HU192587B - Process for concentrating and purifying peptide-type ergot-alkaloides by adsorption - Google Patents

Abstract

Ergot alkaloids of the peptide type are purified and concentrated by adsorbing peptide alkaloids contg. opt. water soluble ergot alkaloids from aq. soln. onto macro-reticular resin and desorbing from the resin first the colouring and fatty matter and finally, opt. selectively the alkaloids.

Description

The present invention relates to a novel process for the enrichment and purification of peptide-type nutmeg alkaloids. Peptide type ergot alkaloids include the components of ergot toxin (ergocornine, α-, and 0-ergocriptine, and ergocristine as well as their C8 epimers), ergotamine, ergosine and epimeye, and simple derivatives thereof, e.g. 2-bromo-a-ergocriptine [Stoll: Helv. Cilia. Acta 26, 1570 (1943) and 28, 1283 (1945), Smith, J. Chem. Soc. 1937, 396, DE 1 926 045], Widespread therapeutic use of peptide alkaloids, particularly for treating vascular disorders and migraine, prolactin. secretion inhibitors, etc. They can be used.

The peptide alkaloids are prepared by extraction of a rye plant infected with a fungal strain, or by fermentation using fungal strains, or more recently by a synthetic route, and the resulting extract (fermentation broth or reaction mixture) is purified by phase exchange (U.S. Pat. No. 171,659, 180,287; and / or by chromatography (Hungarian Patent No. 164,116). In British Patent No. 1,158,380, the crude purification is carried out by phase exchange and the fine purification by chromatography. Since peptide alkaloids are practically insoluble in water, both phase exchange and chromatographic methods involve the use of large volumes of organic solvents. Phase changes have several pH adjustments and there is no purification method that can be used to purify both the fermentative and drug peptide alkaloids.

In our attempts to simplify and purify enrichment-purification, we have found that although peptide alkaloids are practically insoluble in water, they can be adsorbed from aqueous media with high efficiency, e.g. Amberlite type macroreticular resins, e.g. U.S. Patent 3,531,463; and Diaion-type macroreticular resins disclosed in Diaion Manual (Tokyo, 1984, Mitsubishi Chem. Industries Ltd). The peptide alkaloids can then be sorbed from the resin, optionally selectively. Selective desorption is possible in the presence of eluent, whereby the water-soluble nutmeg alkaloids and the peptide alkaloids are dissolved from the resin with different eluents.

By this method, very high (20-60 fold) purification of alkaloids and significant volume reduction (enrichment) can be achieved and the high quality product can be obtained from the eluate by simple methods known per se.

We find the solution surprisingly novel because macroretricular resins have so far been used and recommended only for the purification of bioactive substances that are water soluble and present in water soluble form (U.S. Patent No. 3,531,468). The realization of the possibility of selective desorption is also realized first in the present solution.

The present invention relates to a process for the purification and enrichment of peptide-type nutmeg alkaloids by adsorption, wherein the peptide alkaloids, optionally containing water-soluble nutmeg alkaloids, are bound in an aqueous medium to macroreticular resin and the resin is first dyed. and fatty acids are dissolved in pH 9-14 pH-adjusted water, then the peptide-type nutmeg alkaloids are eluted with a water-miscible organic solvent or, in selective elution, the water-soluble alkaloids with alkanol or aqueous alkanol, then the peptide-2 alkaloids are inorganic or water-miscible organic water-miscible organic solvent.

According to the process of the present invention, peptide-type nutmeg alkaloids are purified or enriched. The peptide alkaloids produced by fermentation may be e.g. ergocristine and its epimer, the preparation of which is described by Amici et al., Appl. Microbiol. 18, 464-468 (1969)] (producing strain: ATCC 20103, deposited at 1968, deposited at Amer. Type Culture Collection, Rockville, Maryland, USA, ergocornine, Oes) 3-ergocriptine and their epimies (producing strains e.g. .: MNG 0088, deposited on January 25, 1972, OKI, Budapest, the application of which strain is described, for example, in Hungarian Patent Application No. 164,816, MNG 00186, deposited on May 9, 1979, OKI, Budapest, the application of which strain Hungarian Patent No. 178,405).

