference: U.S 4,009,262by Boeck and Bery]

Another advantage of this method is that when bicarbonate, such as sodium bicarbonate, is added to juice in an amount of 1 g per liter, the deoxynaracin antibiotic is easily separated from the military in salt form. Preferred solvents for separating antibiotics into mycelium are methanol, although other lower alcohols and ketones are also suitable.

In addition, azeotropic distillation can be conveniently used for the recovery of deoxynacin antibiotics. In this method, an organic solvent which forms a suitable azeotrope with water is added to the aqueous fermentation broth, and the solvent-juice mixture is azeotropically distilled to remove half the water from the juice, leaving a water-solvent mixture. Antibiotics exist in the form of solutions in organic solvents. Insoluble byproducts may be separated by any suitable method such as filtration or centrifugation. Deoxynarcin antibiotics are recovered from the organic solution by known methods, ie by evaporation of the solvent, addition of a nonsolvent, by precipitation formation or extraction.

Organic solvents that form a suitable azeotrope with water to perform the recovery process include butyl alcohol, amyl alcohol, hexyl alcohol, benzine alcohol, butyl acetate, amyl acetate, 1, 2-dichloroethane, 3-pentanone, 2 -Hexanon, benzene, cyclohexanone, toluene, xylene and the like.

(마그네슘 실리케이트, Floridin Co. P.O. BOX 989, Tallahassee, Fla)등과 같은 흡착물질이 편리하게 사용된다.It is particularly advantageous to recover by azeotropic distillation in large scale fermentation processes. The water and solvent in the azeotrope are separated and recycled by known methods and reused. The water thus removed is free of contamination and does not require disposal. Deoxynarcin antibiotics can be further purified through extraction and adsorption. Silica gel, carbon, florisil

Adsorbents such as (magnesium silicate, Floridin Co. PO BOX 989, Tallahassee, Fla) are conveniently used.

In addition, a culture solid containing a medium composition and a mycelium is used without extraction or separation, but is preferably used after removing water as a source of the deoxynacin antibiotic mixture. For example, after the action of the deoxynarcin antibiotic is produced, the culture medium is lyophilized or drum dried to dry and mixed directly into the premixed feed.

In another aspect, after the deoxynacin action in the culture medium has been produced, the hyphae are separated and dried to obtain a premixed, ready-to-use product. When the hyphae are separated in this way, calcium carbonate (about 10 g / l) is added and filtered to obtain a dry product.

Under the conditions of use so far, 20-deoxy-naracin and 20-deoxy-epi-17-naracin are recovered as main components from the above-described Streptomyces oreofaciens strain of NRRL 11181. Small amounts of components are also recovered from S. oreofaciens NRRL 11181, which cannot be identified because the amount is very small. The ratio between the components depends on the fermentation and separation conditions, but more 20-deoxy-epi-17-naracin is recovered than 20-deoxynacin.

20-deoxynaracin and 20-deoxy-epi-17-naracin are separated from each other and separated as individual compounds using known methods such as column chromatography, thin layer chromatography, and flow distribution. For example, column chromatography on silica gel is used to extract 20-deoxy-naracin and 20-deoxy-epi-17-naracin by extracting the column with various solvent mixtures. For example, using a benzene-ethylacetate solvent mixture on a silica gel column, 20-deoxy-epi-17-naracin is first extracted and later 20-deoxynacin is extracted. Thin layer chromatography on silica gel using 100% ethyl acetate solvent is a convenient way to monitor the dissolution process.

The deoxynacin antibiotic according to the invention is an antibacterial agent. For example, the microbiological comparative activity of 20-deoxy-epi-17-naracin (free acid) is described in Table 4. Conventional disk-dispersion methods were used.

TABLE 4

1phenylsilin resistant bacteria

2 methicillin-resistant bacteria

In another aspect, deoxynacin antibiotics inhibit the growth of anaerobes. The minimum inhibitory concentrations (MIC) in which 20-deoxy-epi-17-naracin (free acid) inhibits various anaerobes are measured by standard agar lime assay and summarized in Table 5. Endpoints were read after 24 hours of incubation.

TABLE 5

The action on mycoplasma is another effective action in terms of the antimicrobial effect of deoxynaracin antibiotics. Mycoplasma species known as Pleuuro-Pneumoniae-like (PPLO) microorganisms cause disease in humans and various animals.

