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    • 专利摘要:
    Deoxynarasin antibiotic complex, comprising 20-deoxynarasin and 20-deoxy-epi-17- narasin, is produced by submerged aerobic fermentation of Streptomyces aureofaciens NRRL 11181. 20-Deoxynarasin and 20-deoxy-epi-17- narasin are separated and isolated by chromatography. The deoxynarasin complex, 20-deoxynarasin and 20-deoxy-epi-17-narasin are antibacterial and anticoccidial agents and also increase feed-utilization efficiency in ruminants.
    公开号:SU818492A3
    申请号:SU782676001
    申请日:1978-10-19
    公开日:1981-03-30
    发明作者:Мицуо Накацукаса Вальтер；Дж. Маркони Гари；Нойсс Норберт；Л.Хамилл Роберт
    申请人:Эли Лилли Энд Компани (Фирма)；
    IPC主号:
    专利说明:

    The invention relates to veterinary medicine, in particular to the production of antibiotics. Methods for the preparation of antibiotics are known, for example A-4696, by growing under aerobic conditions Aetinoplahes sp. ATCC 23342 on a medium containing nitrogen sources of nitrogen and mineral salts 1. 20-deoxynarazine and 20-deoxy-epi-17-narasin are newer polyester antibiotics. They are most closely associated with the known polyester antimicrobial narasin 2 and -13}. The method for producing the deoxynarazine antibiotic complex is that Strepto myces aureofaclens NRRZ 11181 is thrown out in a culture medium containing ycBcLHBa sources of carbon, nitrogen and inorganic salts, under conditions of aerobic fermentation during immersion and when the necessary amount of antibiotic activity is reached, antibiotic, antibiotic, antibiotic, antibiotic, antibiotic, is obtained. The complex is removed from the culture fluid in the form of acids and their pharmaceutically acceptable salts. Deoxinarazine antibiotics inhibit the growth of pathogenic organisms for animals and plants. In accordance with one aspect of the invention, deoxinarazine antibiotics are anti-coccidal agents. In addition, deoxynarazine antibiotics increase the digestibility of food in ruminants. The deoxinarazine antibiotic complex includes 20-deoxinarazine and 20-deoxy-epi-D7-narasin i obtained during fermentation as a mixture. 20-deoxynarazine and 20-de-6-ell-17-17-narasin are isolated and separated as individual substances according to the description below. The deoxinarazine complex also contains minor amounts of substances that are removed according to the procedure described below. The deoxinarazine antibiotic complex is soluble in most organic solvents, but is insoluble in water.
    38164924
    20-leoxinarazine has the same zheraein. The structure of 20-deoxynaracn
    absolute configuration / which is presented in the form
    he
    sicarazine and 2 (5-deoxy-epi-17-narasin in various paper chromatographic systems shown in Table 2.
    table 2
    Water: methane: acetone (12: 3: 1),
    adjusted to pH 10.5
    and then diluted to pH
    7.5
    Water: methanol: acetone {12: 3: 1),
    adjusted to pH 10.5
    and then diluted to
    pH 7.5 HC1
    1% metipizob5g, tilketone (MIBK)
    In tab. 3 shows the R values for ETI. Antibiotics in conventional ton. Ethyl acetate Deoxinarazine antibiotics 20-deoxynarazine and 20-deoxy-epi-17-narazine) are soluble in various organic solvents, such as methyl alcohol, ethyl alcohol, dimethylformamide, dimethyl sulfoxide, ethyl acetate, chloroform , acetone and, but only slightly soluble in non-polar organic solvents, such as hexane, and completely insoluble in water. However, it should be borne in mind that the 20-deoxinarazine free acid is unstable in alcohols, as it is converted into 20-deoxy-epi-17-naraeic free acid. For example, 435.1 mg of 20-deoxy-nenaric acid is dissolved in 40 ml of methyl alcohol and incubated at room temperature for 4 hours. Then the solution is evaporated to dryness in vacuo, the residue is dissolved in dioxane and lyophi, 11 0.08 0.21
    0.25 0.21 0.44
    chromatographic (TLC) system using silica gel.
