PRIMYSIN COMPONENTS AND PROCESS FOR THE SEPARATION OF THE ANTIBIOTIC COMPLEX

Abstract: Components of primysin antibiotic complex and the salts thereof and the process for the separation of the individual components by chromatography. A further object of the invention is a synergistic antimicrobial composition containing the said components and a process for the preparation thereof.

Description: PRIMYCIN COMPONENTS AND PROCESS FOR THE SEPARATION OF THE ANTIBIOTIC COMPLEX Technical field The present invention relates to different components of primycin obtained by the separation of primycin-mixture and process for the separation of these components from the antibiotic complex. A further object of the invention is a synergistic pharmaceutical composition comprising the said components and process for the prepara¬ tion thereof. Background art The primycin antibiotic complex can be obtained from the culture of strain Thermopolyspora galeriensis belonging to the species of Streptomycetae, its preparation by fermentation is described by Hungarian patent specifica¬ tion No. 153,593. Primycin is an antibiotic of macrolide type which is characterized in the prior art /J. Che . Soc, Perkin I. page 816, 1974/ by one single formula: /5-/18-( β C-D-arabino- furanosyloxy)-2-butyl-3,7,11,15 ,19,21,23,25,27-nonah ' ydroxy- -4,16,32,34-tetramethyl-l-oxo-oxacyclohexatriaconta-16,32- -diene-35-yl/- -hydroxyhexy_l7-quanidinum sulphate. Primycin is a very effective antibiotic which can be widely used, it is effective first of all against gram- -positive bacterias but also against the polyresistant human pathogenic strains. Resistancy has not developed against it yet. Our former experiments have already referred to the fact that primycin obtained by fermentation is not a homogeneous substance. The recent developments of chromatogr enabled us to submit the primycin substance mixture to a more detailed searching examination. De ta iled description of the invention It was discovered that the primycin obtained by fermentation comprises three major and six minor components and the process of our Invention is suitable for the qualitative and micropreparative separation of these components. Surprisingly we also found that components enhance each other effect, i.e. they show synergistic activity. We targeted our efforts for separating and isolating the different components of the antibiotic complex produced by strain Thermopolyspora galeriensis. Several chromatographi methods were tested and it was found that thin-layer and coloumn chromatography are the most effective for the above purpose. In the field of thin-Layer chromatography /TLC/ the use of commercial chromatographic plates, with silica gel - adsorbent layer thereafter the high performance thin-layer chromatographic plates with suitable solvent system resulted in the separation of the components with surprisingly high resolution. According to a preferred embodiment of the separation process of our invention the stock solution of the fat-free, crude, semi-half or pure product derived from the mycelium of strain Thermopolyspora galeriensis is applied onto a TLC chrcmatoplate, developed, the spots thus obtained are detected and the major and/or minor components of the anti¬ biotic complex are separated. For the preparation of the stock solution of the crude, semi-half or pure product recovered from the culture medium aqueous solutions of alcohols of 1 to carbon atoms or a mixture thereof, preferably a 25 : 25 : 50 by volume mixture of ethanol : n-butanol : water is used. 0.5 to 1 w/v % solution is prepared by the aid of the above mixture and 50 to 200 ,ug of the substance to be examined is applied to the plate. In our experiments first time we used a commercial silica gel chromatographic plate typ rCieselgel 60 ~ 2 - ^ /Size: 20 x 20 cm, thickness of the silica gel layer: 0.2 mm/. Several solvent mixtures were tested on this plate in order to achieve the best separation of the primycin mixture. The best separation can be attained when the eluent used comprise." halogenated hydrocarbon/s/, organic acid/s/, alcchol/s/, water, and preferably aldehyde/s/. The following solvents can be used as eluent: halogenated aliphatic hydrocarbons /e.g. chloroform, di- chloro ethane/, organic aci'ds, preferably aliphatic carboxylic acids of 1 to 4 carbon atoms /e.g. formic , acid, acetic acid/, halogenated aliphatic carboxylic acids /e.g. ono- chloro-, dichloro- and trichloroacetic acid, trifluoro- acetic acid/, aromatic carooxylic acids /e.g. benzoic acid/, organic sulphonic acids /e.g. ethanesulphonic acid, toluene- sulphonic acid/, alcohols, preferably aliphatic straight or branched alcohols of 1 to 10 carbon atoms /e.g. methanol ,- etanol, n-butanol, isopropanol/ , aldehydes, preferably aliphatic aldehydes of I to carbcn atoms /e.g. 36 f_ form¬ aldehyde/ or aqueous mixture thereof. For the spearation o f t e components o the primycin antibiotic complex the following solvent systems proved to be "he most effective: I. the upper layer of 60 : 10 : 30 mixture f ri-ou ar.ol, glacial acetic ncid and water, II. a 75 : 2 : 3 : 15 mixture of n-butanol, ethanol, glacial acetic acid and water, III. the lower layer of a 5 : 30 : 15 : 20 mixture of chloroform, methanol, glacical acetic acid and water, IV. a 50 : 35 : 14 : 1 mixture of chloroform, methanol, formic acid and water, V. a 130 : 53 : 6 : 9 : 3 : 3 mixture of chloroform, methanol, formic acid, water, formaldehyde and ri-butanol, VI. a 160 : 53 : 6 : 9 : 3 : 3 mixture of chloroform, - . - methanol, formic acid, water, formaldehyde and ri- -butanol, VII. 15 parts b weight of mcnochlcroacetic acid dissolved in a 125 : 53 : 9 : 3 : 3 mixture of chloroform, methanol, water, formaldehyde and n-butanol, VIII. 15 : 2 : 1 mixture of the solvent system III, chloro¬ form and methanol. The solvent mixtures I and II are of higher capacity, while solvent mixtures III, IV, V, VI, VII and VIII are more selective and preferably the developing time is shorter which is preferable from the point of view of industrial serial tests. Solvent systems IV, V and VI are chemically metastabile due to the formation of methylfor iate, while solvent III is physically metastabile. According to its isothermic curves system IV can be used up within 16 to 24 hours calculated from its preparation, systems V, VI, VII and VIII can be used at once after adding the components to each other, while system III can be preserved for 1 week if 1 to 2 ; * methanol is added. Solvent systems IV, 7, VI, VII and VIII are preferred. In eluent IV the suitable separation can be achieved if the development is carried out twice as the 3- values of the different ccmponer.ts o f the antibiotic complex are very similar, while even one •development can lead to appropriate separation if solvent systems V, VI or VII are use:.. The most preferred solvent systems are systems V, VI, VII and VIII. Solvent V can be used for normal chromatographic plates for thin layer chromatography, while eluent VI can be applied for HPTLC plates. Solvent system VII is suitable for the impregnated plates, and solvent mixture VIII is preferably used for the Lobar-coloumn. The components of the primycin substance mixture separated by thin layer chromatography can be detected by chemical methods and/or bioautography. For chemical detection sulfuric acidic vanilline, chlorotoluidine, phospho 'molydic acid, benzidine, modified Sakaguchi reagent and/or ethanolic sulfuric acid known from the prior art and/or. heat treatment are the most effective, giving a specific characteristic and sensitive colour reaction. The spots can be located preferably by immersing the development chro atoplates into suitable reagents and after drying the chromatograms can be evaluated by Shimadzu densitometer in some cases. The hioautographic development is carried out as follows: The plate is cut into stripes perpendicularly by the side of the spots after the thin-layer chromatographic development and chemical detection. In the meantime agar plate is made and inoculated by Bacillus subtilis ATCC 6633. The cut band is pressed onto the agar plate after the spots are marked on the opposite side or the stripe is perpendicularly cut into two ' pieces and only one of the pieces is pressed onto the agar sheet. Thereafter the agar plates are incubated at 37 °C for 20 to 24 hours. Upon rising out from the thermosthate the spots where the bacteria could not grow can be well observed. The spots responsible for the inhibition of the growth