Drugs that are effective against microorganisms are particularly needed in the livestock industry. The minimum inhibitory concentration (MIC) of 20-deoxy-epi-17-naracin (free acid) for mycoplasma species determined by the juicy-dilution method in vitro is summarized in Table 6.

TABLE 6

Deoxynacin antibiotics are also antiviral agents. For example, 20-deoxy-epi-17-naracin is active against linobirus type 3, baccinia virus, herpesvirus and influenza A virus, and has been demonstrated in an in vitro plate inhibition test. [Applied Microbiology 9, [1] 66-72 (1961) by Simanoff].

The deoxynaracin antibiotic according to the invention can be administered to a mammal orally, topically or parenterally for the purpose of treating viruses. A useful amount for the prevention or treatment of virus diseases is 1 to 5 mg / kg per kg body weight of the dose varies depending on the virus, and may vary depending on whether the purpose of prevention or treatment. Moreover, solutions containing deoxynacin antibiotics are used to decontaminate habitats where viruses such as polio or herpes are present in vitro, especially with surfactants. Solutions containing 1 to 1500 mcg / ml deoxynaracin antibiotics are effective for viral treatment.

Acute toxicity of 20-deoxy-epi-17-naracin (free acid) and 20-deoxy naracin (Na salt) is expressed in LD50 when they are intraperitoneally administered to mice, 201 mg / kg and 5 mg / kg.

Anticoccidial action is an important property of the deoxynaracin antibiotic of the present invention. For example, in vivo testing, 20-deoxy-naracin (Na salt) and 20-deoxy-epi-17-naracin (free acid) are most prototyping to coccidiosis at concentrations as low as 0.2 ppm. Azoamine is active against Ameria tenella.

Feeding tests in young chickens confirmed that deoxynaracin antibiotics exhibit anticoccinium activity in vivo. In this test, 20-deoxynaracin (Na salt), administered at a concentration of 100 ppm to chicks treated with Ameria tenella, prevents chicks from killing, gains weight and reduces the number of diseases. The results of the test are shown in Table 7.

TABLE 7

To treat or prevent livestock coccinium, an effective amount of deoxynacin antibiotic as an anticoccinium drug is administered orally to birds daily. Deoxynacin antibiotics can be administered in a variety of ways, but with physiologically toxic carriers, especially feeds digested by birds. Several components may be used to determine the appropriate concentration of deoxynacin antibiotics, with a dosage of 0.003 to 0.03% of the non-drug feed being preferred and 0.004 to 0.02%.

The invention also relates to an anticoccidial feed composition for livestock containing 35 to 180 g of deoxynacin antibiotic per tonne of feed.

The effect of enhancing feed utilization in animals is another important property of deoxynaracin antibiotics. For example, deoxynacin antibiotics enhance feed-use effects in ruminants with advanced gastric function.

Hydrocarbon utilization in ruminants is enhanced by treatments that stimulate the stomach of the animal to produce more propionate compounds than acetate or butyrate. Feed utilization can be measured by monitoring the concentration and production of propionate in the stomach. [Reference: u.s 4,038,384]

The results of the test of the 20-deoxynaracin (Na salt) and 20-deoxy-epi-17-narcine (free acid) in vivo results in the ratio of the volatile fatty acid (VFA) concentration in the treated flask and the concentration in the control flask The results are shown in Table 8.

TABLE 8

* According to the following test, the results were statistically valid (P <0.01) [by the two-tailed LSD test (RGD Steel and JH Torrie, "Principles and Procedures of Statistics," McGraw Hill, New York, NY, 1960, P. 106)]

The deoxynaracin antibiotic of the present invention, when administered orally to ruminants at a rate of 0.05 mg / kg / day to 5.0 mg / kg / day, effectively enhances the amount of propionate to enhance feed utilization. The most beneficial results are obtained in amounts of 0.01 mg / kg / day to 2.5 mg / kg / day. Preferred methods of administration of the antibiotics of the present invention are to be administered in combination with animal feed or as tablets, potions, bolus, or capsules. Formulations of such formulations are completed by methods well known in vertebrate pharmaceuticals. Each dosage unit contains a dose directly related to the appropriate daily dose for the animal to be treated.

The present invention relates to feed compositions for use in hypertrophic ruminants such as cattle and sheep. Such feed composition is composed of 1 to 30g of deoxynaracin antibiotics and feed per ton.