Ergotamine and its epimer are obtained by drug extraction (production strain: MNG 00208, deposited on October 8, 1981, OKI, Budapest), the use of which is disclosed in Hungarian Patent Publication No. T / 30266. In fermentation, e.g. the following media may be used:

Composition of medium E: sucrose 100.0 g / 1 potassium dihydrogen phosphate 0.25 g / 1 1-asparagine 10.0 g / 1 magnesium sulfate 0.25 g / L potassium chloride 0,120 g / L Calcium nitrate 1,000 g / 1 iron (II) sulfate 0.02 g / 1 zinc sulphate 0,015 g / L agar-agar (Difco) 18.0 g / 1

The above components are dissolved in 800 ml of water, adjusted to pH 6 with 15% aqueous ammonium hydroxide solution and made up to 1000 ml.

and sterilized at 110 ° C for 25 minutes. Composition of medium F: saccharose 100.0 g / L succinic acid 10.0 g / 1 potassium chloride 0.30 g / 1 potassium dihydrogen phosphate 0.75 g / 1 magnesium sulfate 0.70 g / 1 iron (II) sulfate 0,007 g / L zinc sulphate 0,006 g / 1 manganese sulphate ' 0,005 g / 1 Composition of G medium: saccharose 200.0 g / 1 succinic acid 12.5 g / 1 potassium chloride 0.3 g / 1 potassium dihydrogen phosphate 0.75 g / 1 magnesium sulfate 0.70 g / 1

ferrous sulfate 0.020 g / l zinc sulfate 0.020 g / l manganese sulfate 0.007 g / l

Formulations F and G as described for E medium

preparing medium. Composition of medium H: mannitol 40.0 g / 1 citric acid 7.0 g / 1 corn steep liquor 2.0 g / 1 potassium dihydrogen phosphate 1.0 g / 1 magnesium sulfate 0.3 g / 1 agar-agar (Difco) 25.0 g / l

192 587 pH 5.2 adjusted with concentrated aqueous ammonium hydroxide

Medium I:

trypsin 7.0 g / l citric acid 4.1 g / l potassium dihydrogen phosphate 0.3 g / l magnesium sulfate 0.3 g / l pH 5.7 adjusted with concentrated aqueous ammonium hydroxide

Medium K:

sucrose 100.0 g / l citric acid 10.0 g / l sodium chloride 10.0 g / l potassium dihydrogen phosphate 0.5 g / l pH 5.7 adjusted with concentrated aqueous ammonium hydroxide 15

St medium:

sucrose 100.0 g / l succinic acid 10.0 g / l potassium dihydrogen phosphate 0.25 g / l magnesium sulfate 0.25 g / l ammonium nitrate 1.0 g / l calcium chloride 1.0 g PH 5.2, adjusted with concentrated aqueous ammonium hydroxide, 25

The components of H, J, K and St media are approx. Dissolve in 800 ml of water, adjust to pH, and after sterilization at 110 ° C for 25 minutes after making up to 1000 ml.

The fermentation is carried out in a manner known per se, but also in the examples. Similarly, in the corresponding example, the drug-extraction method known per se is also described for the drug-derived peptide alkaloid.

Aqueous fermentation broths obtained by fermentative or drug extraction, and / or fermentation broths. extract is then called. adsorbed on a macroretricular resin. Macroretricular resin is a type of adsorptive resin based on a synthetic resin (copolymer and adsorptive property due to its high porosity (specific surface area: min 40 m ² 200 m ² / g) and large pore diameter (minimum 30 Å). e.g., Amberite XAD2, XAD4, XAD7 and XAD8 resins (manufactured by Rohm and Haas Co.) and Diaion HP 20, HP 21, EX2O7 and SP 207 resins (manufactured by Mitsubishi Corp.) and 45 Lowatit OC 1031 resin (manufactured by Bayer AG).

The resin can be used in batch operations e.g. by mixing the resin in a medium containing the alkaloids to be purified and then settling or filtering. It can also be used in a resting or moving 50 fluid bed operation, wherein the medium containing the alkaloids to be purified is passed through the resin column. If the medium is a non-harvested or digested cellular juice, it is preferable to use a resin with a higher specific gravity than mycelium. 55

PH of the liquid contacted with the resin

-6, preferably 3.5 -5.5.

