SU663724A1 - Method of obtaining antibiotic - Google Patents

Description

The present invention relates to methods for producing antibiotic i.ociothoracic and viralostatic activists, more specifically to methods for producing antibiotics using Streptotnsces as a species producer. A known method for producing antibiotic by cultivating St eptom ces iotaceii 1212 in a liquid nutrient medium containing carbon, nitrogen and mineral sources salt, with aeration and stirring, followed by separation of the target product from the culture fluid, purification and chromatographic separation of it into active components. However, the resulting antibiotic is not sufficiently active against microplasma viral and tumor diseases l}. The aim of the invention is to obtain a new antibiotic violamycin, active against microplasma B1GrushiNi and tumor diseases. The method of obtaining the antibiotic consists of a B to Si Wff Tkulative producer of the type Streptomsces viotqcensl MET A 6844 under aerobic conditions on a liquid nutrient medium containing sources of carbon, nitrogen and mineral salts during aeration and mixing, followed by separation of the desired product from the culture liquid, purification and its chromatographic separation into active components. The method differs from t & l that the strain Streptoniyces vioCctcens 1 .MET DA 6844 strain is used as the producer of the target product, and the cultivation is carried out at 25-37 ° C for two strains. The strain of the collection of the collections of the Centregoenny Institute of Microbiology and Experimental Therapy in the city of Yen at The German Academy of Sciences in Berlin under number 1 MET IA 6844. The original type was isolated in 1958 from a sample of the Schwelenlenburg land area in the Elkslsben area (Erfurt). The strain belongs to the type of Streptomvces and corresponds to 5 replicates of H1O5 (Russia-Doria, 1891, Kras.Sipnikov, 1941, Vaksman, 1953), given to agree to the study of the strain of type 1SP 5082 (ZMMZ-1, RIA 656 ATCC 15888) in an international project Strf ptomvces of Schliera and Gottlieb (3nt.T Svs-t.Bc3ict.19: 497, 1969). The cultivation of strain 1 META 6868, like its mutants and variants, is carried out under aerobic conditions. Leophilic dried ground spore material of the strain is poured into the appropriate nutrient agar medium, and then & liquid, previously sterilized nutrient media, and the formed mycelium is cultured by the well-known method at a temperature between 25 and 37 ° C (preferably 28 ° C) for 2-6 days (preferably 4 days) with acidity, until the beginning of the fermentation process is within the pH range 6.2 and 7.0, and by the end between 7.5 and 8.3. The nutrient medium is from carbon and nitrogen sources, as well as from inorganic salts. Starch, glucose, glycerin, mannitol, dex rin, sucrose, soybean oil, and soybean can be used as carbon isches. As a nitrogen source, in addition to the above-mentioned nitrogen-containing substrates, dry yeast, meat peptone and casein can also be used. The strain has the following properties: the substrate on different media and at different time intervals is colored red, purple or blue. The pigment that can diffuse into agar medium also has indicator properties: it is red in acidic and red in alkaline, blue. Aerial mycelium is colored in pale pink, pink or a grayish-pink color. Spore chains are spread out in a more or less uniform (in most cases open) spirals on partially long main hyphae. Slorov prickly surface. On peptone obsessive as well. also non-tyrosine-containing pigment, which has a color from brown to dark blue, differs in c. agar The strain grows on the basal medium with the addition of d-glucoa, larabinogal, d-xylose, m-inositol, d-minnit, d-fructose, rhamnose, sucrose and raffinose. The selection of the antibiotic violamish is carried out as follows (see diagram). The antibiotic is extracted from the culture filtrate with organic, non-miscible or slightly miscible, and from the mycelium with organic, water-miscible solvents. From the mycelium extract, the antibiotic is reextracted with the immiscible or low-mixing solvent used to extract the culture filtrate and combined with the corresponding culture filtrate extract. The extract thus obtained is again concentrated, the antibiotic is transferred to the aqueous phase and at pH 3.0-9.0 (preferably 8.2) is extracted again with an organic, water-immiscible solvent, and precipitated from the extract concentrate by mixing with petroleum ether or gasoline with stirring, free from solvent by filtration. The precipitate is dissolved in chloroform, filtered from an insoluble precipitate, the chloroform solution is washed with water and finally mixed with petroleum ether, and fraction A of violamycin precipitates. The fraction A of violamycin is dissolved in lower alcohols, purified on a chromatographic column, for example, using the Sephadex (P) trademark of the Uppsala pharmacy (Sweden) and divided into the first obtained violamycin A and the biamylacid A biologically inactive complex obtained from the column. The fraction B of violamycin is obtained from the wash water of an extract of chloroform, which is subjected to extraction at pH 6-9 in butanol, the extract is concentrated and mixed with petroleum ether, and the fraction B of the violamycin precipitates. It is dissolved in lower alcohols, purified in a chromatographic column using Sephadex (R), and the violamycin B obtained first is separated, then the violamycin Bd obtained, and also the biologically inactive aglycone complex obtained last, Viopamycinone B. The violamycins A, B and Bb are differentiated by the content of sugar compounds and can be divided by paper chromatography into six different components. The hydrolysates of different violamycins give the same aglycone mixture, however, with the climatic difference of the individual components. 5 Preparation of antibiotic Mycelium (M) Extraction with organic water-miscible solvents Extract I Extraction in an aqueous phase Extract II Extraction with an organic, water-immiscible solvent Extract 111 Reex Organic Lamycin Mixing with petroleum ether Crude viamycin A product, -. Dissolving in lower aliphatic alcohols. Violamycin A -t-- solution - Purification on a chromatographic column of olamycin A. Violamycinone A. Antibiotic violamycin A is an amorphous reddish-cocholamycin B complex. Violamycinone B Deposition with petroleum ether or gasoline. chloroform Washing with water fraction A of the violet fraction B of violamycin 4.. 6 ICINA Culture filtrate (CG) Extraction with organic or immiscible or little mixed with water solvents Extract I Extraction in phase Extract And Extraction with organic solvents that are not miscible with water. Extract III Extraction with Bugnol Butanol Extract of Fraction B Mixing with Petroleum Ether Crude Violamycin B Product Dissolving in Lower Aliphatic Alcohols Violamycin B Solution Purification of brown substances on the chromatographic column, it is well soluble in chloroform, acetone and alcohols, little