Swine dysentery is a common disease in the United States and other countries and causes huge losses in pig growth worldwide each year. u.s. No. 3,947,586 reports that polyether antibiotics are effective in preventing and treating dysentery in swine. Because deoxynaracin antibiotics are a new species of polyether antibiotics, they are also effective in preventing and treating dysentery in swine.

In order to prevent and treat (modulate) dysentery in pigs, it is desirable to incorporate an effective amount of deoxynaracin antibiotics into the feed.

The deoxynacin antifungal agent of the present invention is effective in preventing or treating dysentery in swine when orally administered 35 to 150 g of the active compound per ton per pig. The preferred ratio is 100 g of active compound per tonne. Mixed administration with animal feed is the preferred method of administration, but other methods may also be administered as tablets, potions, bolus or capsules. Each dosage unit contains the antibiotic of the present invention directly related to the lower daily dose of the treated animal.

The present invention further relates to a feed composition for pigs containing an effective amount of pig feed and deoxynacin antibiotics. The effective amount is between 35 and 150 g of deoxynarcin antibiotic per tonne of saro.

Deoxynarcin antibiotics are antiviral agents and are also active against anaerobic bacteria, such as Clostrinium perfringens. Therefore, deoxynaracin antibiotics are beneficial for treating and preventing enteric poisoning in chicks, pigs, cattle and sheep, and ruminants.

Some deoxynaracin compounds (20-deoxy-naracin and salts thereof) have ionic bond and ion transfer properties and are therefore ionic binders. (See B.C. Pressman, Alkali Metal Chelators Te Ionophores, in Inorganic "Biochemistry," Volume 1 G.L. Eichhorn, Elsevier, 1973).

This compound is used when it is necessary to selectively remove a particular cation. Examples of such applications are used to remove and recover silver ions from photographic solutions and to remove toxic cations from the waste stream and desalination of seawater prior to disposal of industrial waste streams. The deoxynaracin compound is used as a component of an ion-unique electrode.

(O.Kedem, et al., U.S.Patent 3,753,887).

Such compounds alter the cation permeability of natural and artificial membranes. Therefore, deoxinarasine compounds are used as the composition of membranes used for selective cation transfer to concentration gradients. This property is applicable to the recovery of thick metals and precious metals. [Reference: E.L. Cussler, D. F. Evans, and Sister M.A Matesick, Science 172, 377 (1971)]

In another aspect, 20-deoxynacin and its salts act as inhibitors of the ATPase enzyme. ATP ase, an alkali-metal-private enzyme found in cell membranes, is associated with the energy required for active transport. "Active transport" involves several stages of manipulation that require energy, whereby intracellular and extracellular fluids maintain their composition.

Inhibitors of ATP ase reduce the energy required for active transport. In vivo testing shows that 20-deoxynaracin (Na salt) inhibits cation transfer ATP ase at a ½ effective concentration of 0.065 mcg / ml in liver mitochondria.

20-deoxynacin and its salts are also potent cardiovascular agents. 20-deoxy naracin enhances cardiac contractility when tested using isolated guinea-pick arteries. The response to this test is expressed as a percentage of the highest contractile force induced by (10 ^-4 M) of norepinephrine, where 20-deoxynaracin (Na salt) is 43.8 ± 1.4 n = 4 at 10 ⁵ molar concentrations). % Average (± standard error) results in an increase in shrinkage. [Reference: us 3,985,893]

The present invention includes a method of enhancing the contractile force of the heart muscle by administering an effective amount of warm blooded animals to 20-deoxynacin or a pharmaceutically harmless salt. A toxic effective amount is in the range of 30 to 500 mcg / kg body weight. Preferred doses range from 30 to 100 mcg / kg. In this way, antibiotics are injected parenterally, for example intravenously. A suitable method of administration is the dropping method, in which antibiotics are added to standard intravenous solutions such as dextrose solution.

20-deoxynacin is preferably administered at a dose of less than 100 mcg / kg until an increase in contractile force is preferably observed. Thereafter, the dose of 20-deoxynaracin administered is determined by the rate of infusion necessary to maintain the desired response. As with clinical administration of other muscle contractors, the dose of 20-deoxynaracene administered is dependent on the individual's resistance to 20-deoxynaracene, the characteristics of heart disease, namely the extent of damage to the heart muscle and the age of the patient. It varies according to factors such as physical condition. The treatment method according to the present invention using myocardial myocardial contractor 20-deoxynarasine can be widely used in various clinical conditions which are classified as cardiac shock. This includes myocardial obstruction, congestive heart failure, and postoperative heart shock.