After the adsorption process, the resin is washed first with water, then with alkaline water (pH 11-13), then again with water, and then the peptide alkaloids are eluted from the resin with any of the 60 water-miscible organic solvents commonly used in alkaloid chemistry. As water-miscible organic solvents, alkanols, preferably C 1 -C 6 alkanols, C 1 -C 4 water-soluble aliphatic ketones, but also dimethylsulfoxide, dimethylformamide, acetonitrile .65 and dioxane may be used. The use of methanol, ethanol or isopropanol is preferred. The solvent used may optionally contain up to 50% water and / or 1-5% acid. The acid used is an inorganic acid, e.g. phosphoric acid, sulfuric acid, hydrochloric acid, or a water soluble organic acid such as tartaric acid, citric acid.

If not only the peptide alkaloids but also the water soluble nutmeg lipids present as resin alkaloids were adsorbed on the resin, first, the water soluble alkaloids were eluted first with alkanol, then selectively the peptide alkaloids 2 eluting with a water-miscible organic solvent containing acid or water-miscible organic acid. The organic solvent and acid used may be as detailed above. The water-soluble organic solvent optionally has a max. It also contained 50% water. The amount of water-soluble alkaloids present as an accompanying alkaloid is 25-75% of the total alkaloid content, or eat! less.

The peptide alkaloids are isolated from the eluate in a known manner by evaporation and / or salt formation.

The invention thus provides a process for purifying and enriching peptide-type nutmeg alkaloids by adsorbing peptide alkaloids containing up to 75% water-soluble nutmeg alkaloids in an aqueous medium by methods known per se, and dyeing the resin first from the resin. - and fats are dissolved in 9 - 14pH alkaline water and then

(a) eluting the peptide-type nutmeg alkaloids with a water-miscible organic solvent, optionally containing up to 50% water and / or 1-5% inorganic or water-miscible organic acid, or

(b) in the presence of water-soluble nutmeg alkaloids, the water-soluble alkaloids containing from 1 to 6 carbon atoms of alkanol containing 1 to 6 carbon atoms or optionally containing up to 50% water and then 1 to 5% of inorganic acid or water miscible organic sf cotton and optionally up to 50% water in a water miscible organic solvent.

The invention is illustrated by the following examples, which are not intended to limit the scope of the examples.

Example 1

ATCC 20103 Claviceps purpurea strain cultured on 14-day incubated medium E was washed with 5 ml of physiological saline and homogenized. 2 ml of the homogeneous suspension were inoculated into 400 ml F medium prepared in a 3000 ml Erlenmeyer flask. The culture was shaken at 24 ° C for 3 days and inoculated into 5 to 5 L of medium in two 10 liter glass fermenters. The inoculum culture was incubated for 48-50 hours, aerated at 24 ° C and stirred. Inoculum was sterilized with 100 1G sterilized in 160 L acid-proof steel fermenter. Cultivation was continued with air flow and stirring at 24 ° C for 14 days.

At the end of the fermentation, the pH of the fermentation broth was 49 and its total alkaloid level was 1820 µg / ml. The amount of alkaloid components determined by liquid chromatography is 1460 pg / ml ergocristine, 150 µg / ml ergokinystine, 190 pg / ml ergotamine and 30 pg / ml ergotamine.

Cell lysis of 50 liters of fermentation broth in a high speed mixing horrorogenator (2000 rpm) for 10 minutes

192 587 are done. 50 L of the resulting cellular fermentation broth thus obtained are passed through a series of movable bed rows of columns containing 1.5 kg of EX 207 resin each at a rate of 15 L / h. The resin was flushed with water, a 3% aqueous solution of NaHCO ₃ , and then again with water until neutral or neutral. wash until the wash water becomes colorless and finally elute with 20 L of methanol. The resulting alkaloid is e.g. methanol by evaporation to a solid. The product weighs 77.8 g and has a total alkaloid content of 87%, of which ergocristine 71.0%, ergocristine 6.4%, ergotamine 8.5%, and ergotamine 1.0%. The product contains 13% unidentified ballast. Yield of adsorption-elution on total alkaloid: 74.4%.