    Table 3
    0.29 0.35
    0.74
    0.42 are obtained by licking 417.8 g of 20-deoxy-epi-17-narasinic acid. Examples of suitable alkali metal and alkaline earth metal salts of 20-deoxinarazine and 20-deoxy-epi-17-narasin are sodium, potassium, lithium, cesium, rubidium, barium, calcium and magnesium salts. Among the acceptable amino salts of 20-deoxinarazine and 20-deoxy-epi-17-narasin are the ammonium, primary, secondary, and tertiary C-C alkylammonium and hydroxy Cj-C-alkyl ammonium salts. Aminosomes include those produced by the reaction of 20-deoxinarazine and 20-deoxy-epi-17-narasin with ammonium hydroxide, methylamine, secondary butylamine, isopropylamine, diethylamine, diisopropylamine, triethylamine, 3-amino-1-propanol, and the like. compounds. The cationic salts of the alkali and alkaline earth metals 20-deoxinaraein and 20-deoxy-epi-17-naraein are prepared according to the procedure commonly used for the preparation of cationic salts. For example, the free acid antibiotic is dissolved in a suitable solvent, such as carbon, or aqueous dioxane. Another solution is added to this solution containing a stoichiometric amount of the required inorganic base. The resulting salt is recovered by conventional methods such as filtration or evaporation of the solvent. In a similar way, organic amine salts can be prepared. For example, a gas or liquid amine may be added to a solution of an antibiotic in a suitable solvent, such as acetone, and then the solvent and excess amine may be removed by evaporation. The preferred method of obtaining the desired salt of one of the deoxynarazine antibiotic is the appropriate initial selection of the isolation procedure, for example, adjusting the pH of the broth by adding the appropriate base or adding the corresponding cationic salt to the extraction solvent. When treating an animal, the form of the antibiotic does not matter. In most cases, under the influence of the conditions existing inside the animal, the initial form of the medicine changes. Consequently, for the method of treatment it does not have;, it does not matter that the drug is administered in salt form. However, salt form may be preferred, based on economic, convenience and toxicity. New antibiotics obtained by cultivating the strain of Streptomyces aireofaciens, which produces deoxin razin, under deep aerobic conditions in a suitable culture medium, until essential antibiotic activity is obtained. Antibiotics are extracted using effective methods in alkaline peeling and cleaning, usually using hellah. in the technique. The organism used to produce deoxinarazine antibiotics is obtained by mutation of Streptomyce aureofaciensvNRRZ 8092 (3) under the influence of M-met l-N-nitro-M-nitrboguanadin. The culture, applicable to the preparation of deoxinarazine antibiotics, is deposited and is included in the collection of pure crops of the Northern Regional Research Center of the United States Department of Agriculture, Agricultural Research Service in Peoria, Illinois 61604 under the number HRRZ 11181. For growing Streptomyces aureofaciens NRRZ. The preferred carbohydrate source is tapioca dextrin, although glucose, corn, starch, fruit sugar, mannose, malt sugar, milk sugar and the like can also be used. compound. Other sources of carbon include corn oil, peanut oil, soybean oil, and fish oil. The preferred nitrogen source is a hydrolyzable casein enzyme, although peptones, soybean meal, cottonseed meal, amino acids such as glutamic acid, etc., can also be used. Among the nutrient inorganic salts that can be included in the culture medium , include the usual soluble salts capable of forming sodium, Mg, calcium, ammonium ions, as well as chloride, carbonate sulfate, nitrate, and the like ions. The composition of the culture medium should also include trace elements necessary for the growth and development of organisms. When carrying out the fermentation process on a large scale, it may be necessary to add small amounts (0.2 ml / l) of an anti-foaming agent, such as polypropylene glycol, if foaming is a serious problem. The yield of antibiotic is increased by adding small amounts of oil, such as soy bean oil. In order to obtain significant quantities of deoxinarazine antibiotics, it is preferable to carry out deep aerobic fermentation in fermenters. Smaller amounts of these antibiotics can be prepared in a shaking flask. - Since the time lag in the production of antibiotics is usually associated with the inoculation of large fermenters with spore forms of organisms, it is advisable to use a planting seed culture. The plant inoculum is prepared by sowing: a small volume of the culture medium cnopoBi iMH with the forms of mycelial fragments of the organism in order to obtain a fresh and actively growing culture of the organism. The plant seed is then transferred to large vats. As the culture medium for growing the plant inoculum, the same medium can be used as used for larger-scale fermentation, but other media can also be used. The deoxinarazing organism can grow in the range of about 20 to. The optimum temperature range for the preparation of deoxinarazines is limited to from about 7 to. Sterile air is blown through the medium, the effective growth of the organism is achieved when about 0.1 volume of air is supplied to the fermenter per volume of culture medium per minute. In order to effectively obtain antibiotics, the volume of air supplied during production in the fermenter should be greater than 0.25 volume of culture medium per minute. High concentrations of dissolved oxygen do not inhibit the formation of antibiotics. Obtaining antibiotics should be monitored during the fermentation process, taking samples of the broth or solid extracts of mycelium to determine the antibiotic activity. Bacillus subtilis ATCC 6633 is used to test the proposed antibiotics. A bioassay is conveniently performed using disk paper samples on agar media. The initial pH of the non-seed culture medium varies depending on the medium used. Typically, the pH should be in the range of 6.0 to 7.5, the pH at the end of fermentation is usually slightly higher, in the range of 6.5-8.0. Antibiotic activity is usually detected on the second day of fermentation. The maximum of antibiotic activity is usually reached at about 4–10 days. When deoxinarazine antibiotics are prepared by deep-seated aerobic fermentation, they can be extracted from the enzymatic medium by methods commonly used in fermentation technology. Antibiotics obtained during fermentation are present both in the mycelial mass and in the filtered broth. Maximum extraction of deoxinarazine antibiotics is therefore achieved by the joint implementation of such methods. Such as filtration, extraction and adsorption chromatography. It is preferable to use ethyl acetate, although