The following examples are provided to fully illustrate the invention.

Example 1

A. Shake-flask Fermentation

Cultures of Streptomyces oreofaciens NRRL 11181 are prepared and preserved on agar gradient plates of the following composition.

The inclined surface is inoculated with Streptomyces oreofaciens NRRL 11181 and incubated at 30 ° C. for 7 days. Cover the mature oblique cultures with sterile bovine serum and scrape the spores with a sterile loop. The resulting beef-serum suspension of spores and mycelium fraction is freeze-dried with up to six pellets. The prepared lyophilized pellets are used to inoculate 50 ml of plant medium of the following composition.

Plant medium inoculated in 250 ml Elenmeyer flasks were incubated at 250 ° C. at 30 ° C. for 24 to 48 hours on a 2-inch diameter shake rotator. The above-mentioned culture plants to 50 ml are used to inoculate 250 ml of the following fermentation broth.

Badge I:

Medium II.

Medium III.

Fermentation is carried out for 10 days at 30 ° C. on a 250-RPM rotary shaker with a 2-inch arc.

B. Tank Fermentation Method

Tank fermentation is carried out using the plant and fermentation broth described in part A of shake-flask fermentation. For tank fermentation, 10 ml of plant medium is inoculated into 400 ml of second stage plant medium in 2 l of Ellenmare flask.

After 24 hours of incubation at 30 ° C., 800 ml of the second stage plant medium is inoculated into 100 l of fermentation, medium in 165 l of fermentation tank. After sterilization at 121 ° C. for 45 minutes, the pH of the medium is about 6.8 ± 0.1. Fermentation proceeds at 30 ± 1 ° C. for 10 days. The tank is shaken with a conventional shaker at 300 RPM and passed through sterile air at a rate of 0.5 volume per volume of culture medium per minute.

Example 2

[Isolation of Deoxynaracin Mixture]

Fermented broth (4 L) obtained by the method described in Example 1 using medium II was added to concentrated hydrochloric acid with shaking for 1 hour to bring the pH below 3.0. The resulting solution is filtered with a filter aid (125 g Hiflo Super-Cell, Diatomaceous Earth, Jones-Manville Corporation). The isolated mycelium cake is extracted using a mixer with 2 l of methanol containing 50 g of sodium hydrogen carbonate per liter. The methanol filtrate was evaporated under reduced pressure to give a solution of about 450 ml, the pH of this solution was adjusted to pH 7.5 by addition of concentrated hydrochloric acid. The resulting solution is extracted twice with CHCl ₃ (500 ml). The CHCl ₃ extracts are combined, dried over sodium sulfate and filtered. The filtrate is evaporated under reduced pressure to give a 2.0 g crude deoxynarcin mixture.

Example 3

[Isolation of Deoxynaracin and Epi-Deoxynaracin]

The crude deoxynarcin mixture (2 g) obtained in Example 2 is dissolved in a minimum amount of benzene and applied to a 1.5- × 22 cm column of silica gel (Merck 7729). Separate fractions, solvents and yields are shown in the following table.

* Dried over sodium sulfate, filtered and the filtrate is evaporated to dryness under reduced pressure.

Ethyl acetate solvent system is used to measure the fractions by TLC. Fractions 16-17 (126 mg) contain 20-deoxy-epi-17-naracin and fractions 18-23 contain a mixture of 20-deoxycinaracin and 20-deoxy-epi-17-naracin.

A mixture of 20 deoxynaracin and 20-deoxy-17-epinaracin obtained in fractions 18-23 is chromatographed by preparative TLC using ethyl acetate as solvent system. The mixture (105 mg on one plate and 130 mg on the other plate) is dissolved in a small amount of CH ₂ Cl ₂ and placed on a silica-gel (Merck) preparative plate. After the plate is developed, the two separation materials are observed under ultraviolet light. Each portion representing the two substances is separated from the plate and extracted as CH ₂ Cl ₂ : CH ₃ OH (4: 1). Here, deoxynacin is a part that is slowly secondary to the two components. 7.5 mg of 20-deoxy-epi-17-narcine and 27 mg of deoxynacin are recovered from a plate containing 105 mg of material. 4.0 mg of 20-deoxy-epi-17-narcine and 56 mg of 20-deoxynacin are recovered from a plate containing 130 mg of the mixture.