of the bacteria can be identified on the basis of the marked opposite side of the stripe if the whole tripe was used, or on the basis of the other half-band placed by the side of the original stripe. In order to illustrate the above procedure the figure of the thin layer chromatographic plate obtained after double development with solvent system IV is shown by Figure 1. The different spots obtained after development of the primycin samples obtained from different culture mediums /spots 1 to 4/ well support that primycin is a mixture. The isolated major components A, , B, and C, and the bioautogram of the complex can be also seen on the figure Developing the primycin mixture once with solvent - o - system V the mixture separates into the following components characterized by the R values In brackets A^ /0.29/, A- /0.31/, A 3 /0.33/, C ± /0.35/, C 2 /0.37/, C 3 /0.39 3 1 /0.54 3,, /0.56/, 3 3 /0.53/. Some other, minor, unidentified components can also be traced. The chromatogra is shown by Figure 2. The efficiancy of the separation can be enhanced by using HPTLC plate. Then solvent system Vl is used as eluent in order to provide appropriate separation. Similarly excellent separation can be obtained when -ieselgel 60 F C , chromatographic plate impregnated wit a suitable metal salt, preferably silver salt is used insted of the hardly available and expensive HPTLC plate. In the course of the impregnation the chromatographic plate is plunged into the aqueous and/or organic solvent e.g. acetonitrilic solution of the metal salt, preferably 20 :i aqueous solution is used. Preferably silver salts, e.g. silver nitrate, iron salts, e.g. iron/II/-phosphate can be used as metal salts. The surface humidity of the i pregna ed layer is dried off thereafter the plate is activated at 120 C for a hour with the exclusion of light. For developing the chromatogram solvent systems V and VI are very useful in this case as well, but the best separation can be achieved when solve-nt system VII is used. The efficiancy of the separation is highly enhanced in this system by the fact that the difference of the R f values of the individual components increases. It was found that primycin is a mixture of three major components /A, , B-, , C,/ and more minor components. The individual components crystallize together depending on the solubility conditions. The percentile composition of the antibiotic complex can be influenced by changing the conditions of the fermentation /nutrient medium, cultivating time and temperature, degree of aerating etc./. The advantages of the thin-layer chromatography elution was improved by working out the preparative separation of the primycin mixture with colouπn chroma¬ tographic method. The partition and adsorption coloumn chromatography was tested. According to our invention the partition coloumn chromatographic separation is carried cut by using a solid carrier /Sephadex LH-20/ and a counter current /Droplet Counter Current Chromatography, further is referred as DCCC/. -The same solvent systems can be used for this method as for the thin-layer chromatography, i.e. two layer solvent systems containing organic acids, and/or chlorinated hydro¬ carbons and/or alkanoLs of 1 to 4 carbon atoms and/or water. Solvent system I is preferred /stationary phase/ wherein the solid carrier /Sephaαex H-20/ is swollen for two days, thereafter it is filled into the chromatographic coloumn and wasned with the lower phase of a 30 : 10 : 60 mixture of r.-butanol , glacial acetic JC:_ _ιr.J .-a r /mooile phase/. Primycin is dissolved in a 20 : 1 'lixt.re of t e mobile and stationary pr.ase, filtered _n α ;l..ss filter and applied onto tne coloumn. In.e lower, -jci ' .e pr.ase s used as el-ent preferaoly using a peristaltic tump ir.α a collector. The composition and purity of - _• fractions are controlled oy thin-layer cnro.-.a o.jraony, tne separation is checked by viewing the cnrcmatograms -nder -Itraviolet light at 206 nm. The fractions of tne same composition are combined and exposed to vacuum distillation and the yields are determined. A 90 to 95 % of tne product applied to the coloumn is recovered. The eluate containing mainly major components A, , 3-, and C, is further purified by chromatograpny. Component A, was chromatographed with solvent system I, component 8, was obtained with a solvent contain- ing benzene, ri-butanol, methanol, glacial acetic acid and water, while component C-, was separated by the DCCC method with a solvent system composed from chloroform, methanol, glacial acetic acid and water. It was found that the components of the primycin mixture can be most preferably separated by coloumn chromatography optionally under gentle pressure using the suitable silica gel packing. The following cσloumns were tested: silica gel coloumn filled by ourself; the ready- -to-use Lobar coloumn /Merck/, a coloumn filled with silic gel impregnated with a suitable metal salt /similarly to the thin-layer chromagtopraphic plate/. The efficiancy of the separation is highly enhance if the Lobar coloumn is used or in lack of this coloumn, t coloumn filled with impregnated silica gel instead of a coloumn filled with simple silica gel. A slight overpressu /l.l to 5.0 at / can be useful in the case of the Lobar coloumn as well as the coloumn filled with impregnated gel. In the course of Impregnation the silica gel Is mixed with a suitable metal salt, preferably it is suspend in the solution of the metal salt prepared with an organic solvent, thereafter the solvent is optionally removed and the impregnated silica gel can be used as the packing of the coloumn. The coloumn Is filled with impregnated silica gel suspended in an organic solvent, preferably halogenate aliphatic hydrocarbone /e.g. chloroform/ and compacted by slight overpressure /l.l to 2.5 atm/. If a silver salt is used as metal salt it is desirable to protect the coloumn from light in order to prevent the reduction of the silver salt. The primycin mixture to be separated or the separa ed components thereof is applied onto the top of the colou dissolved in the solvent mixture used as eluent and adsorb by slight overpressure. The substance mixture to be separated can be industrial primycin sulphate or an other primycin salt preferably primycin acetate or primycin monochloroacetate prepared from primycin sulphate according to the Hungarian patent specification Mo. 257/34. Solvent systems V, VI, VII and VIII can be used as solvents. The best separation then can be achieved if solvent mixture V, VI or VIII is used for eluting the Lobar coloumn, while solvent mixture VII is the most prefer¬ able for eluting the coloumn filled with silica gel impregna ed with metal salt. Fractions of the same volume are collected by a constant flow rate and the composition thereof is controll¬ ed by thin layer chromatography. The fractions containing the substance of the same R -. value are combined and the individual components are recovered by solvent precipita¬ tion. a r e s u It of s parα i_r. by coloumn chromatography the following components were isolated: :hinopricin /R, = 0.__3/ ~ 2 = metipricin /?. , = Q.22/ ' "1 = oxicricin /R , =0.35/ C o imioricin /R oxir.e ipricin / f =0.39/ 3- 1 = n. ' -•!■ ■ - i η / -n - . Ό hi ioricin /R. L-.-.0.56/ 3m = himetipricin /R p =0.5δ/ The separated components were tested and the follow¬ ing results were obtained: 1. A, = chinopricin /R~ = 0.29/ Molar weight measured by fast atom bombardment mass spectro etry method /FA3-MS/ is 1078 /Figure 3/. The isolated component A, was found to be chemically uniform by analysing with thin-layer chromatography and FA3-MS. Its structure determined on the basis of the molar weight, of the IR and of the 1H and 13C NMR one- and two dimensional spectras is represented by formula I. 2. A. = midopricin /R- = 0.31/ and A-, = methipricin /R f = 0.33/ Molar weight /FAB-MS/ 1092 and 1106. On the basis of the molar weight, of chemical method and of H and C NMR one and two dimensional spectroscopic examinations. It was found that components A~ and A~ are the homologues of component A, , the side chain of ~ is longer with a ethylene group, while the side chain of A. is longer with an ethylene group than that of A, . C-, = oxypricin /R f 0.35/ Molar weight: 946 /FAB-MS/ On the basis of the above analytical methods compound C, can be represented by the following formula II. /II/ The difference between comp ~ onent C1, and A .. is that instead of the sugar moiety a hydroxyl group is bound to the carbon atom being in position 13. 