satisfactory, as a solvent for separating deoxinarazine antibiotics from either all or from the filtration enzyme broth, although satisfactory results are obtained when using other, # 1X solvents-. A particularly preferred method for isolating deoxinarazine antibiotics is to lower the pH of the whole fermentation broth to about 3.0. With this pH value, deoxinar antibiotics are conveniently separated with a microlysis mass using a filtration method. Another advantageous aspect of this process is the addition of bicarbonate, such as sodium bicarbonate, to the whole broth in an amount of about 1 g / l. Using this method, deoxinarazine antibiotics are conveniently separated from the mycelial mass as a salt. It is preferable to use methyl alcohol as a solvent to separate antibiotics from the mycelial mass, but other lower alcohols and ketones can also be used. Azeotropic distillation can also be successfully used to extract deoxinarazine antibiotics. In accordance with this method, an organic solvent, which forms the corresponding azeotropic mixture with water, is added to the aqueous fermentation broth. The resulting mixture of solvent and broth is subjected to azeotropic distillation in order to remove at least half of the water from the broth and obtain a mixture of water with a solvent in which deoxinarazine antibiotics are dissolved in an organic solvent. Insoluble by-products can be separated by appropriate methods, such as filtration or centrifugation. The deoxanine antibiotics are then removed from the organic solvent using well-known methods, such as evaporating the solvent, precipitating by adding a non-solvent, or extraction. As examples of organic solvents which form azeotropic mixtures with water for carrying out such an extraction procedure, such compounds as butyl, C1MILY, hexyl and benzyl alcohols, acetic acid butyl ester, amyl acetic acid ester, 1,2-dichloroethane, 3-pentanone, 2-hexanone, benzene, cyclohexanone, toluene, xylene, and the like. When performing fermentation processes on a large scale, the extraction is particularly preferably carried out by azeotropic distillation. Water and solvent, distilled from the azeotropic mixture, can be separated by known methods and then recycled for reuse. The water separated in this way does not contain contaminants and does not require the implementation of the wastewater treatment process. Additional purification of deoxinarazine antibiotics includes additional extraction and adsorption. Preferably, materials such as silica gel, coal, florinol and the like are preferably used as the adsorbent. On the other hand, it is possible to use solid culture mass, containing medium components and mycelium, without implementation. . extracting or separating as a source of deoxinarazine antibiotic complex, but preferably after removing water. For example, after adjusting the deoxinarazine antibiotic activity, the culture medium can be dried by lyophilization or on a roller mixer and directly added to the nutrient mixture. In accordance with another aspect of the invention, after the activity of the culture medium has been reached, the mycelium is separated and dried, obtaining a product which can be directly used as a food additive. By separating the mycelium for this application, the calcium carbonate additive (about 10 g / l) facilitates filtration and gives a dried product of higher quality. 20-deoxynarazine and 20-deoxy-epi-17-narasin are separated and targeted as individual compounds, using well-known methods such as column chromatography, thin-layer chromatography, countercurrent distribution, and similar methods. For example, for separation of 20-deoxinarazine and 20-deoxy-epi-17-narasin, silica gel column chromatography is used, using various mixtures of solvents for the elution of the column. Using a mixture of solvents such as benzene and ethyl acetate, in a column of silica gel, first elute with 20-deoxy-epi-17-narasin, and then 20-deoxinarazine. Thin layer chromatography on a silica gel column using pure ethyl acetate is a convenient way to monitor the elution process. Deoxinarazine antibiotics are antimicrobial agents. In tavl Figure 4 shows the relative microbial activity of 20-deoxy-epi-17-narasin (free acid) using the standard disco-diffusion method. Tables a.4. StaphyIOCOCCUS aureus3055 - 16.9 13.8 - "- 3074 19.5 15.4 - - 19.0 17.2 Streptococcus pyogenes (Group A) 12.0 10.0 (Group D) 17.0 14.6 Dtplococcus pneumonia 16.0 13.0 Penicillin-resistant. Methicillin Resistant. in accordance with one aspect of the invention, deoxinarazine antibiotics inhibit the growth of anaerobic bacteria. In tab. Figure 5 shows the minimum total concentrations (MPC) of 20-deoxy-epi-17-narasin (free acid) relative to various anaerobic bacteria. BMDs are determined by standard agar dilution assay. The final values correspond to a 24 hour incubation period. Table 5 Actinomyces israeJil w. 8558 Clostridlum perfriffgens Clostridium septicum 8116 112816 123516 Eubacterium aerofaciens 1302 Peptococcus asaccharojitlcus .g Peptococcus prevo i 12814 Peptos t reptococcus anaerobius 1428 4 Peptostreptococcus intermedi us 12644 792 Propionibacterium acnes Bacteroides fragiBisill128 Activity against mycoplasma is another aspect cents antiikrobnoy deoksinaraeinovyh antibiotic activity. In mycoplasia, known as the pleuro-lung-like (PLPO) organism, it is also a weapon for humans and various animals. Substances active against S1TO organisms, especially in the poultry industry. In tab. 6 summarizes the values of mini-inhibitory concentrations (MPC) of 20-deoxy-epi-17-narasin (free acid) versus the indicated mycoplasma ids determined in vitro on broth dilutions. Table 6 of the IPC, Organism µg / mp M. gal IIsepticum N. hyorhinIS N. synovlae Deoxine antibiotics are also antiviral preparations. For example, a 20-deoxy-epi-17-narasin is active against rhinovirus type 3, a vaccine: vaccine virus, herpes virus, and anfluence L. virus. In accordance with one aspect of the invention, a deoxynarzine antibiotic can be administered to a mammal orally, topically or parenterally to suppress viruses. For preventing or treating diseases, doses are usually applied which vary from about 1 to 5 mg / kg of the body of a mammal, depending on the type of virus and on whether the drug is used prophylactically or therapeutically. Moreover, solutions containing a deoxinarazine antibiotic, preferably together with a surfactant, can be used to deactivate in vitro habitats where viruses such as polio or herpes are present. Solutions containing approximately 1-1500 µg / ml of deoxinarazine antibiotic are effective for suppressing viruses. Acute toxicity of 20-deoxy-epi-17-narasin
    Infected control
    20-deoxinarazine
    (100 million shares)
    Two stands with four chickens in each .. for
    processing.
