HU184123B - Method for yielding heart glycosides from their solutions of aqueous or aqueous-organic solvent - Google Patents

Abstract

The present invention relates to a method for recovering cardiac glycosides from its aqueous or aqueous-organic solvent sides. The method consists in treating the aqueous or aqueous organic solvent solution of the cardiac glycosides with activated carbon and dissolving the carbon glycosides quantitatively bound on the activated carbon with filtration from the carbon with an organic solvent or solvent mixture. The process of the present invention provides for the economical recovery of cardiac glycosides from aqueous or aqueous-organic solvents. -1-

Description

The present invention relates to a process for the recovery of cardiac glycosides from their aqueous or aqueous-organic solvent solutions.

Cardiac glycosides as used herein refer to steroidal glucosides derived from Digital and Strophantus plants, and used in their therapeutic specificity for the heart (see, e.g., Chemistry of Natural Drugs, Vol. II, pp. 184-185, 1969).

Cardiac glycosides are known to be of great therapeutic importance and are essential for pharmacotherapy due to their life-saving effect. Their production is very expensive, so making it more economical is an important task. Cardiac glycosides are exclusively found in plants, e.g. Digital Lanata, Digital Purpurea, Strophantus cumin or Strophantus gralus. Some of the resulting glycosides can be used directly as drugs, while others become therapeutically useful only after prior chemical or microbiological conversion. A number of processes for the above transformations are known in the art. Of these, the most important is the 12 fb hydroxylation of A-series digitalis glycosides. First, Nozakin and coworkers [Agr. Biol. Chem., 29, 783 (1965)] were thus able to prepare digoxin from digitoxin (Japanese Patent Nos. 42-22611, 42-22612, 42-22613, 42-22614, 42-24498).

The digoxin formed is recovered from the fermentation broth by chloroform extraction and purified by column chromatography.

No. 175,474, no. The process according to Hungarian Patent Application No. 4,195,195 also allows the microbiological transformation of digitoxin to digoxin, According to the procedure described in Hungarian Patent No. 5,195, Streptomyces can produce digoxin from lanatoside A. In both processes, the glycosides are recovered by extraction with chloroform or chloroform-methanol.

In addition, other fermentation processes for the conversion of cardiac glycosides are known, but these are not so-called hydroxylation but microbiological down-regulation of primary glycosides. secondary to glucose, glucose or glucose. acetyl group.

For example, U.S. Patent No. 175,601. Hungarian patent application No. 5,198 discloses the possibility of producing digitoxin from lanatoside A. Here again, the recovery of glycosides is carried out with methanol or methanol. chloroform extraction. Finally, no. 2,343,400 is of theoretical importance. U.S. Patent No. 4,600,125, which discloses the conversion of cardiac glyceride into a fermentor-grown plant cell and extracts the resulting products with methanol after evaporation of the fermentation broth to dryness.

Thus, after microbiological conversion, the extraction of cardiac glycosides is practically carried out in each case by direct extraction of the fermentation broths with organic solvents.

Since the glycoside content of fermentation broths is low (0.02-0.5%), however, they are poorly soluble in water-immiscible organic solvents, they can only be recovered from large volumes of fermentation broths using a large amount of solvent.

SUMMARY OF THE INVENTION It is an object of the present invention to provide a processing method which allows the recovery of glycosides from aqueous or aqueous organic solvent solutions of cardiac glycosides without the use of large amounts of solvent.

In order to provide a more economical treatment with various adsorbents, it has been found that when an aqueous or aqueous organic solvent fermentation broth containing a cardiac glycoside is mixed with activated carbon and then filtered, all glycosides are completely bound by carbon and can be completely dissolved from the carbon with an organic solvent.

In the chemical industry, activated carbon is generally used to purify compounds and remove impurities, but it is also known that carbon is one of the most potent apolar adsorbents. Thus, according to the state of the art, it was expected that activated carbon, among the large number of (heterogeneous) components of the fermentation broth, would primarily contain apolar substances, impurities and so on. adsorbs pigments that are poorly water soluble, oils used as antifoam agents.

On the other hand, the underlying realization of the present invention is that, from the above solutions, activated carbon binds first and foremost to semipolar cardiac glycosides in a quantitative manner. However, the organic solvent eluate of the carbon adsorbate contains much less impurities than the extracts obtained by direct organic solvent extraction of fermentation broths in the previously described processes. As a result, further purification of cardiac glycosides can be achieved more easily and at a more favorable yield. Carbon adsorbs glycosides either well or poorly in water to such an extent that the filtrate of the fermentation broth can be discarded without further extraction. However, glycosides can be liberated from carbon using much less solvent than is required to extract the original volume of fermentation broth.