4.C 2 = oxy ipricin /R~ = 0.37/ and C-, = oxy etipricin /R-=0.39 Molar weight /FA3-MS/: 960 and 974. Cn the basis of the above analytical methods it was found that the side chain of component C, is longer with a methylene group, while the side chain cf C-, is longer with an ethylene group than that of C, . 5.3, = hydropricin /R- = 0.54/ Molar weight /FAB-MS/: 930. On the basis of the above analytical methods the compound can be represented by the formula III wherein instead of the sugar moiety a hydrogen is bound to the carbon atom being in position 18. 3,, = hymipricin /R f = 0.56/ and B, = hy etipricin /R-. = 0.53/ Molar weight /FA3-MS/: 944 and 960. Gn the basis of the above analytical methods it was found that the side chain of ccmponent 3_ is longer with a methylene group and the side chain of component B, is longer with an ethylene group than that of 3,. The usual physical constants /melting point, elemental analysis data, optical activity, 'V and IR specra, etc./ are not characteristic for the individual compounds. E.g. the melting point of the single component,; is 160 to 5 170 °C, the melting point of their mixture is 167-168 °C. The elemental analysis data are also not characteristic for the individual components as they are of similar structure, high molar weight, therefore the differences in the elemental analysis data are in the range of measuring 10 error. The main part of the primycin mixture comprises components A, , 3_ and C-, therefore they are .called as major components. The other components being present in smaller amount are called as minor components. The primycin components were isolated in the form 15 of sulfate or other salts different from sulfate, prefer¬ ably acetate or onochlorcacetate depending on the starting primycin mixture. The sulfate salts of the isolated primycin components can be transformed into other salts by the aid of different 20 organic or inorganic acids as it is described in * the Hungarian patent application :.c. 2571/84. The spectra of the antimicrobial activity o the primycin mixture and the three minor compone s /A, , 3. and C-,/ . is shown by Table 0 /see later on pa-^e 1 . . / . _ ~ ~ _< The table contains the MIC values measured on human pathogenic polyresistant strains. The different values refer to the fact that the three major components probab¬ ly exert their antimicrobial activity in a different manner, they attack different points of the microorganism. 30 Surprisingly we found that the components strengthen each other's effect, i.e. they show potenciorative synergysm. On the basis of the synergistic effect and the supposed different attacking point of the major components the following advantageous effects can be 35 achieved by using the composition of the components: 1. By simultaneously attacking the metabolism of the pathogens on different points the "cid-effect" i.e. the perishing of the microorganism can be attained more safely which is much more preferable than the staticus effect, i.e. the simple inhibition of the growth of the microorganisms. 2. If different routes of metabolism are simultaneou ly attacked, the resistance against the active ingredient combination can not develop or slowly develop. 3. The amount of the individual components used can be highly decreased. It results in the advantage that the harmful side-effects diminish or completely disappeare due to the administration of less active ingredient. This ' effect is very advantageous from thereapeutical points of view. When less active ingredient is used the expenses also decrease, which is favourable from economical points of view. In the synergistic composition according to our invention the major components /A-,, B,, C,/ and other, minor components of primycin and derivatives thereof preferably salts can be used. A composition can comprise two or three or more different components. If two major components are used in the composition the weight ratio of the components is in the range of 5 : 95 to 25 : 75, while if three components are used, then the weight ratio of the components is in the range of 4 : 3 : 3 to 7 : 1 : 2. The preferred embodiments of the synergistic compositions according to the invention are illustrated by the following examples, wherein the MIC values measured by using different microorganism strains and the degree of the additive and synergistic effect are also indicated. Under the term "additive effect" that is understood that the effect of the components is summarize thus e.g. by adding the half of the MIC value of the one component to the half of the MIC value of the second component, the MIC value of the combination of the two active ingredients is obtained. When the antimicrobial activity exceeds the additive effect due to the favourable interaction of the components, potentiorative synergis appears and the effect thus obtained is the synergistic effect. The MIC values were determined in Difco-3ouillon culture medium. The evaluation was carried out after incubating at a temperature of 37 °C for 24 hours, and 5 . 10 /ml spores were inoculated. In Examples 13 to 25 the 197 cases expressively illustrate that the composition containing more active ingredients is of synergistic activity, thus only a part or a small portion of original quantity of the active ingredients has to be administered in order to achieve the -same effect. The primycin components accoring to the invention can be administered alone cr mixed with each other, optionally together with other pharmaceutical active ingredients. They can be administered alone, but are generally administered with a pharmaceutical carrier selected on the basis of the route of adminis ration and standard pharmaceutical practice. The active ingredient can be administered in solid dosage forms, such as capsules, tablets, dragees, suppositories, semi-solid forms such as ' ointments cr gels cr liquid dosage forms such as injectable solutions, suspensions or syrups. The preferred dosage forms are the gels, ointments, surgical dusting powders, injec¬ tions, suspensions and combinations of powder a poulles- solvent a poulles. The compositions according to our invention can be administered orally, parenterally, rectally or topically /e.g. ointments/. They contain usual pharmaceutical carriers /e.g. magnesium carbonate, magnesium stearate, starch, talc, water, etc./ or novel carriers, e.g. cyclodextrine and optionally excipients, disintegrating agents, emulsifiers etc. The dosage forms suitable for oral administration may be e.g. tablets, dragees or capsules. The synergistic compositions containing more active ingredient according to the invention can be also used in veterinay therapeutics e.g. in the form of a liquid mixed to the feedstuff. The dosage froms suitable for parenertal administration can be aqueous emulsions, solutions or suspensions. Dusting powders, ointments, aqueous or oily emulsions or bandages impregnated with these emulsions or suspensions or sprays can be used for topical administration. The invention is illustrated by the following, non-limiting examples. - iό - Example 1 Separation of primycin on Kieselgel by thin-layer chromatography by double development 0.5 % solution of primycin-complex is prepared with a 1:1:2 mixture of n-butanol, et_anol and water or methanol. 10 .ul /50 ,ug of active Ingredient/ of the solution is dropped onto the Kieselgel plates /DC- -Plastikfolie, Kieselgel 60 - 5 ^ or DC-Alufolie Kieselgel 60, produced by Merck, layer thickness: 0.2 mm/. Solvent system IV Is used as eluent and the chromatogram is developed for a distance of 16 cm. The development is repeated. The spots are visualized by a colour reaction and the evaluation is carried out by densitometry at 590 n . The biological activity is detected by bioautographic assay. The composition of the culture medium used in the course of the bioautographic assay is as follows: Beef extract /Difco/ 3 g 3acto pepton /Difco/ 5 g Sodium chloride 5 g Disodium hydrogenphosphate 10 g Potassium dihydrogenpho≤phate 1 g 3acto agar /Difco/ 13 g Distilled water to 1000 ml The culture medium is incubated at 121 °C thereafter the pH Is adjusted to 3.0. 