    The maximum possible number is 4.00
    For the prevention or treatment of coccidiosis, poultry are preferably orally administered daily to an effective amount of a deoxinarazine antibiotic. The deoxinarazine antibiotic may be in various forms, but it is most convenient to use it together with physiologically acceptable carriers, preferably with food swallowed by birds. Although various factors should be taken into account when determining the appropriate concentration of the deoxinarazine antibiotic, the amount injected is usually Hc1;
    within the range of 0.003-0.03% by weight of the non-treatable feed, and preferably in the range of 0.004-0.02%.
    The invention also relates to antioxidant food compositions for poultry, including food.
    37,5 114
    4.00
    185 0.13
    /
    for poultry and about 35,160 g of deoxinarazine antibiotic per ton.
    The ability to improve the efficiency of the assimilation of food by animals is another important property of deoxinarazine antibiotics. For example, these antibiotics improve the digestibility of food by ruminants, which have an advanced function of the scar.
    Deoxinarazine antibiotics usually effectively increase the yield of propionate — and, consequently, improve the effectiveness of food when administered to ruminants orally, in an amount of about 0.05–5.0 mg / kg per day. The most impressive results are obtained with the introduction of antibiotics.
    in amounts of about 0.1-2.5 mg / kg in (free acid) and 20-deoxinaraain (sodium salt) when administered intravenously to mice, expressed as Lugo is equal to 201 mg / kg and 5 mg / kg, respectively. Anticoccidial activity is an important property of deoxinarazine. Antibiotics; 20-Deo1 synarazine (sodium salt) and 20-Geoxy-epi-17-narasin (in the form of free acid) are active and compounds in amounts already 0.2 parts per thousand, against Etmeria tenella, the simplest organism, for the most part associated with coccidiosis. Experiments on young chickens show that deoxingphasin antibiotics have anticocidial activity in vivo. 20-Deoxinarazine (sodium salt), administered in the amount of 100 million shares in the food of chickens infected with Eimeria tenel), prevents mortality, improves growth and reduces the rate of lesions in chickens. The results of the experiment are presented in t-abl. 7. Table 7 day. The preferred method of administering antibiotics is to mix them with animal feed. However, POSSIBLE and other their introduction, for example in the form of tablets, jelly, pills, or capsules. The formulation of these various dosage forms can be carried out by methods well known in the veterinary pharmaceutical industry. Each individual dosage unit must contain that amount of compound that is directly related to the daily dose for the sick animal. The invention also relates to food compositions intended for fattening ruminant breeds, such as cattle and sheep. These food compositions include cattle feed and 1–30 g of deoxinarazine antibiotic 2 tons of feed. In pigs, dysentery in pigs is a common disease in OHA and other countries, annually causing a generalized death of cattle in pig farms. Prevention and treatment of dysenter in pigs is preferably carried out by incorporating an effective amount of a deoxynarazine antibiotic into the diet. Deoxynarazine antibiotics are usually effective in preventing or treating dysentery in pigs if they are administered to pigs in an amount of about 35 to 150 grams of active compound per ton of feed. A particularly preferred amount is about 100 g of active compound per ton. The most acceptable method of administering antibiotics to animals is to add the drug to animal feed, although it can also be administered by other means, for example, in the form of tablets, capsules, gel, or pills. Each dosage unit must contain an amount of antibiotic that is directly related to the required daily dose for the animal being treated. V The invention also relates to food compositions for pigs, including a diet for pigs and an effective amount of a deoxinarazine antibiotic. As previously indicated, the effective amount of the antibiotic is typically in the range of about 35-150 g of deoxinarazine antibiotic per ton of feed. Deoxinarazine antibiotics are antivirals and are active against anaerobic bacteria, such as Clostrldlum perfringens. Therefore, Deoxynarazine antibiotics are useful for treating and preventing inflammation of the small intestines in chickens, pigs, cattle and sheep, as well as in the treatment of peritoneal inflammation in ruminants. Some Deoxinarazine compounds (20-deoxinarazine and its salts) exhibit ion-binding and ion-transporting properties and, therefore, are ionoforms. These compounds can be used when it is desirable to carry out the selective removal of a particular cation. Examples of this application are the removal and extraction of silver ions from photographic solutions, the removal of toxic cations from industrial wastewater before such waters enter the environment, and the desalination of seawater. The deoxynarazine compound can be used as one of the components of the ion-specific electrode. These compounds alter the cationic permeability of primrose and artificial membranes. Consequently, the deoxine razine compound can be used as a component of the membrane used for the selective movement of cations against the gradient of concentrates. One of the possible applications of this property is to extract heavy and noble metals in commercial scale. 