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Description

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(71) VEB Arzneimittelverk Dresden, Postfach 89/90, 8122 Radebeul 1, German Democratic Republic of Germany (DD) (72) Klaus-Dieter Volzke, Dresden, Eva Borowski, Radebeul, Klaus Braun,

Radebeul, Klaus Breuel, Dresden, Christoph Dauth, Radebeul, Dieter Erge, Halle (Saale), Werner Gravert, Radebeul, Detlef Gröger, Halle (Saale), Liselotte Höhne, Dresden, Edda Knothe, Radebeul, Monika Miiller, Radebeul, Gisela Nord , Dresden, Rudolf Schirutschke,

Radebeul, German Democratic Republic-Democratic Republic of Germany (DD) (7 ^ 0 Oy Borenius & Co Ab (5I +) Simultaneous production of ergocornine, ergocryptine and ergometrine by fermentation with maximum yields - Samtidig fram-ställning av ergokornin, ergokryptin this method is used for fermentation

The invention relates to a process for the simultaneous industrial production of ergocornine, ergocryptine and ergometrine, which process comprises culturing a new strain of the microorganism Claviceps purpurea (Fr.) immersed in synthetic media, and recovering the ergot alkaloids formed therefrom. These alkaloids are already known for their high therapeutic value, which makes them useful in the treatment of internal diseases, neurological diseases and gynecological diseases.

The industrial production of ergot alkaloids is, as is well known, still widely cultured by cultivating alkali-containing sclerotia in rye using the microorganism Claviceps purpure, which grows as a parasite. In order to avoid the considerable disadvantages associated with this preparation, attempts have long been made to cultivate Claviceps under saprophytic conditions to form an alkaloid.

However, according to general experience, very few 2 5 8 51 4 strains of Claviceps actually cultivated for this purpose are suitable for cultivation in synthetic media.

Due to the correspondingly variable composition of the alkaloid spectrum of these strains, only a part of them form alkaloids belonging to the ergotoxin group, which are particularly valuable peptide-structured ergot toxoids.

Despite the existence of some less detailed strains (cf. HU Patent Publication No. 150,631, DD Patent Publication No. 41,967, GB Patent Publication No. 1,158,380, DE Patent Publication No. 1,806,984; 1,909,216 and U.S. Pat. patent 3,884,762), still has not in any way solved the problem of saprophytic recovery of ergot toxin alkaloids alone, or in combination with other ergot alkaloids on an industrial scale.

One of the preconditions for a high yield of continuous production is that the high-yielding starting material of the stock is continuously available to the greatest extent as an inoculum for the fermentation process, as well as in a methodologically simple and reliable manner. In technical microbiology, the most conventional and advantageous method is to use certain reproductive units (conidia) of the organism to obtain a strain at a high-yielding level by cultivation techniques such as mutagenic treatment or selection of individual spores. without recourse to the very complex and expensive method described in DE-A-1,206,384 and, above all, due to the large number of spores per unit volume, small amounts of seed culture can be inoculated into is disclosed in U.S. Patent 3,884,762.

However, the application of this method requires that the strain has a certain genetic trait, that is, the ability to form a large number of spores with a large and stable ability to biosynthesize the desired alkaloids. Thus, inoculation of the nutrient solution can also take place on an industrial scale with a suspension of conidia, so that a high and repeated alkaloid yield from foreign microorganisms in a free fermentation process is achieved with the aid of 3,585 4 culture. However, it is precisely this property, namely the formation of conidia and the corresponding tendency to alkaloid biosynthesis, that is absent, as can be clearly seen from HU Patent Publication No. 150,631 and Amici et al. (Appl. Microbiology 1_8, (1 969), pp. 484 ... 468), completely or almost entirely from saprophytically cultured strains of the Claviceps purpurea ergotoxin group used to prepare alkaloids, as experience to date has shown that the tendency to form conidia prevents the tendency to form alkaloids. In this case, as is apparent from U.S. Pat. No. 3,884,762, it is necessary to cultivate a large amount of mycelium on solid nutrient media and to homogenize the seed material thus obtained under sterile conditions, which is very disadvantageous for continuous industrial-scale fermentation. In addition, as disclosed in the U.S. patent, when mycelium is used as an inoculation material in the manufacture of ergocornine and ergocryptine, a power loss of about 50% of the strain can occur when moving from a shake flask to a fermentation chamber, even on a small technical scale.