130 ml of culture medium of a temperature of 53 °C are filled to sterile glass dishes /18 x 25 cm/ and 3acillus subtilis ATCC 6633.spore suspension is added in a ratio of 1 : 2,000. Stripes of 5 mm width are cut from the chromatograms, dried solvent-free and superposed to the agar plate and incubated for 18 to 24 hours at a temperature of 37 °C. Example 2 Separation of the primycin mixture on Kieselgel by single thin-layer chromatographic development The procedure of Example 1 is followed except th solvent mixture V is used as eluent therefore the development is carried out once. Example 3 Separation of the primycin mixture on HPTLC plat with single development 4 ,ul /20 ,ug active ingredient/ of the 0.5 % solution prepared according to Example 1 is applied onto a HPTLC /Merck/ plate and developed for a distance of- 18 cm. The further steps are the same as described in Example 1. Example 4 Separation of primycin mixture on impregnated Kieselgel plate with single development The procedure of Example 1 is followed except that solvent mixture VII is used for the development and the Kieselgel 60 2 --, plate is impregnated with a metal salt according to the method described hereinabove before use. The further steps are the same as described in Example 1. Example 5 Separation of the primycin mixture on preparative thin layer chromatographic plate 2 ml of the 1 % solution of the sample to be - 13 - examined is applied onto a ?3C-Fertigplatten, Kieselgel 60 F- CΛ /Merck/ plate.. The plates are developed by solvent system IV more times. If a 3-3 cm wide stripe on the left and the right side of the glass plates is plunged into re-heated Sakaguchi reagent, the spots appeared as vivid red spots at room temperature. The distinct fractions are scraped off and eluted In a micro- percolator with 10 o acetic acidic methanol until no reaction can be observed upon adding sulphuric acidic vanilline. The eluate is distilled in vacuo, the dry residue is dissolved in solvent system I and extracted with distilled water of fifth part volume four times. The components of the antibiotic are in the upper layer, while the "layer dead weight" is in the lower phase. The components obtained by evaporating the " upper phase are further purified according to a method of Example 1. Example 6 Separation of the components "A" of primycin by partition coloumn cnromatography Solvent system used: stationary phase: solvent systeml mobile phase: the lower phase of a 30:10:60 mixture of n-butanol, glacial acetic acid and water Preparation of the coloumn: 720 g of Sephadex LH-20 gel is swollen in 3 1 of solvent system I for 1 day. The suspension is filled to a chromatographic coloumn /58 x 1150 mm/, settled, thereafter washed with 1.5 1 of mobile phase. Chromatographic procedure: 2 g of primycin are dissolved in a mixture of 300 ml of mobile phase and 15 ml of stationary phase, the solvent is filtered and applied onto the coloumn. The elution is carried out by the aid of the mobile phase with a flow rate of 50 to 60 drops/minute. Generally fractions of 17 ml volume /=800 drops/ are collected and the elution is proceeded until 300 to 400 fractions are obtained. The fixed phased Sephadex LH-20 coloumn can be used up for 3 to 4 times if the phase is changed. The gel can be regenerated as follows: it is suspended in acetone, filtered, refluxed for 2 hours with acetone in the vapour space thereafter dried. The substances being in the different fractions are identified -by thin-layer chromatography and UV spectroscophy. Isolation of the components: The fractions which are found to comprise the same substance on the basis of the analysis data /UV and TLC/ are combined. The residue obtained after evaporation of the solvent is dissolved in a 2:1 mixture of methanol and benzene and evaporated again. Thus 743 mg- of a mixture of the components A and a mixed fraction of 570 mg comprising components A and C are obtained. Components B remain on the coloumn in this system, they can be dissolved by solvent system I, 480 mg of components B can be obtained. A further chromatography of component mixture A under the above conditions results in major component A, in a purity higher than 98 %. Example 7 Separation of components B of primycin by partitio coloumn chromatography The components B obtained as described in Example 6 are further purified with a chromatographic method described hereinabove except that the so-called "3-selective" solvent system is used as eluent. Thus major component B, can be obtained with a purity higher than 96 %. The composition of the "3-selective" solvent system is as follows: stationary phase: the upper phase of a 1:41:10:5:43 mixture of benzene, n-butanol, methanol glacial acetic acid, water, mobile phase: the lower phase of a 1:24:10:5:60 mixture of benzene, n-butanol, methanol, glacial acetic acid and water Example 8 Separation of the components of primycin by DCCC /Droplet Counter Current Chromatography/ The mixed fractions obtained in Example 6 are further purified by DCCC. The solvent system used is as follows: stationary phase: the lower phase of a 26:39:9:26 mixture of chloroform, methanol, glacial acetic acid and water, mobile phase: the upper layer of the same mixture Elation: The solvent system is stirred for 2 hours, left to stand for 16 hours and the phases are separated. The DCCC instrument /ID=2.0 mm, 300 tubes/ is filled with the lower phase /stationary phase/. 130 mg of the substance to be examined are dissolved in 12 ml of upper phase /mobile phase/, filtered and pumped to the container. The elution is carried out by the aid of the upper phase, under an overpressure of 1.7 to 1.8 atm with a flow rate of 15 ml/hour and fractions of 3.7 ml volume /=220 drops/ are collected. Thus major component C, is obtained in a purity of 90 % . 2 ϊ Example 9 Separation of the components of primycin by adsσrptive coloumn chromatography on silica gel impregnated with a metal salt Preparation of the coloumn: In a solution of 40 g of silver nitrate and 500 ml of acetonitrile 200 g of Kieselgel HF ,-, silica gel are suspended thereafter the solvent is evaporated in vacuo. The impregnated silica gel is suspended in 500 ml of chloroform and filled into the chromatographic coloumn. In order to promote the settling and the compaction a slight overpressure /l.l to 2.5 atm/ is used until the height of the packing bed settles. In order to avoid the reduction of silver nitrate due to light the coloumn is covered with aluminium foil. Chromatographic procedure: 400 mg of primycin are dissolved in 12 ml of solvent system VII freshly prepared. The solution is adsorbed by the aid of slight overpressure /l.l atm/ onto the packing bed. 30 ml of eluent /solvent system VII/ are applied onto the top of the packing and the elution is started with a flow rate of 1.1 ml/minute under an overpressure of 1.5 to 2.5 atm. Working up: The substances being in the fractions are identified by thin-layer chromatography. 10 ,ul of each fractions are applied to the starting points of Kieselgel 60 p ,-, /Merck/ plates and developed for a distance of 16 cm in an unsaturated chamber with solvent system V. The spots are located by development with chlorotolidin reagent and/or sulfuric acid and/or heat treatment after drying with cold air. The fractions comprising the same substance are combined and the primycin component is precipitated by adding ether. The solid substance precipitated is filtered off and washed with some acetonitrile and ether. The residue Is dissolved in the upper layer of a 4:1:5 mixture of n-butanol, acetic acid and water and washed with 5 ml of water three times. The solvent is evaporated from the substance being in the separated upper layer under vacuo, the residue is triturated with ether, filtered and dried under vacuo. Thus 135 mg of component A, = chinopricin, 30 mg of component A- = midoprxcin, 40 mg of component A-. = metipricin, 20 mg of component C, = oxypricin, 20 mg of component B, = hydroprrcin are obtained. Remark: the major part of the silver nitrate being on the coloumn protected from light can be recovered by washing with acetonitril and used up again. Example 10 Separation of the components of primycin by absorption coloumn chromatography en ready-made Lobar coloumn The coloumn used: Lobar C /Merck/ coloumn filled with LiChroprep, preferably two or more colourans are joined. Chromatographic method: The procedure of Example 9 is followed except that solvent system VIII is used as eluent. 