20-Deoxinarazine and its salts are also active inhibitors of the ATP-ase enzyme, ATP-ase, an enzyme sensitive to alkali metals, has been found in It is involved in energy expenditure for active transport. Active transport refers to a series of energy consuming processes by which the composition of intracellular and extracellular fluids is maintained. Inhibitors. reduce the energy required for active transport. In experiments outside the body ( in vitro) it has been shown that 20-deoxinarazine; (sodium salt) suppresses cation-transporting ATP-ase in hepatic mitochondria at a half effective concentration of 0.065 mg / ml. 20-Deoxynarazine and its salts are also effective preparations for tonicity of the heart. In experiments on an isolated atrial of the guinea pig, 20-deoxinarazine, for example, increases the contraction of the heart. Therefore, the invention also includes a method to increase. nor the force of contraction of the heart muscle of a warm-blooded mammal, according to which an effective non-toxic dose of a 20-deoxynarazine dose of a pharmaceutically acceptable salt is administered. The effective non-toxic dose is in the range of about 30-500 µg / kg of live weight. Preferred range:; pH doses are limited to about 30,100 µg / kg body weight. For the method under consideration, the antibiotic is administered parenterally, for example, intravenously. A suitable method of administration is the drip method, in which the antibiotic is incorporated into a standard intravenous solution, such as a glucose solution. 20-Deoxinarazine is preferably administered in doses below about 100 µg / kg until the desired force of contraction is observed. The amount of 20-deoxinarazine injected can then be controlled by the feed rate so as to provide the desired reaction. As in the case of the clinical use of other inotropic drugs, the dose of 20 deoxinarazine may vary for a given clinical case depending on such factors as the individual, susceptibility to 20-deoxyrazin, the nature of heart disorders, i.e. the degree of damage to the heart muscle , age and general physical condition of the patient. The proposed method for producing an antibiotic complex, including the use of a positive inotropic agent 20-deoxinarazine, can be used in a number of clinical situations that are widely classified as cardiogenic shock. These include, for example, myocardial infarction, postoperative cardiogenic shock, and progressive heart failure. Example 1.A. Enzyme in inoculated flask. Prepare a culture of S t reptomy.ces aureofaciens NRRZ 11181 and store it on beveled agar having the following composition, g: CANNED MgSO ;, - UNDO MNDMO ,, UNPO ZnSO. 7HBO Glucose Up to 1 liter Deioniaiova: pH of water (unregulated) Oblique agar seeded with Streptomyces aureofaciens NRRZ 11181 and infected cleaved agar are incubated at 30 ° C for up to 7 days. The mature culture is coated sterilely, spores are removed with beef serum and a sterile loop. The resulting beef serum suspension of spores and mycelial fragments is lyophilized to a maximum of six granules. One granule prepared in this way is used to inoculate 50 ml of the plant medium of the following composition, g: Glucose20 Soybean Flour15 Liquid after the maceration of corn with CaCO2. Tap water pH is adjusted to 6.5 by addition. sodium hydroxide. The infected plant medium in a Lenmeyer flask with a volume of ml is incubated at 30 ° C for approximately 24-28 hours on a rocking chair with a rotation speed of 250 rpm. Incubironic plant medium prepared according to the description of ml) is used to inoculate the MP of one of the enzymatic media. Wednesday 1 has the following composition, g: Tapioca dextrin 60 Enzyme hydrolyzed casein 6 Enzymatic casein hydrolyzate 2 CACOp, 2 MgSOi, 7H, .0O, B Port wine molasses with treacle15 Purified soybean oil 0.5 ml / l Water tap Up to 1 l pH ( unregulated) 6.6 Stadex 11, A.E. Steli, Illinois. Amber is out. Amber Laboratories, Juni 1ata Viscontin. Sf. D Amin A of firms Sheffield Chemical, Norwich, New York. Wednesday 2 has the following composition, g: Tapioca dextrin Glucose Enzyme hydrolyzed casein Enzymatic casein hydrolyzate Yeast extract .CaCO,. 7Н „0 Port wine molasses with molasses 15 Oily soybean oil 5.0 ml / l Tap water Up to 1 liter of pH (unregulated) 6.4 XX, XXX Correspond to the values indicated in the note for medium 1. Wednesday 3 has the following composition, g: Soyeva flour, 25 Glucose .20 CaCO-2.2.0 MadbO JUNDO1,0 Purified soybean oil 20 ml Methyloleate 20 ml Pe50ts, 6 MnCJ. "THttO0.3 Ascorbic acid 0.018 Dewirnisirone water to 1 liter of pH (unregulated) 6.5 Fermentation of grating for 10 days at a rocking chair at a rotation speed of rpm. B. Fermentation in the fermenter.