Example 4

[Isolation of 20-deoxynaracin (variation)]

Hydrochloric acid is added to the fermented broth (95l) to adjust the pH to 3 and shake for 1 hour. Filter aid (Hyflo Supercel, 3%) is added and the juice is filtered.

The separated hyphae cake is extracted twice with 45 l of acetone containing 50 g of sodium bicarbonate per liter. The acetone extracts are combined and concentrated under reduced pressure to give an aqueous solution of 10 l. The pH of this solution is adjusted to pH 8.0 with 5N hydrochloric acid and the resulting solution is extracted three times with 1/2 volume of CH ₂ Cl ₂ . The CH ₂ Cl ₂ extracts are combined and evaporated under reduced pressure to give an oily residue. Hexane; Methanol: Take in 500 ml of the upper side of the solvent system of water (10: 7: 1), and extract this six times into 300 ml of the solvent system lower side. The extracts are combined and concentrated in vacuo to dissolve the residue in dioxane and freeze-dry the dioxane solution to give 19.4 g of deoxynacin mixture.

In the same way, several samples obtained were collected (40 g), dissolved in toluene and filled into a liquid chromatography column (waters Associates prep LC / system 500) silica gel column. Toluene collected at a flow rate of 250 ml / min; Elution is carried out as a solvent of ethyl acetate (9: 1). Fractional contents are monitored by TLC. Fractions 37 to 52 are collected, concentrated under reduced pressure, redissolved in dioxane and lyophilized to yield 7.8 g of 20-deoxynacin-rich purified material.

This material is chromatographed again on Waters Preparative Liquid Chromatography./System 500. Fraction 50 toluene; After eluting with a solvent of ethyl acetate (9: 1), the eluting solvent is changed to 100% ethyl acetate. Collecting fractions 51 to 53 and evaporating under reduced pressure yields a residue, which is dissolved in dioxane and lyophilized to yield 1.12 g of 20-deoxynarcin as the sodium salt.

Example 5

[Preparation of 20-deoxynaracin free acid]

20-deoxynacin sodium salt (200 mg) is dissolved in ethyl acetate (10 ml). The solution is washed with 0.1 N hydrochloric acid (10 ml) and washed twice with water (5 ml). The organic layer obtained is evaporated to dryness to afford a residue, which is redissolved in dioxane and freeze dried to afford 139.6 mg of 20-deoxy-narcine free acid as a white solid.

Example 6

[For treatment of coccidiosis]

In order to speed up weight gain, a balanced high-energy feed used in chick feed is prepared with the following prescription:

The deoxynaracin antibiotic mixture, 20-deoxy-naracin or 20-deoxy-epi-17-naracin (about 0.01% by weight) is mixed with the feed in a standard feed mix. The chicks fed this feed along with livium water are protected from comsidium.

Example 7

[Improved cattle-positive feed]

A balanced high-grain cow-shea feed is:

* Content per pound: 2,00,000 IU, vitamin A; Soybean Feed with 227,200 IU Vitamin D ₂ and 385.7 g of 1% oil

** Corn distillate containing 20,000 I.U of d-alpha-tocophenyl acetate per pound

The deoxynaracin antibiotic mixture, 20-deoxy-naracin or 20-deoxy-epi-17-naracin (about 0.004% by weight) is mixed with the feed according to standard techniques. Compress the mixed feed and pelletize it. One animal digests an average of 15 pounds of feed per day, so 300 mg of antibiotics per animal per day.

Example 8

[Improved Pig Feed]

The entire mixture is prepared by standard methods using the following ingredients.

The entire mixture is added to pig feed and standard feed-mixing methods are used to ensure that the final product contains 100 g / t of active compound.

Claims (1)

Under immersion aerobic conditions, Streptomyces oreofaciens, which has the same biosynthesis as Streptomyces oreofaciens NRRL 11181, in a medium containing hydrocarbons, nitrogen and inorganic salts as an assimilation source until an amount of antimicrobial activity is actually produced. Incubating and recovering the antibiotic mixture in the form of an acid or a pharmaceutically acceptable salt thereof, 20-deoxy-epi-17 of formula (I) and (II) A process for preparing a deoxynaracin antibiotic mixture consisting of narcin.