3400 mg of primycin are dissolved in 40 ml of solvent system VIII, thereafter this solution is applied onto the top of a coloumn system containing four coloumns connected in series. Working up: The fractions comprising the same substances are combined, r-butanol in a quantity corresponding to 10 % of total volume of the fractions is added and the solutions are evaporated in vacuo at a maximal temperature of 45 C. The solid residue is triturated with ether and filtered. Thus 1100 mg of component A, = chinopricin, 110 mg of component A ? = midopricin, 80 mg of component A., = metipricin, 110 g of component C, = oxypricin, 20 mg of component C~ = oxymipricin, 15 mg of component Cm = oxymetipricin, ' 150 mg of component B, = hydropridin, ' 50 g of component B p = hymiprlcin, 40 g of component B_» = hymetipricin are obtained. Example 11 Preparation of the sulfate salts of the components of primycin The procedure of Example 10 is followed except that 5 ml of methanol and 0.3 ml of IN sulfuric acid are added to the combined fractions. The precipitation is completed by adding 300 ml of ether. Thus the primycin components of Example 10 are obtained in the form of sulfate salt. Example 12 Preparation of the salts of the components of primycin different from sulfate From the sulfate salts of the components of primycin other salts different from the sulfate salt are prepared by adding barium salts according to the process of Hungarian patent application No. 2571/84. Thus the salts of the primycin components different from sulfate are obtained in the form of e.g. formiate, acetate, raonochloroacetate, oxalate, benzoate, tosilate, perchlorate, chloride etc. The antimicrobial activity of the primycin complex and the components thereof Is tested on different micro¬ organism strains. The results are listed in Table 0. Table 0 The antimicrobial spectra of the major components of primycin /Inoculation: 5 x 10 5/ml, incubation at 37 Co for 24 hours/ Human pathogenic polyresistant MIC. /,ug/ml/ strains Major Components 1./ Staphylococcus aureus CCM.335. 0.25 0.075 o 2./ Staphylococcus aureus DSM.20231 0.05 0.05 1 3./ Staphylococcus aureus CCM.2317. 0.25 0.05 1 4./ Staphylococcus aureus CCM.2326. 0.25 0.05 25 5./ Staphylococcus aureus CCM.2514 0.25 0.05 1 6./ Staphylococcus aureus CCM.2515. 0.25 0.025 1 7./ Staphylococcus epidermidis 0.75 0.025 25 CCM.2271 8./ Staphylococcus aureus Smith 0.25 0.05 0.5 9./ Streptococcus faecalis CCM.1875. 2.5 0.75 2.5 10./ Streptococcus agalactiae CCM.5153. 0.75 0.075 0.5 11./ Streptococcus agalactiae CCM.5534. 1.0 0.25 1.0 12./ Streptococcus disgalactiae ATTC.9926 2.5 0.5 2.5 /Contn. of Table 0/ Human pathogenic polyresistant MIC. / /U /ml/ strains Major Components A- B, 13./ Bacillus subtilis ATTC.6633. 0.25 0.025 0.1 14./ Bacillus cereus CCM.2010. 0.25 0.025 0.25 15./ Bacillus licheniformis CCM.2182. 0.25 0.025 0.25 16./ Bacillus licheniformis CCM.2205. 0.25. 0.025 0.25 17./ Bacillus subtilis CCM.1718. 0.25 0.05 0.25 18./ Listeria monocytogenes CCM.5576. 0.5 0.075 0.25 19-/ Micrococcus flavus ATCC.10240 . 0.075 0.01 0.05 20./ Micrococcus luteus DSM.20030. 0.05 0.01 0.075 21./ Sporosarcina ureae DSM.317 0.025 0.01 0.025 22./ Pseudomonas aeruginosa CCM.1960. 25 25 25 23./ Pseudomonas fluorescens CCM.2115. 25 25 25 24./ Pseudomonas acidoverans CCM.283 25 25 25 25./ Proteus vulgaris CCM.1799. 50 50 50 26./ Shigella sonnei CCM.1373. 50 50 50 27./ Salmonella typhimurium CCM.5445. 50 50 50 23./ Escherichia coli DSM.30038. 50 50 50 Table 0/a The antimicrobial spectra of the primycin antibiotic complex for human pathogenic microorganism strains Strains MIC / /Ug/ml / 1./ Bacillus subtilis ATCC.6633. 0.02 2./ 3acillus subtilis CCM.1718 0.1 3./ 3acillus cereus CCM.2010 0.1 4./ Bacillus licheniformis CCM.2182 0.075 5./ 3acillus licheniformis CCM.2205 0.075 6./ Staphylococcus aureus CCM.385. 0.25 "7 . / Staphylococcus aureus CCM.2317 0.1 3./ Staphylococcus aureus CCM.2514 0.25 9./ Staphylococcus aureus CCM.2515 0.25 10./ Streptococcus faecalis CCM.1875 0.5 11./ Streptococcus agalactiae CCM.5534. , 0.5 12./ Streptococcus dysgalactiae CCM.5548. 0.5 13./ Streptococcus agalactiae CCM.5153. 0.5 14./ Mycobacterium tuberculosis typ. hu H-, 7 Rv. 0.05 15./ Mycobacterium tuberculosis typ. bovin Ξ- 0.3-1.0 16./ Mycobacterium tuberculosis 607 0.5 17./ Micrococcus flavus ATCC.10240 0.075 18./ Pseudomonas aeruginosa CCM.1960. 25 19./ Pseudomonas fluorescens CCM.2115. 25 20./ Pseudomonas acidoverans CCM.283. 25 21./ Pseudomonas pictorum CCM.284 10 22./ Proteus vulgaris CCM.1799. 25 " 23./ Shigella sonnei CCM.1373. 25 24./ Salmonella typhi-murium CCM.5445. 50 25./ Salmonella cholerae-sus Inst. CCM.5438 50 Pasteur strain 2 1 - /Contn. of Table 0/a/ Strains MIC / / ug/ml / 26./ Escherichia coli DSM.30033 50 27./ Escherichia haemolyticus CCM.5863 50 28./ Escherichia coli /ATCC11775, CCM.5172 50 cystitis/ 10 29./ Escherichia coli lysegenic, CCM.130 50 colicinogenic 30./ Escherichia coli OTKI.17963 25 31./ Escherichia coli OTKI.23473 50 32./ Klebsiella pneumoniae CCM.1843 50 15 33./ Serratia Marcescens CCM.303 25 34./ Pasteurella multocida CCM.5419 25 35./ Vibrio parahaemolyticus CCM.5938 25 36./ Candida albicans CBS.562 25 20 37./ Candida tropicalis CBS. 33 25 38./ Candida pseudotropicalis 372 5 39./ Candida krusei K-47 10 40./ Cryptococcus neoformans -lό 5 41./ Saccharomyces cerevisiae OKI.1232 10 __ Example 13 • Synergistic composition No. I 30 n /the number of the components/ = 2 the ratio of the components : A, 3 1 = 75:25 The results are summarized in Table I Explanation of the table: Coloumn I : the MIC value of component A, / ,ug/ml /, 5 Coloumn II : the MIC value of component 3, - 23 - Coloumn III the MIC value of the composition comprising components A, and B, Coloumn IV the amount of A, being in the composition related to the MIC value of A 1 /%/ Coloumn V the amount of B, being in the composition related to the MIC value Of B- j^ /%/ Coloumn VI /IV + V//n Coloumn VII the additive portion of the antimicrobia activity /IV + V/ Coloumn VIII the synergistic portion of the antimicrobial activity /100 - VII/ Table I The sign Experimental result;s of the strains examined I II III IV V VI ι/II VIII 1 0.25 0.5 0.2 60 10 35 70 30 ' 6 0.25 0.1 0.06 18 15 16.5 33 67 8 0.25 0.5 0.2 60 10 35 70 30 10 0.75 0.5 0.4 40 20 30 60 40 11 1 1 0.8 60 20 35 80 20 12 2.5 2.5 1 30 10 20 40 60 13 0.25 0.1 0.08 24 20 22 44 56 14 0.25 0.25 0.1 30 10 20 40 60 15 0.25 0.25 0.08 24 8 16 32 68 16 0.25 0.25 0.08 24 8 16 32 68 17 0.25 0.25 0.05 12 5 8.5 17 83 18 0.5 0.25 0.2 30 20 25 50 50 19 0.075 0.05 0.02 20 10 15 30 70 Example 14 Synergistic composition No. II n = 2 the ratio of the components: A-, : 3, = 50:5 The antimicrobial activity of the composition is summarized in Table II Table II - The sign of the strains I II III IV V VI VII VIII examined 1 0.25 0.5 0.2 40 20 30 60 40 . 0.25 0.25 0.08 16 16 16 32 63 5 0.25 0.1 0.08 16 40 28 DO 4 6 0.25 0.1 0.04 8 20 14 23 72 7 0.75 0.25 0.06 4 12 8 16 34 3 0.25 0.5 0.2 40 20 30 60 40 9 2.5 2.5 1 20 20 20 40 60 10 0.75 0.5 0.03 5.3 3 ό.6 13.3 36.7 11 1 1 0.6- 30 30 30 60 40 12 2.5 2.5 0.3 16 16 16 32 63 13 0.25 0.1 0.03 16 40 23 56 44 14 0.25 0.25 0.1 20 20 ' 20 40 60 15 0.25 0.25 0.06 12 12 12 24 76 16 0.25 0.25 0.06 12 12 12 24 76 17 0.25 0.25 0.05 10 10 10 20 80 19 0.075 0.05 0.03 20 30 25 50 50 20 0.05 0.075 0.03 30 20 25 50 50 Example 15 Synergistic composition No. Ill n = 2 the ratio of the components: A. : B, = 25:75 The experimental results of the antimicrobial activity are summarized in Table III. /The abbreviations are the same as in Table I./ Table III - The sign of the strains I II III IV V VI VII VIII examined 1 0.25 0.5 0.2 20 30 25 50 50 4 0.25 0.25 0.1 25 30 27.5 55 45 6 0.25 .0.1 0.03 3 22.5 11.7 25.5 74.5 l 0.75 0.25 0.1 3.3 30 16.7 33.3 66.7 3 0.25 0.5 0.2 20 30 25 50 50 9 2.5 2.5 1 10 30 20 40 60 10 0.75 0.5 0.2 6.7 30 18.4 36.7 63.3 11 1 1 0.6 15 45 30 60 40 13 - . 25 0.1 0.05 5 37.5 21.2 42.5 57.5 14 Z . 25 0.25 0.1 10 30 20 40 60 15 0.25 0.25 0.03 8 24 16 32 63 16 0.25 0.25 0.08 8 24 16 32 63 17 0.25 0.25 0.06 10 18 12 24 76 19 0.075 0.05 0.03 10 45 27.5 55 45 20 0.05 0.075 0.03 15 30 22.5 45 55 Example 16 Synergis t ic composi tion No . IV n = 2 the ratio of the components: '1 ' "1 75:25 The results relating to the antimicrobial activity of the composition are summarized in Table IV. /The abbreviations are the same as in Example 13 except that component C, is used instead of 3,/ Table IV The sign of the strains I II III IV V VI VII VIII examined 0.25 0.075 0.03 9 10 9.5 19 31 . 2 0.05 0.05 0.03 45 15 30 60 40 3 0.25 0.05 0.02 6 10 8 16 34 4 0.25 0.05 0.03 9 15 12 24 76 5 0.25 0.05 0.C4 12 20 16 32 63 o * - 0.25 0.025 0.02 0 20 13 26 74 7 0.75 0.025 0.01 1 10 5.5 11 39 8 0.25 0.05 0.01 3 5 . 