It is also apparent from said publication that when these ergotoxin-producing microorganisms are used, only a small part of the ergotoxin developed in the cells is transferred to the culture filtrate during fermentation.

Thus, in order to completely recover the alkaloids formed, both the culture filtrate and the mycelium must be treated separately. The high proportion of pigments and useless substances formed in the biosynthesis of ergot alkaloids using a submerged culture requires very complex treatment methods and the yield obtained is not satisfactory. Thus, according to GB Patent No. 1,158,380, the culture filtrate obtained must be extracted with chloroform at pH 9, the extract thus obtained must be extracted with aqueous tartaric acid, the aqueous extract thus obtained must be re-extracted with chloroform at pH 9, and this extract must be combined with a correspondingly complex and purified mycelium extract, after which the combined extracts must be evaporated and the crude alkaloid bases precipitated with hexane. The precipitate thus obtained is stripped of ergotamine sulphate with glacial acetic acid, methanol and sulfuric acid, the residue is evaporated, its aqueous solution, 58514 is re-extracted with chloroform at pH 9, the concentrated chloroform extract is chromatographed on a 100-fold amount of silica gel and finally isolated. More ergotamine is obtained from the second chromatographic fraction by evaporation and dissolving the residue in aqueous acetone.

Post-treatment according to the general method described in GB Patent No. 1,158,380 and U.S. Patent No. 3,485,722 to isolate ergot peptide alkaloids was not successful when culturing the novel Claviceps purpurea strain described in more detail below. The strong tendency of culture filtrates and mycelium extracts to form emulsions during extraction with chloroform had a disturbing effect. It was also not possible to convert ergotoxin to a crystalline base or to achieve the yields mentioned in the above-mentioned patents using non-crystalline pure or crude alkaloid concentrates.

According to DD Patent No. 41,967, an extract containing both ergotoxin and ergometrine obtained from a broth containing a low concentration of alkaloids is increasingly chromatographed in a manner known per se using increasingly polar eluting means and the individual alkaloids are crystallized in a manner known per se. However, it is well known that elution with methanolic solvents results in harmfully high pigment concentrations of ergo-metrin.

The object of the invention is to improve the industrial production of ergocornine, ergocryptine and in particular ergometrine. It is an object of the invention to provide a suitable method for this.

It has been found that a strain of Claviceps purpurea (Fr.) (Tul) which, in synthetic nutrient media containing inorganic nitrogen compounds, sugars, organic acids and conventional inorganic salts, forms a substantial ratio of both the hitherto unknown high level of alkaloid synthesis for industrial use and the ability to form a very advantageous number of spores in liquid media under certain conditions for fermentation needs, with a high ability to lead to the synthetic formation of alkaloids.

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In addition, the ergotoxin formed is present on the sixth and seventh days of cultivation, i.e. at the maximum time of alkaloid formation, for the most part, and ergometrine is virtually completely contained in the culture medium surrounding the mycelium.

The invention is therefore characterized in that a new strain of Claviceps purpurea (Fr.) Tul. IMET PA 130 under saprophytic conditions to form conidia, these conidia are used to inoculate a nutrient solution containing sucrose, inorganic nitrogen and other additives known per se, culturing from the culture broth with ethyl acetate or acetone to which a hydrocarbon mixture, especially petroleum ether-xylene mixture has been added, the extract is purified by switching the phase between the acidic aqueous phase and the organic phase, the product thus obtained is concentrated, xylene is crystallized from a highly pure, epimer-free ergocornine-ergocryptine mixture as an aromatic adduct. When the culture broth is diluted before extraction, on the one hand, some harmful by-products are produced less than usual and, on the other hand, surprisingly, a considerable increase in the amount of ergotoxin contained in the culture filtrate is also observed.