According to our studies, any carbon (plant, bone) carbon of any origin can be used to bind glycosides.

Surprisingly, no significant difference was found between the specific surface area of the carbon species and the extent of adsorption, since the glycosides were already adsorbed quantitatively from the fermentation broth even at relatively low application rates of 500-600 m ² / g specific surface area. Although, as stated above, the extent of adsorption is not significantly affected by the specific surface area of the carbon and, in parallel, the particle size, the amount of filter aid (e.g. Hyflo, Perlit) added to facilitate filtration may need to be selected occasionally. Elution may be carried out after drying the carbon filter at 160 ° C, or more preferably directly from the wet carbon. If the carbon fermentation broth is boiled before filtration, the use of filtration aids may be partially or completely avoided.

Accordingly, the present invention provides a process for the recovery of cardiac glycosides from their aqueous or aqueous-organic solvent solutions which comprises converting the cardiac glycosides in aqueous or aqueous-organic solvent solution into activated carbon. and filtering quantitatively bound cardiac glycosides on activated carbon after filtration with an organic solvent or solvent mixture. According to a preferred embodiment of the process according to the invention, the aqueous or aqueous organic solvent solution, which may be filtered fermentation broth or any other solution containing cardiac glycosides, is 3 to 20 times, preferably 10 to 10 times the content of cardiac glycoside. with activated charcoal of a weight of 1 to 2 hours. After filtration with Hyflo supercell or filter perlite, the carbon-bound glycosides are preferably extracted several times with a suitable solvent or solvent mixture with chloroform-alcohol. Further purification of the residue obtained after concentration of the combined extracts to dryness can be carried out by chromatography or, e.g. the method disclosed in Hungarian Patent Application Publication No. T / 24320 can be used advantageously.

It is also possible, in particular in the processing of fermentation broths having a high content of cardiac glycosides, when part of the material is already bound to the mycelium and / or the it is present in the fermentation broth that prior to filtration of the mycelium, the fermentation broth is mixed with 10-20% methanol or ethanol to form the precipitate. mycelium-bound glucoside, and then filter the fermentation broth. Subsequent carbon adsorption is not reduced by the presence of an organic solvent. The eluting solvent may be any water-immiscible or immiscible organic solvent for the cardiac glycosides or mixtures thereof, preferably chloroform-methanol (7: 3). The elution may be carried out simultaneously with the total amount of eluting solvent, or preferably in portions thereof, with two to three elutions successively successively filtered.

The main advantage of the process according to the invention over the purely solvent extraction method is that it significantly reduces the solvent requirement and thus reduces the loss of solvent usage and evaporation cost.

Another great advantage of our process is that the operations required to obtain the cardiac glycosides are simple. The process requires little material. The yield reaches or exceeds the direct solvent processing.

Filtration is easy with large-scale equipment. The glucosides are completely adsorbed from the solution. Large amounts of solvent can be avoided. High volume equipment is not required. Difficulties with possible emulsion formation can be eliminated. Activated carbon can be reused after regeneration (eg with hydrochloric acid).

The process of the invention is illustrated by the following embodiments. In the examples, the quantitative analytical determination of glycosides is described in 176.249. s. was performed according to the dixantylurea method disclosed in Hungarian Patent Application, or by thin-layer chromatography according to Hörhammer et al. Hörhammer, H. Wagner, H. König Deut. Apothcker Ztg. 103, 502 (1963)].

Example 1 A solution of 40 mg of lanatoside A in 40 ml of methanol, 40 mg of digitoxin dissolved in 1 ml of tetrahydrofurfuryl alcohol and 40 mg of digoxin dissolved in 1 ml of dimethylformamide was added to 100 ml of water with stirring. To the aqueous solution was added 1 g of activated carbon (REANAL, Budapest) and stirred at room temperature for 2 hours. The suspension is then filtered through MN 640 W paper (Macherey-Nagel, DÜREN, Federal Republic of Germany) and the filtrate and filtrate are extracted three times with 30 ml of a 7: 3 mixture of chloroform-ethanol. The appropriate extracts are combined and their glycoside content determined. No glucosides were detected in the filtrate by the dixantylurea method, while 37 mg of lanatoside A, 38 mg of digitoxin and 39 mg of digoxin were measured in the filtrate extract.