    Using the plant and enzyme media described in section A for fermentation in a flask on a rocking chair, fermentation is carried out in a fermenter. For fermentation, 10 ml of the plant medium are taken into the fermenter and the second stage is inoculated with 400 m of the plant medium in a 2 liter Eudermeyer flask. After incubation for 24 hours at 800 ml of plant medium from the second stage, the enzyme medium in 165 liters of the fermenter was sown in 100 liters. The pH of the medium after sterilization at 121 ° C for 45 minutes is approximately 6.8 + 0.1. Fermentation lasts 10 days at 30 ± 1 ° C. The tank was flushed with sterile air at a flow rate of 0.5 volumes of air per volume of culture medium per minute by mixing with standard agitators at a speed of 300 rpm.
    Example 2. The separation of the deoxinarazine complex.
    The pH of the whole enzymatic broth (4 l), prepared by the method described in Example 1, using medium 2, is lowered by adding concentrated hydrochloric acid, while stirring for an hour. The resulting solution is filtered through a layer of fine grain 600 ml 300 ml 300 ml 900 ml 2.0 l 300 ml 450 ml 450 ml 1.2 l 1.2 l 1.0 l 1.0 l 1.5 l 1.5 l 450 ml 900 ml 1.0 l 30.0 ml
    nisty material (125 g of the Guatau Super-Sel brand of Jones-Menville diatomaceous earth). The filtered mycelium is extracted in separate portions using a stirrer. Extrusion is carried out in total 2 liters of methyl alcohol, which contains 50 g of NaHCOft per liter. The methanol filtrate is evaporated in vacuo to a volume of about 450 ml. The pH of this solution is adjusted to 7.5 by the addition of concentrated hydrochloric acid. The irradiated solution is extracted twice with chloroform (500 ml). The chloroform extracts are combined, dried over sodium sulfate, and filtered. 5 The filtrate was evaporated in vacuo to give 2.0 g of crude deoxinarazine complex.
    PRI me R 3. Separation of deoxinarazine and epi-deoxinarazine.
    2 g of the crude deoxynarazine complex prepared in accordance with Example 2 is dissolved in a small amount of benzene and passed through a silica gel column (Merck 7.729} 22 cm long to a digester
    5 1.5 cm
    Enhanced fractions, solvents used and quantitative yields are listed in Table. eight.
    Table8
    4: 1
    85 5 7
    Same same
    18
    22
    7
    22 9 7 5
    12
    3: 1
    14
     28 The same
    100
    26
    70
    54,
    12
    100%
    Ethyl acetate. Obtained after drying and evaporation. Fractions were monitored by thin layer chromatography using ethyl acetate as a solvent. Fractions 16-17 (126 mg) contain 20-deoxy-epi-17-narasin, and fractions 18-23 contain a mixture of 20-deoxynarazine and 20-deoxy-epi-17-narasin. A mixture of 20-deoxinarazine and 20-deoxy-epi-17-narasin a, present in fractions 18-23, is subjected to chromatographic separation according to the method of preparative thin-layer chromatography. Acetic acid ethyl ester is used as a solvent. The mixture (105 mg on one plate and 130 mg on another) was dissolved in a small amount of dichloromethane and applied onto a silica gel (Merck) preparative sheet. After the sheet has been developed, two separate materials are observed in ultraviolet light. Each of the areas occupied by the two substances is removed from the sheet and extracted with a mixture of dichloromethane and methyl alcohol (4: 1). In this system, deoxinarazine is a slower moving component than the other two. 7.5 mg of 20-deoxy-epi-17-narasin and 27 mg of dioxinarazine are isolated from a sheet containing 105 mg of material. From a sheet containing 130 mg of the mixture, 4.0 mg of 20-deoxy-epi-17-narasin and 56 mg of 20-deoxynarazine are obtained. Example 4. Another method for isolating 2P-deoxinarazine. The whole enzymatic broth (95 ml) is adjusted to pH 3 by the addition of hydrochloric acid, followed by stirring for an hour. A fine-grained powder (grade 1 Hufflo-Super-Sel, 3%) is added and the broth is filtered. The separated mycelial filter cake is extracted twice with about 45 ml of acetone, which contains 50 g of sodium bicarbonate per liter. The acetone extracts are combined and concentrated in vacuo to give about 10 L of aqueous solution. The pH of this solution is adjusted to 8.0 by the addition of 5N hydrochloric acid, and the resulting solution is extracted three times with half volumes of sodium sulfate 1d sodium sulfate and the filtrate to dryness in vacuo. tana. The dichloromethane extracts are combined and evaporated in vacuo to give an oily residue. This residue is dissolved in 500 ml of the upper phase of a mixture of hexane-methyl alcohol-water (10: 7: 1). The upper phase