Depending on the residual alkaloid content, the biomass obtained after filtration can be extracted with acetone, the clear extract obtained by filtration can be purified after adjusting its pH to 2-3 with aqueous solutions of acids, especially phosphoric acid, in hydrocarbon mixtures such as petroleum ether or xylene. and further extracting the separated aqueous phase after adjusting its pH to 8-9 with aqueous ammonia, lower alkyl carboxylic acid esters, especially ethyl acetate, and washing the resulting extract with water.

A new cultured strain with hitherto unknown characteristics for the production on an industrial scale of technically advantageous and economically viable ergot alkaloids, in particular ergot toxin alkaloids and ergometrine, has been deposited at 5851 4 s with the Zentralinstitut fUr 130.

This strain was selectively isolated by selecting a culture from sclerotia of the test culture area that was mutagenically treated. The morphological properties of this strain are summarized in Table 1, and the strain has the following advantages over the previously known methods for the simultaneous production of ergocornine, ergocryptine and ergometrine by fermentation: In 7 days under technical fermentation conditions at a yield level of about 1300 mg ergotoxin and 550 mg ergometrine per liter of broth.

2. The ergotoxin formed is found in a considerable amount in the fermentation medium surrounding the mycelium, so that the separation of alkaloids can take place under better starting conditions compared to previously known methods.

3. Solid or liquid media form, under certain conditions, a large number of spores which can also be used to synthesize alkaloids in technical fermenters. These new properties systematically allow simpler and more reliable than previous methods for long-term preservation of spore lyophilization, as well as standardized and less susceptible to microorganisms inoculation of nutrient solution on a technical scale and thus provide a better repeatability of the overall method.

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table 1

Morphological properties of the microorganism Claviceps purpurea, strain IMET PA 130 on various agar media after culturing this strain for 14 days in petri dishes at 24 ° C Medium 1 3% sucrose 0.001% FeSO 4 · 7H 2 O 0.3% NaNO 2 1% corn extract 0.1% K 2 HPO 2 + 3% agar 0.05% MgSO 4 · 7H 2 O pH 6.8 ... 6.9

Cluster shape:

Well-developed dense irregular and strongly wrinkled clusters, the wrong side smooth, not rising from the bottom of the plate. Diameter of clusters: 1.0. ..1.5 cm Color of clusters:

Top light brown ^ purple, edge white, wrong side brown, edge light brown.

Average number of spores: e 43.6 x 10 conidia / cluster Medium 2 15% beer wort 0.1% ammonium citrate 0.5% yeast extract 3% agar 0.5% (NHu) 2PO 4 pH 6.0%

Cluster shape:

Center elevated, crater-like recess; edge radially folded, grown through agar, cluster rising from bottom of plate. Diameter of clusters: 2 .. .3 cm Color of clusters:

Top and bottom light gray.

Central itiömäärä:

C

34.1 x 10 conidia / cluster β 58514 tab. 1 cont.

Medium 3 2% glucose 50% potato aqueous extract 3% agar pH 6.0

Cluster shape:

Center elevated, crater-like recess; grown through agar, a cluster rising from the bottom of the plate.

Cluster diameter: 1.5 .. .2.0 cm Cluster color: top gray-purple, wrong side gray-brown Average number of spores: g 3.3 x 10 conidia / cluster.

Microscopic characteristics of conidia and fungal filaments:

In all cases, the conidia have the shape of an ellipsoid with dimensions of 6 ... 10 μm (long axis), resp. 4 ... 8 pm (short axis).

The fungal filaments of the culture medium are up to 200 μm in length and 4 to 8 μm in diameter. In the case of bulging hyphae, the diameter is 8 .. .16 pm. There are air straps with a length of more than 200 pm and a diameter of only 2 ... 4 pm.

The invention is illustrated in more detail below by means of some exemplary embodiments.