If desired, the product may be, e.g. to form the active ingredient as follows: Dissolve in 5% phosphoric acid water, then selectively extract ergocristine with benzene at pH 3.8-4 to give 64.2 g of ergocristine benzene crystal after evaporation.

Example 2

The culture of Claviceps purpurene variant strain deposited under number MNG 0088 was grown on 200 ml of medium I in 750 ml Erlenmeyer flask and incubated at 24 ° C for 3 days. The resulting culture was inoculated into 5 L K medium in a 10 L laboratory fermenter and incubated for 3 days at 24 ° C and incubated with aeration. The inoculum thus obtained is inoculated onto 1001 St medium in an acid-resistant mixer fermenter and the culture is fermented with stirring and aeration at 24 ° C for 6 days. After the fermentation has stopped, the pH of the fermentation broth is 4.5 and its total alkaloid level is 1.00 mg / ml. The amount of alkaloid components determined by liquid chromatography is 0.435 mg / ml ergocornin and epimer 0.262 mg / ml ergocriptine and epimer, 0.123 mg / ml / 3-ergocriptine and epimeye and 0.280 mg / ml ergometrine and epimer. Cell lysis of 50 liters of fermentation broth in a high speed mixer (2000 rpm) was performed for 10 minutes.

The resulting 50 L of digested cellular fermentation broth was passed through a mobile bed column containing 1.0-1.10 kg EX 207 resin at a rate of 18 L / h. The resin was washed as in Example 1 and the alkaloids were eluted from the resin with 20 L of isopropanol. (The concentration of alkaline alkaloids is 2.508 mg / ml, including the proportion of alkaloids relative to the starting fermentation broth.) The alkaline alkaloidal content of the 20 L isopropanol solution is thus 50.1 g, which corresponds to a yield of 91.2% in the adsorption-adsorption process. The isopropanol solution of the alkaloids is evaporated to dissolve the alkaloids in acidic water.

The separation of alkaloids from the acidic aqueous solution thus obtained is known in the art, e.g. Ergocornine, α- and β-ergocriptine and their epimers are extracted at pH 5 with the same solvent and the ergometrine and its epimer are extracted at pH 8. Ergocornine ergocriptine was isolated as benzene crystal (32.7 g) and ergometrine as bimaleate salt (15.1 g).

Example 3

50 liters of digested cellular fermentation broth prepared in Example 2 containing 0.435 mg / ml of ergocornine and its epimer,

Contains 0.262 mg / ml ergocriptine α and its epimer, 0.123 mg / ml ergocriptine and its epimer and 0.280 mg / ml ergometrine and epimer, passing it through a movable bed column containing 2.0 kg of EX 207 resin at 15 1 / h speed. The resin was washed as described in Example 1. From the resin, ergometrine and its epimer are first eluted with 10 l of methanol (The eluate contains 1.090 mg / ml of ergometrine and ergometrinin, and ergotoxin-type alkaloids in an amount of 0.054 mg / ml), and secondly, 10 l of 2% phosphoric acid. (85%) containing methanol. The second eluate fraction has a total ergot toxin content of 3.31 mg / ml and contains only traces of ergometrine. The yield in the adsorption-elution step was 77.9% for ergometrine and 80.8% for ergotoxins.

Ergometrine as bimaleate was obtained by evaporating the first methanolic eluate fraction to 500 ml at 40 ° C in vacuo and adding to the concentrate 6.0 g of maleic acid previously dissolved in 25 ml of methanol.

Yield: 14.22 g of crude ergometrine melea (70.5% alkaloid).

Evaporation of the phosphoric acid-methanol eluate fraction to dryness yielded 43.8 g of an ergot toxin phosphate precipitate (alkaloid content 75.5%) which could be converted to 28.9 g of ergocornine ergocriptine in a known manner by benzene crystal. (Alkaloid content is 80%.)