is extracted six times with 300 ml portions of the lower phase. The extracts were combined and concentrated in vacuo to give a residue. This residue was dissolved in dioxane, and the dioxane solution was lyophilized to obtain 19.4 g of deoxinarazine complex. Several samples obtained in a similar manner were combined (40 g), dissolved in toluene, and introduced into a silica gel column in a liquid chromatograph (preparative liquid chromatograph / system 500). The column was eluted with a solvent mixture of toluene-ethyl acetate (9: 1) at a rate of 250 ml / min, collecting fractions of 250 ml. The composition of the fractions is controlled by thin layer chromatography. Fractions 37-52 are combined and concentrated in vacuo to give a residue, which is dissolved in dioxane and lyophilized. The result is 7.8 g of purified material enriched in 20-deoxinarazine. This material is re-passed through a chromatograph (preparative waters system 500 of Waters, similar to that described above). After elution of 50 fractions with toluene system; ethyl acetate (9: 1), the elution solvent is replaced with 100% ethyl ® Fractions 51-53 are combined and evaporated in vacuo to give a residue, which is dissolved in dioxane and lyophilized. The result is 1.12 g of 20-deoxinarazine in the form of its sodium salt. Example 5. Obtaining 20-deoxynazoic free acid. 200 mg of the sodium salt of 20-deoxinarazine are dissolved in ethyl acetate (10 ml), the solution is rolled with O, 1N hydrochloric acid (10 ml) and then twice with water (5 ml). The resulting organic layer was evaporated to dryness to give a residue, which was redissolved in dioxane and lyophilized to give 139.6 mg of 20-deoxynarazic acid as a white solid.
    Example 6: Chicken diet for suppressing coccidiosis.
    Ground Yellow Corn
    Soybean flour, solvent extracted, peeled, finely ground 50% protein
    Animal fat (beef tallow)
    Dried fish meal with bard
    {60% protein)
    Corn bard
    Dicalcium Phosphate, Food Grade Calcium Carbonate
    A mixture of vitamins (vitamins A, O, K
    and choline, nicotinic acid,
    paigogenic acid, riboflavin,
    biotin, glucose as a filler
    Cooking salt
    (MnSO, ZnO, K & FeSO, CaCOj)
    2-amino-4-hydroxybutyric acid. (Methionine oxy-analog)
    Following the standard feed mixing technology, deoxinarazine JJOMplex, 20-deoxinarazine or 20-deoxy-epi-17-narasin (approximately 0.01 wt.% Are mixed with the above ration. 40 Chickens that receive this ration with ad Cibitum water are protected from coccidiosis a.
    Example 7: Improved cattle ration.
    Prepare a balanced diet for cattle given in Table. ten.
    Table 10
    According to the table. 9, a balanced high-calorie diet is prepared for feeding chickens in order to obtain a fast gain.
    T a blitz 9
    454
    280.6 59
    45.4 36.3 16.3 7.3
    4.5
    2.72
    0.91
    0.91
    50
     Content per pound: 40 2000000 units. vitamin A, 227200 units vitamin D, and 385.7 g of soybean feed with the addition of 1% oil. Dried corn pellet with bard containing 2000Q units. tocopheryl acetate alpha65 per pound.
    Following the standard procedure, the following is prepared: a mixture of deoxinarazine anti-Siotic complex, 20-deoxynaraein or 20-deoxy-epi-17-naraein (approximately 0.004 wt. With this diet) The feed is granulated. The average daily amount of this mixture given to an animal is 15 pounds, which corresponds to daily dose of antibiotic per animal equal to 300 mg.
    Example 8. Improved diet for sweats.
    A mixture of the following composition is prepared in a standard way, g / kg: Active compound150.0
    Calcium silicate 20.0
    sn
    The fact is that growing Streptomyces aureof ciens NRRZ1118i in a culture medium containing digestible sources of carbon, nitrogen and inorganic salts, under conditions of aerobic fermentation during immersion and when the required amount of antibiotic activity is reached, the antibiotic complex is removed from the culture fluid as
    Calcium carbonate (flour Oster Shell)
    830.0
    Total weight 1000 g. This mixture is added to an industrial pig feed using a standard mixing procedure. The final content of the active compound is 100 g / t.
    Forko la invention
    A method for producing a deoxinarazine antibiotic complex containing 20-deoxinarazine of the formula and 20-deoxy-epi-17-narasin of the formula
    CH,
    acids and their pharmaceutically acceptable salts.
    Sources of information taken into account in the examination
    1. Patent of the USSR No. 403133, cl. From 12/9/02, 1973.
    2. Patent ON 4035481, cl. 424-122,12.07.77.
    3.Latent USA 4038384, CL. 424-122,26.07.77.