Example 1

To cultivate conidia spores, a suspension of IMET PA 130 Claviceps purpurea spores lyophilized in skim milk is prepared and transferred to 3% agar culture medium (pH = 6.0) containing 15% beer wort, 0.5% yeast extract, 0.5% ( NH 2 O 2 PO 4 and 0.1% ammonium citrate, and cultured as an oblique agar culture for 14 days at 19 ° C. According to this method, 1x10 spores / culture vessels can be placed in 100 ml wide-necked flasks as inoculation material for submerged culture, with which up to 120 l of nutrient solution can be inoculated if necessary.

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Example 2

The inoculum is prepared using a suspension of spores cultured according to Example 1, which is transferred at a concentration of 1 x 10 5 spores / ml to a liquid medium containing 1% wort and 2% yeast extract. The culture takes place at 22 ° C using 500 ml round-bottomed flasks with 120 ml of liquid medium placed in a rotary shaker. The culture contains, after 14 days, 6x10 spores / flask, which are used as inoculum for the fermentation of the alkaloid.

Example 3

The inoculation spores are prepared as in Example 1.

The culture medium used is 3% agar medium (pH 6.8 to 6.9) with 3% sucrose, 0.3% NaNO 2, 0.1% 1 H 2 O 2, 0.05%

MgSO 4 • 7H 2 O, 0.05% KCl, 0.001% FeSO 4 • 7H 2 O and 1% corn extract water. Using narrow-necked flasks in 500 ml industrial glass, an oblique agar culture is obtained after 14 days of culture at 24 ° C g in 3x10 spores / culture vessels, which are used as starting material for submerged culture for the synthetic production of alkaloids.

Example 4

According to Example 1, the spores of a cultured inoculum culture are suspended and some of them are used as inoculum in equal amounts in vertical round 500 ml flasks, each containing 120 ml of preculture medium (10% sucrose, 1.5% ammonium citrate, 0.1% CaiNO 2). 0.025% KH 2 PO 4, 0.01% HCl, 0.03% MgSO 4, 0.001% FeSO 4, 0.0004% ZnSO 4, distilled water, pH 5.5, sterilization 30 minutes at a pressure of about 50 KPa. The flasks are shaken at 24 ° C on a rotary shaker rotating at 175 rpm. After 7 days, the contents of the flasks are inoculated 1:10 into 300 ml Erlenmeyer flasks, each containing 120 ml of the main culture medium (20% sucrose, 1% ammonium itrate, 0.1% Ca 2 NO 4, 0.025% KH 2 PO ^, 0.01% KCl, 0.03% MgSO 4, 0.001% FeSO 4, 0.0004% ZnSO 4, distilled water, pH = 5.5, sterilization for 30 minutes at a pressure of about 50 MPa.These flasks are shaken under the same conditions as above.

10 5851 4 After 14 days, 2470 mg of alkaloid / 1 l of culture medium, calculated as ergotoxin maleate, are detected by the Van Urk reaction. The alkaloids are extracted with methylene chloride using the method of Hohmann & Rocheimeyer (Arch. Pharmazie 297 (1964), p. 186/187), separated by thin layer chromatography using diatomaceous earth saturated with formamide and determined by UV spectrophotometry. 1280 mg of ergotoxin and 340 mg of ergoraethrin / liter are obtained, the remainder being divided into two hitherto unknown natural peptide alkaloids and simple ergoline derivatives, such as agroclavine, zanoclavine, etc.

In parallel, the determination of the mycelium and filtrate separated by suction filtration shows that the filtrate completely contains ergometrine and in addition 90% of the total ergotoxin formed.