663724

7. soluble in benzene and insoluble in ether and water. Violamycin A has the properties of an indicator and, in acidic solutions, has a red color, and in a slit 6 h1X solutions is blue. Both the fresh fermentation broths of the violamycin formers and the antibiotic isolated from them inhibit the growth of gram-proliferative bacteria and microbacteria, to a lesser extent they inhibit the growth of gram-negative bacteria, on the other hand, yeast and fungi do not experience an antibiotic effect. Violamycin exhibits cytostatic as well as cancerostatic activity against placental lh cells of the Migo (in test tubes) and against experimentally induced tumors in mice. Studies on the kanderostatic effect of the antibiotic violamycin. In the first stage of the cancerostatic Test (experimental department of 11 METs, Yen), vyolamycin raw appears in mice that have been experimentally diagnosed with leukemia L 1210 and LATI, as well as sarcoma 18O (activity against sarcoma 180). A single injection of 5 and 2.5 µg of viiol iiina raw 20 g mouse weight inhibits the growth of solid tumors compared with the untreated control

The lowest inhibitory concentration

8 Mgc / ml per 48.9 and, accordingly, a copy of rum by 53.6%. Bacterial viruses, as well as representatives of animal viruses, suspend their growth under the influence of intracellular penetration of ant and ibiotics without simultaneously blocking the exchange of host cell metabolism. Violamyiine is particularly active in cell division and metabolism in the resting state of mycoplasma species. Investigation of the bacteriostatic and bactericidal action of myam-plasma violamycin antifibiotik. The effect of the antibiotic is checked on two of the most prominent episodes in agriculture, especially in animal husbandry and cattle breeding. Violamiains A and B are characterized by a striking bacteriostatic strength and, at the same time, bactericidal activity against Mljcoptasnia ctE-tisepticum (Je strain) and scopCa5mo io .ftlimS-Comparative study of the biological activity of the antibiotic violamycin A and B. on nutrient broths, the next spectrum of action (see table).