8 92 9 2.5 0.75 0.8 24 26. ι 6 25.3 50.6 49.4 10 0.75 0.075 0.03 3 10 6.5 13 37 11 1 0.25 0.2 15 20 17.5 35 65 12 2.5 0.5 0.4 12 20 16 32 68 13 0.25 0.025 0.02 6 20 13 26 74 14 0.25 0.025 0.03 9 20 13 26 74 15 0.25 0.025 0.03 9 30 19.5 39 61 16 0.25 0.025 0.03 9 30 19.5 39 61 17 0.25 0.05 0.03 9 15 12 24 76 18 0.5 0.075 0.06 9 20 14.5 29 71 19 0.0750.01 0.02 20 50 35 70 30 20 0.05 0.01 0.02 30 50 40 80 20 Example 17 Synergistic composition No. V n = 2 the ratio of the components 1 :C 1 = 50:50 The results relating to the antimicrobial activity of the composition are summarized in Table V. /The abbreviations are the same as in Example 13 except that component C. is used instead of 3./ Table V The sign of the strains I II III IV V VI VII VIII examined 1 0.25 0.075 0.04 3 26. 6 17. .3 34. 6 63.4 2 0.05 0.05 0.02 20 20 20 40 60 3 0.25 0.05 0.02 4 20 12 24 ( ' O 4 0.25 0.05 0.02 4 20 12 24 76 0.25 0.05 0.02 4 20 12 24 76 o r 0.25 0.025 0.01 2 20 11 22 73 7 0.75 0.025 0.02 1.3 40 20.o _- 41. 3 53.7 8 0.25 0.05 0.01 2 10 6 12 38 10 0.75 0.075 0.02 1.3 13. 3 7. 3 14. 6 33.4 12 2.5 0.5 0.6 12 60 36 72 23 13 0.25 0.025 0.02 4 40 22 44 56 14 0.25 0.025 0.02 4 40 22 44 56 15 0.25 0.025 0.02 4 40 22 44 56 16 0.25 0.025 0.02 4 40 22 44 56 17 0.25 0.05 0.04 8 40 24 48 52 18 0.5 0.075 0.1 10 66. .6 38. ,3 76. 6 23.4 o o Example 18 Synergistic composition No. VI n = 2 the ratio of the components = A. : C, = 25:75 The results relating to the antimicrobial activity of the composition are summarized in Table VI. /The abbreviations are the same as in Example 13 except that component C, is used instead of B,/ Table VI The sign of the strains I II III IV V . VI VII VIII examined 3 0.25 0.05 0.05 5 75 40 30 20 2 2 . 5 0 . 5 0 . 6 6 90 48 96 4 Example 19 Synergistic composition No. VII n = 2 the ratio of the components: C, : 3, = 75:25 The results relating to the antimicrobial activity of the composition are summarized in Table VII. /The abbreviations are the same as in Example 13 except that component C, is used instead of component A, ./ Table VII The sign of the strains I II III IV V VI VII VIII examined 1 0.075 0.5 0.04 40 2 21 42 58 2 0.1 0.5 0.02 15 1 8 16 34 3 0.05 0.1 0.02 30 5 17.5 35 65 4 0.05 0.25 0.03 45 3 24 48 52 5 0.05 0.1 0.03 45 7.5 26.3 52.5 47.5 6 0.025 0.1 0.02 60 5 32.5. 65 35 3 0.05 0.5 0.02 30 1 15.5 31 69 10 0.075 0.5 0.06 60 3 31.5 63 37 11 0.25 1 0.2 60 5 32.5 65 35 12 0.5 2.5 0.04 6 0.4 3.2 6.4 93.6 17 0.05 0.25 0.02 30 2 16 32 68 Example 20 Synergistic composition No . VIII n = 2 the ratio of the components: C-,:3. = 50 : 50 The results relating to the antimicrobial activity of the composition are summarized in Table VIII. /The abbreviations are the same as in Example 13 except that component C, is used instead of component A../ Table VIII The sign of the strains I II III IV V VI VII VIII examined 1 0.075 0.5 0.08 53.3 8 30.6 61.3 38.7 2 0.05 0.1 0.03 30 15 22.5 45 55 3 0.05 0.1 0.03 30 15 22.5 45 55 4 0.05 0.25 0.03 30 6 18 36 64 5 0.05 0.1 0.03 30 15 22.5 45 55 6 0.025 0.1 0.02 40 10 25 50 50 7 0.025 0.25 0.02 40 4 22 44 66 8 0.05 0.5 0.03 30 3 16.5 33 67 9 0.75 2.5 0.8 53.3 16 34.6 69.3 30.7 10 0.075 0.5 0.1 66.6 10 33.3 76.6 23.4 11 0.25 1 0.06 12 3 7.5 15 85 12 0.5 2.5 0.03 3 0. 6 1.8 3.6 96.4 13 0.025 0.1 0.02 40 10 25 50 50 15 0.025 0.25 0.02 40 4 22 44 66 16 0.025 0.25 0.02 40 4 22 44 66 17 0.05 0.25 0.02 20 4 12 24 76 18 0.075 0.25 0.08 53.3 10 34.6 69.3 30.7 Example 21 Synergistic composition No. IX n = 2 the ratio of the components: C, : B, = 25:75 The results relating to the antimicrobial activity of the composition are summarized in Table IX. /The abbreviations are the same as in Example 13 except that component C, is used instead of component A,./ Table IX The sign of the strains I II III IV V VI VII VIII examined 1 0.075 0.5 0.1 33.3 15 24.1 48.3 51.7 2 0.05 0.5 0.04 20 6 13 26 74 3 0.05 0.1 0.04 20 30 25 50 50_ 4 0.05 0.25 0.08 40 8 24 48 52 6 0.025 0.1 0.02 20 15 17.5 35 65 7 0.025 0.25 0.03 30 9 19.5 39 61 8 0.05 0.5 0.06 30 9 19.5 39 61 11 0.25 1 0.4 40 30 35 70 30 12 0.5 2.5 0.1 5 3 4 8 92 13 0.025 0.1 0.02 20 15 17.5 35 65 14 0.025 0.25 0.03 30 9 19.5 39 61 15 0.025 0.25 0.03 30 9 19.5 39 61 16 0.025 0.25 0.03 30 9 19.5 39 61 17 0.05 0.25 0.03 15 9 12 24 76 18 0.075 0.25 0.1 33.3 30 31.6 63.3 36.7 19 0.01 0.05 0.02 50 30 40 30 20 20 0.01 0.075 0.02 50 20 35 70 30 Example 22 Synergistic composition No . X n = 3 the ratio of the components "1 ' w l B L = 40:30:30 The results relating to the antimicrobial activity of the composition are summarized in Table X. The abbreviations of Table X are as follows: Coloumn I MIC value of component A, / .ug/ml / - J ( - Coloumn II MIC value of component C 1, / /.ug°/ml / Coloumn III MIC value c_ " component B, / ,ug/ml / Coloumn IV MIC value of the composition comprising components A, + B, + C, Coloumn V the amount of A, being in the composition related to the MIC value of component A. /%/ Coloumn VI the amount of C, being in the composition related to the MIC value of component C. /%/ Coloumn VII the amount of component 3, being in the composition related to the MIC value of component B. /%/ Coloumn VIII /V+VI+VII/ /n Coloumn IX the additive portion of the antimicrobial activity /V+VI-t-VII/ Coloumn X the synergistic portion of the antimicrobial activity /100 % - IX/ Table X The sign of the strains I II III IV V VI VII VIII IX examined 1 0.25 0.075 0.5 0.1 16 40 6 20.6 62 38 2 0.05 0.05 0.1 0.05 40 30 15 28.3 35 15 5 0.25 0.05 0.1 0.05 8 30 15 17.6 53 47 6 0.25 0.025 0.1 0.03 4.336 9 16.6 49.8 50 7 0.75 0.025 0.25 0.05 2.660 6 22.8 68.631.4 8 0.25 0.05 0.5 0.08 12.848 4.321.8 65.634.4 10 0.75 0.075 0.5 0.08 4.232 4.813.6 41 59 11 1 0.25 1 0.08 3.2 9.6 2.4 5.1 15.284.8 12 2.5 0.5 1 0.6 9.636 7.217.6 52.347.2, 13 0.25 0.025 2.5 0.3 4.836 9 16.6 49.350.2 - 33 - /Contn. of Table X/ The sing of the strains I II III IV V VI VII VIII IX examined 14 0.25 0.025 0.1 0.03 4.3 36 3.6 14.3 44.4 55.6 15 0.25 0.025 0.25 0.03 4.3 36 3.6 14.8 44.4 55.6 16 0.25 0.025 0.25 0.05 3 60 6 24.6 74 26 17 0.25 0.05 0.25 0.02 3.2 12 2.4 5.8 17.6 82.4 20 0.05 0.01 0.0750.01 8 30 4 14 42 58 Example 23 Synergistic compositicn Mo. XI n = 3 the ratio of the components: A : Z : 3 = 50:25:25 The results relating to the antimicrobial activity of the composition are summarized in Table XI. /The abbreviations of the table are the same as in Example 22./ - j y - Table XI The sign of the strains II III IV VI VII VIII IX examined 1 0.250.075 0.5 0.1 20 33.3 5 19.4 58.341.7 3 0.250.05 0.1 0.08 16 40 20 25.3 76 24 4 0.250.G5 0.25 0.1 20 50 10 26.6 30 20 5 0.250.05 0.1 0.04 8 20 10 12.6 38 62 6 0.250.025 0.1 0.04 8 40 10 19.3 58 42 7 0.750.025 0.25 0.05 3.350 5 19.4 58.341.7 8 0.250.05 0.5 0.05 10 25 2.5 12.5 37.5 62.5 9 2.5 0.75 2.5 1 20 33.3 10 21.1 63.336.7.- 10 0.750.075 0.5 0.1 6.633.3 5 14.9 44.955.1 11 1 0.2 1 0.4 20 40 10 23.3 70 30 12 2.5 0.5 2.5 0.4 8 20 4 10.6 32 68 13 0.250.025 0.1 0.04 8 40 10 19.3 58 42 14 0.250.025 0.25 0.C6 12 60 6 26 78 22 15 0.250.025 0.25 0.03 6 30 3 13 39 61 16 0.250.025 0.25 0.05 10 50 5 21.6 65 35 17 0.250.05 0.25 0.05 10 25 5 13.3 40 60 13 0.5 0.075 0.25 0.08 3 26.6 8 14.2 42.6 57.3 19 0.075 αoi 0.05 0.0213.350 10 24.4 73.326.7 20 0.C50.01 0.075 0.0220 50 6.6 25.5 76.623.4 «-. Example 24 _> Synergistic composition No . XII n = 3 the ratio of the components: A : C : 3 = 60:20:20 The results relating to the antimicrobial activity of the composition are summarized in Table XII. /The abbreviations of the table are the same as in Example 22./ Tabli e XII The sign of the strains I II III IV V VI VII VIII IX X examined 1 0.25 0.075 0.5 0.08 19.2 21.3 3.2 14.6 43.7 56.3 4 0.25 0.05 0.25 0.1 24 40 8 24 72 28 5 0.25 0.05 0.1 0.04 9.6 16 7.3 11 32.9 67.1 6 0.25 0.025 0.1 0.03 7.2 24 6 12.4 37.2 62.8 7 0.75 0.025 0.25 0.05 4 40 4 24 48 52 . - 8 0.25 0.05 0.5 0.04 9.6 16 1.6 9.1 27.2 72.8 9 2.5 0.75 2.5 1 24 26.7 3 19.6 58.7 41.3 10 0.75 0.075 0.5 0.03 2.4 8 1.2 3.9 11.6 38.4 11 1 0.25 1 0.4 24 32 8 21.3 64 36 - 12 2.5 0.5 2.5 0.4 9.6 16 3.2 9.6 28.8 71.2 13 0.25 0.025 0.1 0.04 9.6 32 8 16.5 49.6 50.4 14 0.25 0.025 0.25 0.04 9 -6 32 3.2 14.9 44.3 55.2 15 0.25 0.025 0.25 0.05 12 40 4 18.6 56 44 16 0.25 0.025 0.25 0.04 9.6 32 3.2 14.9 44.8 55.2 17 0.25 0.05 0.25 0.04 9.6 16 3.2 9.6 28.3 71.2 19 0.075 0.01 0.05 0.02 16 40 3 21.3 64 36 20 0.05 0.01 0.075 0.02 24 40 5.3 23.1 69.3 30.7 Example 25 Synergistic composition No. XIII n = 3 the ratio of the components: A, : C, : B, = 70:10:20 The results relating to the antimicrobial activity of the composition are summarized in Table XIII. /The abbreviations of the table are the same as in Example 22./ Table XI II The sign of the strains I II III IV V VI VII VIII IX X examined 2 0.05 0.05 0.1 0.04 56 8 8 . 