Example 3

A 10-liter glass fermentor is inoculated in a submerged spore culture as in Example 2. Each fermentation vessel contains 1.3 l of nutrient solution A with 10% sucrose, 1.5% ammonium citrate, 0.05% KHgPO 4 and 0.03% MgSO 4, 0.001% PeSO 4 and 0, 0004% ZnSO 4 in tap water (pH 5.5, sterilization for 30 minutes at a pressure of about 50 kPa, fermentation at 24 ° C with stirring at 400 rpm and aeration of 0.3 l of air / min for 7 days). afterwards, transfer the contents of one fermentation vessel to an 18 liter stainless steel fermentation vessel containing 10 l of nutrient solution B containing 10% sucrose, 1.4% citric acid, 1.0% arammonium hydroxide (25%), 0.05% Ga (NO2) 2, 0.025% KH 2 PO 4, 0.01% KCl, 0.03% MgSO 4, 0.001% FeSO 4 and 0.0004% ZnSO 4 in tap water, pH 5.5. ° C, mix at 360 rpm and aerate at 2.5 l of air / min, after which, on the fifth day, half of the contents of the fermenter are used to recover 40 l of nutrient solution C in a 63 liter stainless steel fermentor. is 20% sucrose, 1.75% citric acid, 0.075% KH 2 PO 4, 0.02% KCl, 0.03% MgSO 4 and 0.003% FeSO 4, 0.0012% ZnSO 4 in tap water. Ammonium hydroxide is added to pH 5.3, sterilized for 60 min at 110 ° C. The culture is stirred for 7 days at 24 ° C at 300 rpm and aerated with 27 liters of air / min, adding antifoam if necessary. At the end of the culture period, the culture solution contains a total of 2640 mg of alkaloids, 5851 4 per 11 liters. Analysis according to Example 4 shows that this amount is 1300 mg of ergotoxin and 550 mg of ergometrine.

Example 6

According to Example 3, the total number of spores of the cultured inoculum culture is evenly divided into two 18 liter stainless steel fermenters containing 10 l of nutrient solution A according to Example 5. The fermentors are stirred for 7 days at 24 ° C at 360 rpm and aerated at 2.5 liters. / min. The contents of these two fermentors are then transferred to a 250 liter stainless steel fermentor containing 180 L of nutrient solution B of Example 5 and cultured at 24 ° C with stirring at 160 rpm and aeration at 0.5 liters of air / min. After 5 days, the culture is transferred to a 2000 liter stainless steel fermentor containing 1400 l of nutrient solution C according to Example 5, to which 0.0005% NiSO 4 has been added. The culture is carried out at 24 ° C by stirring at 160 rpm and aerating with 0.6 liters of air per liter of nutrient solution per minute. If necessary, an anti-foaming agent is added to all fermenters. On the seventh day, the culture contains all 2430 mg of alkaloids / liter of nutrient broth. According to the analytical assay described in Example 4, 1150 mg of ergotoxin and 490 mg of ergometrine / liter of nutrient solution are obtained.

Example 7 200 l of a culture filtrate obtained from a corresponding amount of culture broth prepared according to Example 6, diluted by adding water with stirring in a volume ratio of 1: 1 before separation of the mycelium, is basified with 25% aqueous ammonia to pH 8.5 and extracted with ergotoxin. alkaloids and ergometrine in 50 liters of ethyl acetate using a two-stage countercurrent extraction apparatus. The ethyl acetate extract is concentrated under reduced pressure at a maximum of 30 ° C to 1:20 of the initial volume, then 2 volumes of chloroform are added to the evaporation residue, and the reaction mixture is allowed to stand at 4 ° C for 15 hours to crystallize the adduct of ergometrine and chloroform. 35.4 g of a yellowish adduct of ergometrine and chloroform in a yield of 75%.

12 5 85 1 4

Dissolve 35.4 g of the adduct of ergometrine and chloroform in 3.1 liters of acetone with activated carbon under heating, add to the filtrate a calculated amount of maleic acid acetone solution, separate the crystalline ergometrine dimaleinate formed by suction and dry this product at 70 ° C. The alkaloid salt is obtained as a thin-layer chromatographically pure, white-yellowish crystalline powder in 90% yields, with a purity of 98-100%. Specific rotation Cet) = +50 ° C (c = 1 in water).

