CS202641B1 - Method of transformation of the digitoxygenine glycosides - Google Patents

Abstract

Digitoxigenin glycosides (I) are converted into digoxin (II) and gitoxin (III) by exposing a (I)-contg starting material, pref. freshly gathered, comminuted digitalis plants, to the action of the enzyme system inherent in the digitalis plant, at 20-45 degrees C for >=60 hrs with exclusion of air and/or with passage of N2 and CO2, and working up the converted material in known manner to give the glycosides. The fermentation may be followed by high-temp drying. Alternatively, the starting material can be reacted with extracts of fresh, lyophilised or dried (at 35 degrees C) digitalis plants purified over a crosslinked dextran gel, under the same conditions (but at 30-35 degrees C) for 24 hrs.

Description

The present invention relates to a process for converting digitoxigenin glycosides to dioxin or gitoxin by targeted feamentation using a native or extracted drug feed system.

In the production of cardiac glycosides from the species Digitalis and e, they mainly isolate Lanatoside C, which is the most important substance for therapeutic use, and the secondary glycoside digoxin. In addition, glycosides of the A and B series, which are also present in Diggtalis purpura, are still present in Diggtalis lanata plants. However, these glycosides are only of limited use. Therefore, a number of attempts have been made to increase the proportion of digiooxigenta glycosides. Three ways were proposed for this purpose.

The content of these substances can be increased, for example, by targeted breeding, which can in particular reduce the proportion of glycosides of the A series.

^{78 3/1} 965 ^/) were responsible jnaTC ^h after publication cel ^{E r P} series ition, týbaj ^ i ^ is plteměny digitox ^.mu.g compound per digoxigenin, digitoxin convert 29% yield to digoxin by a specific strain Streptcmyces.

202 641

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A third possibility of increasing the proportion of digoxigenin glycosides is that the plants are treated in a certain way after harvesting. For example, Pitra'a spol. suggested by the harvest of the harvested plants to increase Lanatoside C - in the dried metteia. (CSSR patent δ 123 570, Pitra I., Kučera M. and Starý F., published on Aug. 8, 1963 / July 15 - 7, 1967).

Pitra and epoo. [(Pitra I., Sterba I., Horak, P., Pr ^{of about} chá ka - V, Pharmacia, '. ²⁷ 77 ^{9/1971 /)} ^ y ^ pisi another possible way of increasing the proportion ^- Lanattoeidu C. - that ^the blades. Jul ^sucks the harvested plant Digitalia lanata stored '7 days at 25 ° C and 100% relative vlhkooti in a well ventilated area, thereby increasing the proportion of desired compounds Hsty the lavages stored ^{d p} s te ^p ture of 40 ° C, ^No. IMZ ^d to preserve them. When tepntáta ¹ 5 ° ^C increases the ^G lykosidů ^- very ^p omau ^p s te ^p ture of 30 ° C - is even failure to ^the tateč> · Ny, while ^p s ^t e ^p lot ^of 4 ° C occurs in the ^P r ^UB EHU ² days pri- further storage at ^- 38 ° C to U conversion of ^p flax lanatoside 0- to digoxin.

In the case of irretracted space, Lanatoeid C is formed under the above conditions, and in poorly ventilated space, Lrnnatoside C is rapidly degraded to digoxin. A possible cause - of this phenomenon - is a lack of moolcular oxygen. The authors cite the growth conditions of plant cells, the water content and the size of the pressure as essential factors for the formation of Lfaiatoside C.

The disadvantages of the above process are that the formation of Lrniatoside C can only be achieved on the isolated leaves and not on the whole plant, and also that 9 days is required to produce the drug obahuiddd digoxin. Furthermore, it is not technically possible to achieve the above environmental conditions. The process is not economically advantageous and therefore encounters difficulties. It would therefore be desirable to provide a process which - in isolating umooňoval -Lanatosidu C or remeasured lanatoside A digoxin or digitoxin to glykoaidy, UH1 p ^ ^ i ^ Ln ^ _l ISI for therapeutic purposes - to achieve this conversion or fermentation of fresh plant .