    权利要求:
    Claims (1)
    [1]
    Claim
    A method of obtaining a deoxynarasin antibiotic complex containing 20-deoxinarasine of the formula T and 20-deoxy-epi-17-naraein of the formula II comprising growing Streptomyces aureofaciens NRRZ 11181 in a culture medium containing assimilable sources of carbon, nitrogen and inorganic salts, under conditions aerobic fermentation upon immersion and upon reaching the required value of antibiotic activity, the antibiotic complex is extracted from the culture fluid in the form of acids and their pharmaceutically acceptable salts.
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    同族专利:
    公开号 | 公开日
    ATA750778A|1980-10-15|
    CA1119541A|1982-03-09|
    FI783174A|1979-04-21|
    FI58791C|1981-04-10|
    IL55738D0|1978-12-17|
    BE871274A|1979-04-17|
    DE2861882D1|1982-07-22|
    FR2406639B1|1981-08-14|
    EP0001709B1|1982-06-02|
    PL117570B1|1981-08-31|
    IL55738A|1981-10-30|
    JPS6244553B2|1987-09-21|
    IT1101615B|1985-10-07|
    IE782078L|1979-04-20|
    HU179463B|1982-10-28|
    FR2406639A1|1979-05-18|
    PT68661A|1978-11-01|
    US4141907A|1979-02-27|
    IE47665B1|1984-05-16|
    GB2006774B|1982-09-02|
    MY8500595A|1985-12-31|
    GB2006774A|1979-05-10|
    AU519966B2|1982-01-07|
    EP0001709A3|1979-05-16|
    IT7828926D0|1978-10-19|
    YU241578A|1982-10-31|
    ZA785804B|1980-05-28|
    PL210410A1|1979-10-22|
    ES474327A1|1979-11-01|
    GR71734B|1983-06-22|
    DD139520A5|1980-01-09|
    DK465978A|1979-06-15|
    PH14339A|1981-05-29|
    AU4077678A|1980-04-24|
    FI58791B|1980-12-31|
    NO783545L|1979-04-23|
    AR217869A1|1980-04-30|
    EP0001709A2|1979-05-02|
    LU80389D|1979-03-19|
    CH636903A5|1983-06-30|
    CS204037B2|1981-03-31|
    MX5603E|1983-11-07|
    AT362499B|1981-05-25|
    NZ188670A|1981-05-29|
    RO77417A|1981-11-04|
    JPS5470276A|1979-06-05|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    US3753887A|1969-06-04|1973-08-21|Hydronautics|Alkali metal specific measuring electrode|
    US3947586A|1974-05-20|1976-03-30|Hoffmann-La Roche Inc.|Method of combatting swine dysentery|
    US4038384A|1974-06-10|1977-07-26|Eli Lilly And Company|Antibiotic a-28086 and process for production thereof|
    US4035481A|1975-04-21|1977-07-12|Eli Lilly And Company|Antibiotic A-28086 and process for production thereof|
    US3985893A|1974-10-29|1976-10-12|Eli Lilly And Company|Method for treating cardiac insufficiency with antibiotic A-23187|
    US4009262A|1975-04-21|1977-02-22|Eli Lilly And Company|Antibiotic a-28086 recovery process|GB2024806B|1978-03-16|1982-05-06|Taisho Pharma Co Ltd|Onomycin derivatives|
    IE49936B1|1979-07-11|1986-01-08|Int Minerals & Chem Corp|Zinc-containing antibiotic agents|
    US4309504A|1980-01-28|1982-01-05|Eli Lilly And Company|Process for preparing narasin|
    US4394377A|1981-07-31|1983-07-19|SyntexInc.|Ruminant animal performance by co-administering choline and propionate enchancers|
    US4625041A|1984-02-17|1986-11-25|Pfizer Inc.|Acidic polycyclic ether antibiotic|
    US4859599A|1984-10-11|1989-08-22|The Dow Chemical Company|Antibiotic A26201-1 and antibiotic A26201-2 produced by a novel strain of actinoplanes|
    US4613503A|1984-10-11|1986-09-23|The Dow Chemical Company|Antibiotic A26201-1 and antibiotic A26201-2 produced by a novel strain of actinoplanes|
    US5041374A|1986-05-29|1991-08-20|International Minerals & Chemical Corp.|Polyether antibiotic recovery and purification|
    US5049495A|1986-05-29|1991-09-17|International Minerals & Chemical Corp.|Fermentation method for producing polyether antibiotics|
    US5047338A|1986-05-29|1991-09-10|International Minerals & Chemical Corp.|Polyester Antibiotic preparation|
    US20080161324A1|2006-09-14|2008-07-03|Johansen Lisa M|Compositions and methods for treatment of viral diseases|
    CN108743905A|2018-08-24|2018-11-06|河南科技大学第附属医院|A kind of Chinese medicine preparation and preparation method thereof for treating flu|
    法律状态:
    优先权:
    申请号 | 申请日 | 专利标题
    US05/844,087|US4141907A|1977-10-20|1977-10-20|Deoxynarasin antibiotics|
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