CS240345B1 - The method of isolation of tylosin antibiotic - Google Patents

Abstract

The invention provides a method for isolating the antibiotic tylosin from the filtrate of the fermentation broth after the end of the cultivation of the production strain Streptomyces fradiae, or from other aqueous solutions of tylosin, which is characterized in that the filtrate of the fermentation broth, or another aqueous solution of tylosin is extracted with butyl acetate, which contains 1 to 30% by volume of another organic solvent that is sparingly soluble in water, preferably butanol, from which tylosin is precipitated as a salt, e.g. with an acetone solution of tartaric acid. The obtained tylosin-tartrate can be used directly for the preparation of medicinal preparations, or it is converted by known methods into tylosin-base, which is used for the preparation of injections!.

Description

The invention provides a method for isolating the antibiotic tylosin from the filtrate of the fermentation broth, after cultivation of the production strain Streptomyces fradiae, or from other aqueous tylosin solutions.

Tylosin is a macrolide antibiotic of the general formula C45H77NO17. It is poorly basic in nature, poorly soluble in water, well soluble in polar and semi-polar organic solvents, and insoluble in non-polar solvents such as hexane or petroleum ether. Aqueous tylosin solutions are still between pH 5.5 and 8.5. By acid hydrolysis, tylosin breaks down into biologically similar active desmycosine.

With acids tylosin forms salts soluble in water and in polar organic solvents.

Tylosin is an antibiotic used in veterinary practice, particularly to suppress infections caused by gram-positive bacteria and mycoplasmosis (Ose E. E., J. Am. Vet. A. 137, 421-423, 1980). Tylosine alone is particularly useful in the treatment of dysentery and pneumonia in farm animals (Curtins R, A .: Canad. Vet. J. 3, 285-288, 1962; Doornebal, H .: Canad. J. Comp. Med. Sci. 29, 179). —182, 1965; Schovánek V .: Veterinary 25, 505-507, 1975). Combinations of tylosin with other antibacterial scavengers such as sulfonamides have also been widely used (Ose EE: Vet. Med. Smal. Anim. Clin. 68, 539-543, 1975; Reichel, F .: Biol. Chem. Animal Nutrition 10, 343-350, 1974), furazolidine (US 3, 690, 869), colistin (Ger. Offen 2, 327, 392), as well as inert additives such as sodium sulfate (US 3, 932, 995, polyoxyethylene esters (Brit. 1, 219, 785) or with urea (Ger. Offen 1, 812, 471.) For the preparation of medicinal products either tylosin in the form of a base or in the form of salts such as tartrate, chloride, phosphate or sulphate is used (Bruszewski W Wet., 6, 349-352 (1972) .Technical tylosin prepared by drying a fermentation pod (Dvořák, J., Straková, J., Ševčík, B .: Biol. Chem. Vet. 18, 77-78 (1982).

Up to now, tylosin was isolated from the fermentation pod by filtering off the mycelium, extracting the filtrate with chloroform and, after distilling off the chloroform under reduced pressure, from the oily residue of tylosin precipitated with hexane or petroleum ether (US 3,178,341). The tylosin thus prepared had to be recrystallized or converted to a salt prior to further use, such as the tartrate from which pure tylosin base was prepared (Hamill R.L .: Antib. And Chemother. 11, 328-334, 1961). A 40% tylosin preparation can be prepared by salting tylosin from a deproteinized fermentation pod filtrate, or by extracting tylosin with alcohols from a sprayed fermentation broth, e.g. 90% propanol (Aut. Certificate 197 600). Tylosin of similar purity can also be obtained by azeotropically distilling water from the filtrate of a fermentation pod with, for example, n-hexyl alcohol (Ger. Offen 2,054,085). Tylosin in the form of tylosin tartrate can be prepared of high purity by extracting tylosin from the filtrate of the fermentation pod by extraction into butyl acetate (AO No. 231 279). The disadvantage of this procedure is the necessity to choose multiple extraction of the filtrate, as in the single-stage extraction! Butyl acetate extracts tylosin in a small yield. With extraction of Ί000 ml of fermentation tray filtrate with 200 ml of butyl acetate at pH 9.0 and 25 ° C, only about 53% tylosin is extracted into the organic phase. With higher yield, tylosin can be extracted with chloroform-ethyl acetate, but due to the toxicity of chloroform, the utility of this method on an industrial scale is very limited.

It has been found that tylosin can be obtained in high purity and extract from the fermentation pod filtrate, or from other aqueous solutions such as e.g. An aqueous solution of technical tylosin phosphate according to the invention, characterized in that the filtrate of the fermentation pod or another aqueous solution of tylosin is extracted with butyl acetate containing 1 to 30% by volume of ethyl acetate. a water-soluble alcohol such as amyl alcohol, isoamyl alcohol, isobutyl alcohol, benzyl alcohol (preferably 10% v / v butanol) or ketone such as e.g. methylisobutyl ketone, methyl ethyl ketone and the like. The advantage of this process is that a single-stage extraction of tylosin by this process is sufficient to extract tylosin in a high yield. Extraction with 1000 ml of fermentation tray filtrate with 180 ml of butyl acetate and 20 ml of benzyl alcohol at pH 9.0 and at a temperature of 20 ° C is extracted into the organic phase of 91% tylosin.

Unlike chloroform extracts, the tylosin extracts thus obtained can be directly precipitated with an acetonic acid solution, e.g. 1 wt. vol. with a tartaric acid solution to obtain a good crystallizing sol-tylosin tartrate. This can either be used directly for the preparation of medicaments, or converted to tylosin base by conventional procedures. The advantage of this process is that tylosin is obtained in high yield and purity from the fermentation pod filtrate using nontoxic and readily regenerating solvents.

The following example illustrates the process according to the invention but does not limit it in any way.

To 500 ml of the fermentation plate filtrate with an efficiency of 7,630 µg / ml was added 100 ml of butyl acetate and 25 ml of butanol. The mixture was adjusted to pH 9.0 with vigorous stirring with stirring and allowed to stir for 15 minutes. After this time, the organic layer was separated to give 125 ml of an organic phase with an efficiency of 27,750 µg / ml which was 91 pens. extraction yield. The organic extract obtained is then added dropwise to 300 ml of acetone, in which 3.5 g of tartaric acid is dissolved. After one hour of vigorous stirring after addition of all the organic extract, the precipitated tylosin tartrate is filtered off, washed with 30 ml of acetone and air-dried at room temperature to give 4.1 g of tylosin tartrate having an efficiency of 850 m. U./mg, which is 86% recovery calculated on the fermentation broth filtrate.

Claims (1)

240345 to 300 ml of acetone in which 3.5 g of tartaric acid was dissolved. After an hour of vigorous stirring after the addition of the entire amount of the organic extract, the precipitated tylosin tartrate is filtered off, washed with 30 ml of acetone and dried in the air at room temperature to give 4.1 g of tylosin tartrate with an efficiency of 850 IU / mg, a 86% yield. , calculated on the filtrate of the fermentation broth. BACKGROUND OF THE INVENTION The method of isolating the tylosin antibiotic from the fermentation filter filtrate after the culturing of the Streptomycesfradiae production strain or from other aqueous thiosin solutions is characterized in that the filtrate of the fermentation broth or other aqueous solution of thylazine is extracted butyl acetate, which contains from 1 to 30% by volume of a water-soluble organic solvent, with preference of butanol and from the extract, the tylosin is precipitated as a salt with an acetic acid solution, preferably 1% by weight. vol. solution of tartaric acid in acetone.