MvcopEasnia gatEieepticum

0.030.15

strain 56

/ W jcopEasma iiiorViinis

0.150.1519.00.76 The results in the table show that violamycin A is bacteriostatic more active against M.gaBEiseptncum (Strain S6) than violamycin. B, but violamycin B exhibits a stronger bactericidal activity against M, 111O111115, it is violamycin A. Both violamycin differ from the previously known natural 760.76 materials that have activity against mycoplasma with their hundreds of times greater bactericidal action. Such biological activity against representatives of mycoplasmas, which can cause painful symptoms in domestic animals, has not yet been described for anthracycline antibiogics, which is particularly valuable. Example 1. Two vessels with a straight neck with a capacity of 500 ml each contain 80 ml of the next culture medium, weight. %: Glucose1.5 Soev flour1 5 Sodium chloride, 5 Calcium carbonateOD Primary phosphate Acid Rest Water, o. Sterilization: 35 min at 110 C. After sterilization, pH 6.9. The culture medium in the vessels with the straight neck is inoculated with a spore and mycelial suspension, obtained with a sterile isotonic solution in the salt of the culture-forming violaam 10–14 days old, grown in a test tube on the following nutrient medium,%: Oat flour 0 Ovs Nash flakes, ground1,0 Agar-agar2,0 Piped waterEverything Sterilization: 35 minutes at 120 ° C, natural acidity. Grown cultures were incubated for 48 hours at 28 ° C in an apparatus with shaking (180 rpm), 8 mm of the thus obtained culture are used to inoculate one vessel with a straight neck, which contains 80 m of the next medium, weight. %: Glucose2.0 Soev. Muka2.0 Sodium Chloride0.5 Calcium carbonate0.3 Tap water Remaining Sterilization: 35 minutes at 115 C. After sterilization, pH 6.3. Temperatures during fermentation are 28 ° C, shaking frequency 180 rpm. After 72 hours of fermentation, the highest violamycin content is 125 µg / ml. Activity determination is carried out by known methods. Example 2. 80 ml of the liquid medium indicated in Example 1, containing the vessel with a straight mouthful of 5.0 ml, are inoculated with the producer strain violamycin. from the culture medium as described in Example 1. They are cultivated for 24 hours at 28 ° C in an apparatus with shaking (180 rpm). 12 ml of the nutrient broth thus obtained is used to inoculate a 400 ml bottle of the same pre-culture medium contained in a two-liter glass flask. The next 24 hours are cultivated at 28 ° C and with shaking (180 rpm), the resulting 400 ml broth culture is used to inoculate 2 O of the medium described in Example 1. This medium is contained in a 32-liter glass fermenter. During the fermentation, which was carried out at a stirring rate of 40O rpm and with an air supply of 15 liters / min, the pricing is controlled by the addition of small amounts of sunflower oil or other silicone-containing antifoam agents. The pH of the 20 liter culture solution was adjusted to 5.0 with hydrochloric acid and after the separation of the mycelium, 18 l of cultured filtrate was obtained. The butanol extract of the culture filtrate is concentrated under vacuum at a ratio of 1:10 of the original volume and combined with the butanol extract of the mycelium extract concentrate. Pre-mycelium is mixed twice with methanol in a ratio of 1: 6. The combined extraction under reduced pressure is concentrated to 1/20 of the volume of methanol, and the resulting aqueous solution is exhaustively extracted with butanol. The combined butanol extracts together with the butanol extract of the culture filtrate are adjusted to 1/25 of the volume of the culture solution. From the final concentrate is obtained by adding three times the volume of petroleum ether ZOO g of biologically active raw material (A 6844) per 1 m culture medium, Example 3. 200 g of raw material A 6844 are suspended in 5 l of chloroform and after 1 hour of stirring the solution is separated from the insoluble material. The chloroform solution is washed five to six times in half a volume of water and, by that, completely freed from water-soluble pigments. After drying, the chloroform solution is concentrated under reduced pressure to 1/5 O of the initial volume and stirred with a tenfold amount of petroleum ether. In this case, the fraction A of violamycin is biologically active. Fraction A is filtered off, washed with pure petroleum ether and. drying & s under reduced pressure and in the coil above the concentrated sulfuric acid. Fractional A yield is 16 g. Violamic fraction A is separated by chromatography on a column for antibiotic violamycin A and the biological unfavorable chromoform complex violamycinone A. From 10 g of viralamcin fraccappa A, 5 g of viamycinin A is obtained; Chloroform wash water with pH = 1.5% after pH = 8.5, extraction with bu. tanol. After concentration of the butanoic guy extract and after its research institute with petroleum ether, the resulting precipitate is filtered and dried. The output of violamycin fraction B is 25 g per 200 g of raw material A 6844, Fraction B, similarly to fraction A, can be divided into antibiotic violamycin B and the biological unfinished chromophore complex violamiinone B. From 10 g of fraction B, 9.5 g actibiotimo are obtained. ) |: a violamycin B. 4 12 and 3 about b p. Formula A method of obtaining an antibiotic by cultured by the producer Streplomices NloCotcCrtS in a liquid nutrient medium containing sources of carbon, nitrogen and mineral salts, with aeration and stirring, followed by separation of the target product from the culture fluid, purification and chromatographic separation of it into active components, In contrast, in order to obtain a new antibiotic, violamycin, which is active against microplasma, viral and neoplastic diseases, the Stre strain is taken as a producer of the antibiotic. ptomices vnptacensi M ET 1A at 84, and cultivation was carried out at 25-37 C for two to six years. Sources of information taken into account in the examination of l.Kroi5-i8mkov N .A-ei AC. Aci-inottraces synthesiring- qnli-xiVoe antibioticus. Jou) of Aniibioticus.Ser.QjVoE. XHI / No. 1, I960.