24 72 28 3 0.25 0.05 0.1 0.1 28 20 20 22.6 68 32 4 0.25 0.05 0.25 0.1 28 20 3 18.6 56 44 5 0.25 0.05 0.1 0.05 14 10 10 11.3 34 66 6 0.25 0.025 0.1 0.03 8.4 12 6 3.3 26.4 73.6 7 0.75 0.025 0.25 0.04 3.7 16 3.2 7.6 22.9 77.1 8 0.25 0.05 0.5 0.08 22.4 16 3.2 13.3 41.6 58.4 10 0.75 0.075 0.5 0.2 18.6 26.6 8 17.7 53.2 46.3 11 1 0.25 1 0.4 28 16 8 17.3 52 48 12 2.5 0.5 2.5 0.6 16.3 12 4.8 11.2 33.6 66.4 13 0.25 0.025 0.1 0.03 8.4 12 o 8.3 26.4 73.6 14 0.25 0.025 0.25 0.04 11.2 16 3.2 10.1 30.4 69.6 15 0.25 0.025 0.25 0.06 16.3 24 4.3 15.2 45.6 54.4 16 0.25 0.025 0.25 0.03 8.4 12 2.4 7.6 22.3 77.2 17 0.25 0.5 0.25 0.04 11.2 8 3.2 7.4 22.4 77.6 18 0.5 0.075 0.25 0.2 28 26.6 16 23.5 70.6 29.4 19 0.075 0.01 0.05 0.02 18.6 20 8 15.3 46.6 53.4 20 0.05 0.01 0.075 0.01 14 10 2.6 8.8 26.6 73.4 The following examples relate to pharmaceutical compositions comprising the individual components of primycine or the synergistic mixture thereof as active ingredient. The pharmaceutical compositions are prepared in a manner known per se. Under the term "active ingredient" the isolated components of primicin or a mixture thereof is understood Example 26 Dusting powder Mo. I Active ingredient 1.0 g Cyclodextrine 9.0 g 10.0 g Example 27 Dusting powder No. II Active ingredient 1.0 g Amylum ncn mucilaginosu 9-0 g 10.0 g Example- 23 Dusting powder No. Ill Active ingredient 1.0 g Aetheroleum lavandulae 0.02 g Acid silicicum colloidale 0.10 g Magnesium stearinicum 0.10 g Zincum oxidatum 0.20 g Bolus alba 0.50 g Magnesium carbonicum 1.00 g hydroxidatum Cyclodextrine 7.08 g 10.0 g Example 29 Sprayable dusting powder Active ingredient 0.20 g Isopropylmyristate 1.00 g Freon 11/12 5050 /carrier gas/ 93.80 g 100.00 g Example 30 Aqueous aerosol Active ingredient 0.5 g Ethanol /62.5 % by volume/ 10.0 g water 39.5 g 50.0 g 100.0 g Example 31 Sprayable όurgical plaster Active ingredient 0.1 g polyvinyl-pirrolidone 1.50 g ethanol /62.5 % by volume/ 48.40 g propellant gas 50.00 g Example 32 Gel Active ingredient 0.20 g Lidocain 2.00 g Chlorophill 0.005 g Menthol 0.20 g Carbopol 940 1.20 g Triethanolamine 1.50 g Tween 20 1.50 g Isoadipat 5.00 g Ethanol 96 % 50.00 g Aqua dest. ad 100.00 g The ingredients are mixed and filled into a flash Example 33 Ointment Active Ingredient 2.00 o Oleum paraffin! 33.0 g Vaseliήu album 31.0 s Ethanol 96 % 20.0 g Tween 60 9 .0 o σ Wool fat 5 .0 ε 100.0 g The ingredients are mixed and filled into a bottle or jar. Example 34 Eye-salve Active ingredient 0.04 g Ethanol 62.5 by volume 0.08 g Distilled water • 3.16 g Beeswax 300.00 g Colesterine 25.00 g Sterile castor oil to 1000.0 g. Example 35 Ointment washable by water No. 1 Active ingredient 2.0 g Tween 60 5.0 g Liquid paraffine 5.0 g Cetylstearylalcohol 15.0 g White vaseline 25.0 g Distilled water to 100.0 g Example 36 Ointment washable by water No. II Active ingredient 0.100 g Ethanol 62.5 % by volume 2.000 g Water 7.900 g Sorboxaethene stearate 3.600 g Liquid paraffine 3.600 g Cetylstearylalcohol 10.800 g White vaseline 18.000 g Propyl-paraoxybenzoate 0.054 g Methylparaoxybenzoate 0.126 g Ethanol, 96 % by volume 2.930 g Lidocainum chloride 1.000 Distilled water to 100.000 g Example 37 Eye-salve Active ingredient 0.02 g Sodium hydrogencarbonate 18.00 g Viscous solvent /4 g of ethylcellulose + 3.5 g of NaCl + distilled water ad 510 g/ 510.00 g Phenyl-mercury-borate, /0.1 %/ 15.1 g distilled water to 1000.0 og Example 38 Oily eye-salve Active ingredient 0.02 g Ethanol, 62.5 % by volume 0.40 g - 4 o - Distilled water 1.58 g Cholesterine 25.00 g Sterile castor oil to 100.00 g Example 39 Vaginal suppository Active ingredient 0.02 g Ethanol, 62.5 % by volume 0.262 g Gelatine 1.40 Sodium-acetate 0.26 g Glycerine 4.50 g Distilled water to 9-50 g Example 40 Antimicrobial bandage A solution of the isolated components of primicin or a mixture thereof is made preferably with ethanol and bandages, e.g. mull-sheet Is saturated with it, thereafter the bandage is packed and sterilized in a usual way. The sterile, antimicrobial bandage thus prepared can be used up at any time.

Claims: Patent Claims 1. The components of the primycin antibiotic complex. 5 2. Component A, of primycin /chinopricin/ of the formula I having an R f value of 0.29 developed on thin-layer chromatoplate with a combined solvent system and having a molar weight of 1078 determined by FAB-MS method. 20 3. Component A p of primycin /midopricin/, homologue of component A, having an R f value of 0.31 developed on thin-layer chromatoplate with a combined solvent system and having a molar weight of 1092 determined by FAB-MS method. ' . 4. Component A^ of primycin /methipricin/, homologue of component A, having an R f value of 0.33 developed on thin-layer chromatoplate with a combined solvent system and having a molar weight of 1106 determined by FAB-MS method. 0 5. Component C, of primycin /oxypricin/ of the formula II having an R f value of 0.35 developed on thin-layer chromatoplate with a combined solvent system and having a molar weight of 946 determined by FAB-MS method. 6. Component C p of primycin /oxyn-άpricin/, homologue of component C,, having an R~-value of 0.37 developed on thin-layer chromatoplate with a combined solvent system and having a molar weight of 960 determined by FAB-MS method. 7. Component C_ of primycin /oxymetiprlcin/, homologue of component C, , having an R- value of 0.39 developed on thin-layer chromatoplate with a combined solvent system and having a molar weight of 974 determined by FAB-MS method. 3. Component B, of primycin /hydropricin/ of the formula III having an R value of 0.54 developed on thin-layer chromatoplate with a combined solvent system and having a molar weight of 930 determined by FA3-MS method. 9. Component B p of primycin /hymipricin/, homologue of component B-, , having an R- value of 0.56 developed on thin-layer chromatoplate with a combined solvent system and having a molar weight of 944 determined by FAB-MS method. 10. Component B., of primycin /hyraetipricin/ homologue of component B., having an R f value of 0.58 developed on thin-layer chromatoplate with a combined solvent system and having a molar weight of 960 determined by FA3-MS method. 11. Process for the separation of the primycin antibiotic complex w h i c h c o m p r i s e s separating the components of the crude, semi-half or pure products obtained in the course of the preparation of primycin by fermentation, by once- or twice developing thin-layer-chromatography and/or coloumn chromatography with a combined solvent system and if desired isolating the individual components. 12. The process as claimed in Claim 11 h i c h c o m p r i s e s . using tnin-layer chromatography for the separation of the primycin components. 13. The process as claimed in either Claim 11 or Claim 12 w h i c h c o m p r i s e s dissolving the antibiotic complex to be separated in a 0.5 to 1.0 % mixture of alcohols of 1 to 4 carbon atoms and/or water and developing on a thin-layer- or containing high pressured thin-layer chromatoplate silica gel, preferably silica gel impregnated with a metal salt as solid carrier. 14. The process as claimed in Claim 11 w h i c h c o m p r i s e s using partition or adsorption coloumn chromatography for the separation of the primycin components. 15. The process as claimed either in Claim 11 or Claim 14 w h i c h c o m p r i s e s using a ready-to-use Lobar-type coloumn or a chromatographic coloumn packed with silica gel impregnated with a metal salt, preferably with silver and/or iron salt in course of the adsorption chromatography. 16. The process as claimed either in Claim 11 or Claim 14 w h i c h c o m p r i s e s using a solid carrier, preferably Sephadex LH-20 in course of partition chromatography and/or droplet counter current chromatography. 17. The process as claimed in any of Claims 11 to 16 w h i c h c o m r i s e s using a mixture of halogenated hydrocarbons, preferably chloroform, alcohols, preferably methanol, ethanol, butanol, isopropanol, organic acids, preferably formic . acid, acetic acid, monochloroacetlc. acid, aldehydes, preferably formaldehydes and water as eluent. 18. The process as claimed in Claim 17 h i c h c o p r i s e s using the upper layer of a 60:10:30 mixture of ti- utanol, galcial acetic acid and water /solvent system 1/ as eluent. 19. The process as claimed in Claim 17 i c h c o m p r i s e s a 75 : 2 : 8 : 15 mixture of n-butanol, methanol, glacial acetic acid and water /solvent system 11/ as eluent. 