Unlike results which - given Pitra et al., It has now been found that in - hermetically sealed containers which are preferably filled with nitrogen or carbon taličitým j ^e can be ^- achieved - in - fresh, drcenýta ROE ^t L ^N -Ac ^lp RI ^- Temp @ ^{20 -,} and ^Z 45 ⁱⁿ average ^of hu ~ b ^of at least 60 hours outside -úplné odbowtaí native lanatoside C the conversion still large proportion digitoxiipninových glycosides - to digoxin and partly on -gitoxin ^{enzymaaické-hydroxy;} \ · · Cheap. It also implies that for synthesis. The digoxigenic glycosides do not need to be maintained, as reported by Pitra et al., as the basic living conditions of the cell-like cells, but that these conditions may to some extent be greater.

Accordingly, the present invention provides a process for the conversion of digitosigenin glycosides from Diggtalis lanata and -Diggialis purpures, such as Lanatoside A, acetyldigitoxin and digitoxin, to digoxin, acetyldigoxin and optionally gitoxin, by fermentation, characterized in that fresh, crushed Diggialis plants are lanata or Diggtalis purpurea undergoes fexmenbation. at a temperature of 20 to 45 ° C for at least 60 hours in the absence of air and / or in an atmosphere of nitrogen or dioxide, whereupon the glycoaid-enriched drug is preserved.

202 641 The advantages of the process according to the invention are that it can be carried out in one step in a substantially shorter time and - under readily achievable conditions by post-mortem biosynthesis of digoxin and gitoxin from digitoxigenin glycosides while deburring native Lanatoside C to digoxin. B to gitoxin, thereby achieving a higher content of digoxigenin substances relative to the content of digiooxigenic glycosides originally present in the fresh plant. In conjunction with subsequent high temperature preservation according to U.S. Pat. No. 157,619, a substantially economical method of digoxin production is achieved.

The invention will be illustrated by the following examples.

Example 1

Freshly. harvested digitalis leaves containing. The digoxin glycosides, calculated on digoxin in dry matter, are cut into pieces approximately 1 cm in size and stored in a closed silo at a height. 1-2 m · is then brought into the silo uiičitý carbon or nitrogen .and material is left in the silo 4 - days · Temperature of cut sheets in the EEE ^r d ^about BE should be in ^the dimension ^ i ^: ^I 20 to 30 ° C then se m ^ riá !. it is dried at high temperature, so it contains 0.51% digoxin and vinegar, and 0.09% digitoxin and acetyldigitoxin when converted to digoxin.

Example 2 Freshly harvested digitalis leaves are treated with water or buffer to a thick slurry. 10 kg of this slurry, ie about 1 pc of dry matter, are mixed with 5 g of Lanatoside A, desaeetyllanatoside A, - digitoxin or acetyldigitoxin and incubated for 4 days _; at a temperature of 20 to 30 ° C in a nitrogen or carbon dioxide atmosphere with stirring in a closed vessel. After summer, 70 to 80% of the added digitoxigenin glycosides are digested to digoxin or acetyldigoxin.

Under the same conditions, the fractions of Lanatoside A resulting from the preparation of Lanatoside C can be converted to digoxigenic glycosides.

To isolate diioxigeninových glycosides - the slurry is pressed and the resulting maatriál is once more extracted with ^about 5 ^and 10 nfoobnjfa mntóstvím yo a ^d 40 ° C. ^In odné solutions ^- went ^{to her} and někooikrát chlorofrmem shaken with chloroform and the solution evaporated to dryness. After desacetylation of the currently obtained Acetyldigoxin with sodium carbonate in a mixture of water and alcohol, the crude digoxin, which still contains a small amount of digitoxin and gitoxin, is further purified in a known manner.

Claims (1)

SUBJECT OF THE INVENTION A process for converting digitoxigenin glycoaids from Digitalis lanata and Digiialis purpurea, such as Lrnintoside A, acetyldigitoxin and digitoxin, to digoxin, acetyldigoxin and optionally gitoxin, by fermentation; fresh, crushed Digitalis * lanata or Digitalis purpurea plants are subjected to ferraenbed at 20 to -45 ° C for at least 60 hours in the absence of air and / or in a nitrogen atmosphere or utility, whereupon the drug enriched with glycoaids is consumed.