Example 2

All proceed as in Example 1, but adding the cardiac glycosides instead of water to a mixture of 70 ml water and 30 ml methanol. The filtrate extract was found to contain no glucosides, while the filtrate extract contained 37 mg of lanatoside A, 38 mg of digitoxin and 39 mg of digoxin.

Example 3

Dissolve 100 mg of K-strophantin in 100 ml of water. To the solution was added 1 g of activated carbon and stirred for 2 hours at room temperature. The slurry was then filtered through MN 640 W paper, and the filtrate was extracted three times with 10 mL portions of chloroform-methanol (7: 3). The combined extracts were evaporated to give 96 mg of K-strophantin.

Example 4

Streptomyces purpurascens strain KA-82 (MNG 177) grown on potato dextrose slant agar for 1 week was inoculated with 1 ml of 10 ml of sterile water in 100 ml of MK medium in a 500 ml Erlenmeyer flask. Composition of MK medium:

% hazelnut flour% corn starch pH 7.0 before sterilization.

Sterilize at 121 ° C for 60 minutes.

The flask was shaken at 32 ° C on a rotary shaker at 300 rpm for 2.5 days and then 5 ml of the culture was inoculated with 100 ml of MK medium containing 100 mg of lanatoside A in 1 ml of methanol. After further shaking for 5 days, the fermentation broth is filtered on a filter canvas, 1 g of activated carbon is added to the filtrate and the mixture is stirred for 2 hours at room temperature and then filtered again. The charcoal filtrate was extracted three times with 7-7 ml of chloroform-ethanol (7: 3)

ι «i g;

s mixture. The cardiac glycoside content of the combined extract was determined by dixantylurea to 8 mg lanatoside A, 10 mg 7 (1-hydroxy-digitoxin, 40 mg digoxin, 15 mg 7-hydroxy-digoxin and 2 mg digitoxin). No glucoside was detected in the post-filtrate filtrate.

Example 5

All proceed as in Example 4, but flask 1 was Streptomyces purpurascens KA-26 (MNG 179), flask 2 was Streptomyces purpurascens KA-43 (MNG 178), and flask 3 was Streptomyces purpurascens KC-157 (MNG 176). Among other cardiac glycosides, digoxin 34 mg, 35 mg, 38 mg, and 38 mg were detected in other flasks.

Example 6

300 ml of a 2-day shake culture of Streptomyces purpurascens KA-82 (MNG 177) on MK medium were inoculated with 5 liters of MK medium containing 9 g of lanatoside A and 20 ml of palm oil in a 10 liter laboratory fermenter. The fermentor was flushed with 5 liters / min of sterile air in a 32 ° C water bath and 350 / min for one day and then for 4 days. Incubate at 500 rpm for a total of 5 days. The vernal juice is then filtered through a canvas, the mycelium is washed with 1 liter of tap water and filtered. The combined hairs were stirred with 55 g of carbon for 1 hour and then 36 g of filter perl (KOSZIG, Tapolca) were stirred for an additional 10 minutes and filtered on paper. Discard the filtrate, which does not contain glycosides as defined by dixantylurea, and extract the wet filter perlite with 250 ml to 250 ml of a 7: 3 mixture of chloroform-methanol and filter three times. The extracts were combined, the aqueous phase was separated off, and the solvent mixture was evaporated to dryness under vacuum at 40 ° C. 5 g of product containing 36% digoxin are obtained. The phytochemical constants of digoxin obtained after purification according to Hungarian Patent Application Publication No. T / 24320 are the same as described in the literature.

Example 7

All proceed as in Example 6, but the mycelium was washed with 1 liter of 20% methanol. This gives 5.8 g of product containing 37% digoxin.

Claims (4)

Patent claims CLAIMS 1. A process for the recovery of cardiac glycosides from their aqueous or aqueous organic solvent solutions, comprising treating the cardiac glycosides in aqueous or aqueous-organic solvent with activated carbon and quantitatively After filtration, the bound cardiac glycosides are dissolved from the carbon in an organic solvent or mixture of solvents. The method of claim 1. characterized in that the aqueous solution is a fermentation broth of Streptomyces species containing cardiac glycidide. 30 3. The process according to claim 1, wherein the aqueous organic solvent is a ferric juice of Streptomyces species containing cardiac glycoside and lower alcohol. 4. A method according to any one of claims 1 to 6 35, characterized in that after filtration, the cardiac glycosides are liberated from the carbon with a mixture of chloroform-lower alcohol.