20. The process as claimed in Claim 17 w h i c h c o m p r i s e s using the lower layer of a 45 : 30 : 15 : 20 mixture of chloroform, methanol, glacial acetic acid and water /solvent system III/ as eluent. 21. The process as claimed in Claim 17 i c h c o m p r i s e s using a 50 : 35 : 14 : mixture of chloroform, methanol, formic acid and water /solvent system IV/ as eluent. 22. The process as claimed in Claim 17 h i c h c o m p r i s e s using a 130 : 53 : 6 : 9 : 3 : 3 mixture of chloroform, methanol, formic acid, water, formaldehyde and -butanol /solvent system V/ as eluent. 23. The process as claimed in Claim 17 w h i c h c o m p r i s e s using a 160 : 53 : 6 : 9 : 3 : 3 mixture of chloroform, methanol, formic acid, water, formaldehyde and n_-butanol /solvent system VI/ as eluent. 24. The process as claimed in Claim 17 w h i c h c o m p r i s e s using 15 parts by weight of monochloroacetic acid dissolved in a 125 : 53 : 9 : 3 : 3 mixture of chloroform, methanol, water, formaldehyde and n_-butanol /solvent system VII/ ■ as eluent. 25. The process as claimed in Claim 17 w h i c h c o p r i s e s using 15 : 2 : 1 mixture of the solvent system III, chloroform and methanol /solvent system VIII/ as eluent. 26. The process as claimed either in Claim 11 or Claim 14 h i c h c o p r i s e s zvr i.^ out the coloumn chromatography under a pressure of 1 to 5 atm, preferably overpressure. 27. The process as claimed in any of Claims 11 to 26 w h i c h c o m p r i s e s combining the fractions containing the components having the same R~ value and recovering the primycin components by precipitating with a solvent, preferably ether or by evaporation. 28. The process as claimed in any of Claims 11 to 27 w h i c h c o m p r i s e s recovering chinopricin /component A, of primycin/ having an R f value of 0.29 developed on thin-layer chromatoplate with a combined solvent system. 29. The process as claimed in any of Claims 11 to 27 w h i c h c o m p r i s e s recovering midopricin /component A ? of primycin/ having an R f value of 0.31 developed on thin-layer chromatoplate with a combined solvent system. 30. The process as claimed in any of Claims 11 to 27 w h i c h - c o m p r i s e s recovering methipricin /component A-. of primycin/ having an R f value of 0.33 developed on thin-layer chromatoplate with a combined solvent system. 31. The process as claimed in any of Claims 11 to 27 w h i c h -c o p r i s e s recovering oxypricin /component C. of primycin/ having an R-. value of 0.35 developed on thin-layer chromatoplate with a combined solvent system. 32. The process as claimed in any of Claims 11 to 27 w h i c c o m p r i s e s recovering oxymipricin /component C~ of primycin/ having an ^. value of 0.37 developed on thin-layer chromatoplate with a combined solvent system. 33. The process as claimed in any of Claims 11 to 27 h i c h c o m p r i s e s recovering oxymethipricin /component C-, of primycin/ having an H, value of 0.39 developed on thin-layer chromatoplate with a combined solvent system. 34. The process as claimed in any of Claims 11 to 27 w h i c h c o m p r i s e s recovering hydropricin /component B, of primycin/ having an R f value of 0.54 developed on thin-layer chromatoplate with a combined solvent system. 35. The process as claimed in any of Claims 11 to 27 w h i c h c o m p r i s e s recovering hymipricin /component B_ of primycin/ having an f value of 0.56 developed on thin-layer chromatoplate with a combined solvent system. 36. The process as claimed in any of Claims 11 to 27 w h i c h c o m p r i s e s recovering hymethipricin /component 3., of primycin/ having an e ¬ value of 0.53 developed on thin-layer chromatoplate with a combined solvent system. 37. The process as claimed in any of Claims 27 to 36 w h i c h c o p r i s e s recovering the components of primycin in the form of __. salt, preferably in the form of acetate, onochloroacetate, sulphate or formiate salt. 38. The process as claimed either in Claim 11 or Claim 37 w h i c h c o m p r i s e s forming salts different from sulfate from the sulfate salts of the components of primycin by adding barium salts of organic or inorganic acids different from sulfuric acid. 39. The process as claimed either in Claim 11 or Claim 37 w h i c h c o m p r i s e s forming the sulfate salts of the components of primycin recovered in the form different from sulfate by adding sulfuric acid. 40. The salts of the components of primycin formed with organic acids, preferably aliphatic carboxylic acids of 1 to 16 carbon atoms, halogenated aliphatic carboxylic acids, aromatic carboxylic acids, substituted aromatic carboxylic acids, organic sulfonic acids, or inorganic acids, preferably hydrogen halogenids or sulfuric acid. 41. The acetate, monochloroacetate, sulfate and formiate salts of component A, of primycin /chinopricin/ /R f = 0.29/. 42. The acetate, monochloroacetate, sulfate, formiate salts of component A- of primycin /midopricin/ /R f = 0.31/. 43. The acetate, monochloroacetate, sulfate, formiate salts of component A-, of primycin /methipricin/ /R f = 0.33/. 44. The acetate, raonochloracetate, sulfate, formiate salts of component C, of primycin /oxypricin/ /R f = 0.35/. 45. The acetate, monochloroacetate, sulfate, formiate salts of component C~ of primycin /oxymipricin/ /R f ~ 0.37/. 46. The acetate, monochloroacetate, sulfate, formiate salts of component C-, of primycin " /oxymethipricin/ /R f = 0.39/. 47. The acetate, monochloroacetate, sulfate, formiate salts of component 3, of primycin /hydropricin/ /R f = 0.54/. 43. The acetate, monochloroacetate, sulfate, formiate salts of component B p of primycin /hymipricin/ /R f = 0.56/. 49. The acetate, monochloroacetate, sulfate, formiate salts of component B-, of primycin /hymethipricin/ /R f = 0.53/. 50. Process for the preparation of an antibiotic composition w h i c h c o r i s e s mixing 0.001 to 10w% of two or more components of primycine, preferably major components of primycin /components A, , 3, C,/alone or completed with the minor components and/or the derivatives of the components, preferably salts, optionally with other antimicrobial active ingredients, and forming a pharmaceutical composition by adding 99-999 to ~ Q '~ -- of pharmaceutically acceptable carriers and/or excipients and/or diluting agents. 51. The process as claimed in Claim 50 w h i c h c o m p r i s e s mixing the primycin - ~ - components in a weight ratio of 5:95 to 75:25 if the active ingredient comprises two primycin components. 52. The process as claimed in Claim 50 w h i c h c o p r i s e s mixing the primycin components in a weight ratio of 4:3:3 to 7:2:1 if the active ingredient comprises three primycin components. 53. The process as claimed .either in Claim 50 or Claim 51 h i c h c o m p r i s e s using the combination of components A, and C, as active ingredient. 54. The process as claimed in either Claim 50 or Claim 51 w h i c h c o m p r i s e s using the combination of components A, and B, as active ingredient. 55. The process as claimed in either Claim 50 or Claim 51 w h i c h c o m p r i s e s using the combination of components C, and B, as active ingredien . 56. The process as claimed in either Claim 50 or Claim 52 w h i c h c o m p r i s e s using the combination of components A, , B, and C, as active ingredient . 57. The process as claimed in any of Claims 50 to 56 w h i c h . c o p r i s e s forming the composition in the form of dusting powders, aerosols, gels, ointments, eye-drops or other dosage forms suitable for topical application. 58. The process as claimed in any of Claims 50 to 56 w h i c h c o m p r i s e s forming an injectable composition preferably suspension or injectable solution or a combination of powder ampoulle - solvent ampoulle. 59. The process as claimed in any of Claims 50 to 56 w h i c h c o m p r i s e s preparing antimicrobial bandages. - DO - 60. Antibiotic composition w h i c h c o m p r i s e s 0.001 to 10 % of one or more components of primycin optionally with other antimicrobial agents and 99.999 to 90 % of pharmaceutically acceptable carriers and/or diluents and/or excipients.

Inventors: DEKANY GYULA, FRANK JUDIT, HORVATH GABOR, KULCSAR GABOR, SZILAGYI IMRE, DEKANY, GYULA, FRANK, JUDIT, HORVATH, GABOR, KULCSAR, GABOR, SZILAGYI, IMRE
Applicants: CHINOIN GYOGYSZER ES VEGYESZET, CHINOIN GYOGYSZER ES VEGYESZETI TERMEKEK GYARA RT
Application year: 1985
Main classification: C07H17
IPC codes: C07H17, C12P19