Example 4

The culture of Claviceps purpurea variant strain deposited under number MNG 00186 was seeded in 200 ml K medium in 750 ml Erlenmeyer flask and incubated at 24 ° C for 3 days. The resulting culture was inoculated into a 5 L K sterilized medium in a 10 L glass fermenter. Inoculation cultures are aerated at 24 ° C and stirred for 3 days. The resulting culture was inoculated with 100 L of St medium medium sterilized in an acid-resistant steel fermenter. Cultivation was continued with air flow and stirring at 24 ° C for 6 days. At the end of the culture, the fermentation broth has a pH of 5.2 and contains the following alkaloids: 0.351 mg / ml ergocriptine and epimer, 0.137 mg / ml ergocriptine and epimer and 0.152 mg / ml / 3-ergocriptine and epimer.

50 liters of fermentation broth is disclosed in U.S. Patent No. 183,266. According to the Hungarian patent, extraction is carried out with 30 L of ethyl acetate and the extract is concentrated to 10 L under vacuum. The concentrate contains 2,431 mg / ml of ergot toxin. The concentrate was then extracted with 85: 10: 5 water-formamide-phosphoric acid (85%) and the resulting 30 L acidic aqueous extract (alkaloid content 7.7 mg / ml) containing 3 x 150 g of HP 20 resin each. The resin is washed with 1.0 l of 0.25% sodium hydroxide water and then with water until the last portion is neutral. The alkaloids were eluted from the resin with ethanol containing 1.2 L of 2% phosphoric acid (85%). The eluate has an alkaline content of 16.95 mg / ml. The efficiency of the adsorption-elution step is 88.2%.

The recovery of ergocornine-ergocriptine is known in the art, e.g. in accordance with Hungarian Patent Application No. 180,056.

-4192 5S7 .5, Example

1000 g of ergotamine-type drug product (total alkaloid content 3.10 mg / g, ergotamine 2.48 mg / g, ergotamine 0.37 mg / g ergometrine, ergometrinin 0.25 mg / g) 4000 ml of ethyl acetate and 250 ml of concentrated aqueous ammonium -hydroxide solution. The drug was filtered off and the procedure was repeated twice with 2000 ml of ethyl acetate. :

The combined extracts were extracted with water (3 x 145 mL) containing 3% tartaric acid. The extracts were combined and washed with 2 x 200 mL of carbon tetrachloride to completely remove traces of ethyl acetate.

The resulting 4! for aqueous tartaric acid liquid (pH =

3.5, total alkaloid content: 0.758 mg / ml, including ergotamine 0.584 mg / ml, ergotamine 0.113 mg / ml, ergometrin and ergometrinin 0.061 mg / ml. 250 g of XAD 7 resin were added to the flask and stirred for 30 minutes. The resin is then filtered off and washed with 0.25% aqueous ammonium hydroxide and water. The alkaloids are eluted from the resin with 1000 ml of methanol.

The total alkaloid content of the eluate is 2.35 mg / ml, including 1.81 mg / ml ergotamine, 0.35 mg / ml ergotamine, 0.19 mg / ml ergometrine and ergomethrin.

The yield of the adsorption-elution process is 77.5%. 25

The methanol eluate is evaporated to dryness under reduced pressure and the residue, e.g. according to Hungarian Patent 171659, ergotamine tartrate salt was filtered.

Claims (1)

A process for purifying and enriching peptide-type nutmeg alkaloids by adsorption, characterized in that peptide alkaloids containing up to 75% water-soluble nutmeg alkaloids are optionally bound in an aqueous medium by a macroretricular resin, and dyes and fats are dissolved in pH 9-14 alkaline water and then (a) eluting the peptide-type nutmeg alkaloids with a water-miscible organic solvent optionally containing up to 50% water and / or 1-5% inorganic or water-miscible organic acid, or (b) the presence of water-soluble mother-of-rye alkaloids on aliphatic alkali containing from 1 to 6 carbon atoms of water-soluble alkaloids or from 1 to 6 alkenols optionally containing up to 50% water, followed by peptide-type alkaloids from 1 to 5% inorganic acid or In this case, elute with a water miscible organic solvent containing up to 50% water.