The resulting ergotoxin-containing filtrate is further concentrated to a volume of 0.297 liters, after which a) the column is chromatographed using 20 times the amount of neutral activity grade I alumina, calculated on the total amount of alkaloid concentrate, and the ethyl acetates are eluted with 3.8 liters of the same solvent. The eluate is then evaporated to dryness under reduced pressure and at a maximum of 30 ° C to give 74.2 g of an evaporation residue which is dissolved in 0.212 liters of benzene and left to stand for 15 hours at 4 ° C so that the residue becomes an adduct of ergot toxin and benzene. after which the formed crystals are separated, washed with benzene and dried in a vacuum oven. 55.2 g of a pure crystalline adduct of ergotoxin and benzene with a purity of 95% are thus obtained by thin layer chromatography. Specific rotation (a) ^ = -183 ° (c = 1.8 in CHCl 3), or b) mixed with 20 times the amount of neutral activity grade I alumina, calculated on the total amount of alkaloid concentrate, until the mixture is homogeneous in color. After 15 minutes, the reaction mixture is extracted three times with 3.82 liters of ethyl acetate each time, after which the combined extracts are evaporated to dryness at 30 [deg.] C. and worked up as in (a) to give 2.5 g of thin white chromatographically pure ergotoxin and benzene. adduct product with a purity of 95%, specific rotation (α) E = = -183 ° (c = 1.8 in CHCl3).

(c) column chromatography using five times the neutral activity grade I alumina, based on the total alkaloid content of the alkaloid concentrate, and addition of activated carbon (adsorbent ratio: alumina: activated carbon = 5: 1) and ethyl acetate, eluting with 4 liters of the same solvent, followed by further elution. ) to give '55 g of a thin-layer chromatographically pure white crystalline adduct of ergotoxin and benzene with a purity of 96%. Specific rotation (ct) E 0 = -183 ° (c = 1.8 in CHCl 3).

(d) mixing using five times the amount of neutral activity grade I alumina on the column, based on the total alkaloid content of the alkaloid concentrate, and adding activated carbon (adsorbent ratio: alumina activated carbon = 5: 1) and eluting on a stirrer with 3 liters of ethyl acetate ethyl acetate, the combined extracts are evaporated to dryness at 30 [deg.] C., followed by further work-up as described in (a) to give 55 g of thin-layer chromatographically pure white crystalline adduct of ergotoxin and benzene with a purity of 96%. Specific rotation (α) p = -183 ° (c = 1.8 in CHCl 3).

Mix 55.2 g of the adduct of ergotoxin and benzene with slightly heating to 830 ml of absolute ethanol, add a calculated amount of 0.5 molar ethanolic solution of ethanesulfonic acid (abs) and then 4.2 l of anhydrous ether, separate the crystals formed by suction and dry at 70 ° C: 95% yield of pure white crystalline ergotoxin ethanesulfonate with a purity of 98-100%.

Example 8

12.0 kg of mycelium prepared according to Example 6 of a new strain, IMET PA 130 Claviceps purpurea, obtained by filtration of an equivalent amount of culture broth are extracted within 3 hours at 20 ° C with 18 liters of acetone, after which the pH of the filtered acetone-containing aqueous extract is adjusted with phosphoric acid to 2 to 3 and degrease and purify twice by extracting with 17 liters of petroleum ether and 11 liters of a mixture of petroleum ether and xylene (1: 1 by volume). The alkali-containing heavy phase obtained is extracted to pH 8 to 9 (pH adjusted with ammonia water) with a total of 7 liters of ethyl acetate, after which the 1: 1 v / v water-washed upper phase is evaporated at 30 ° C to a volume of 0.70 liter and 1.40 liters are added. 1 chloroform, after which the reaction mixture was allowed to stand overnight at 4 ° C, and the precipitated slurry was separated.

14 5851 4

The filtrate is concentrated under the above conditions to a volume of 0.180 liters, chromatographed on 1100 kg of activity grade I alumina and eluted with 2 liters of ethyl acetate, the eluate obtained is evaporated to dryness under reduced pressure and the residue 100 is dissolved in any toluene (or benzene or xylene). The reaction mixture is allowed to stand overnight at H ° C, after which the precipitate is separated off and dried in a vacuum oven to give 14.0 g of a white crystalline adduct of ergotoxin and toluene having a purity of 98-100%.

Example 9

Extract 10.0 l of the culture broth prepared according to Example 5 with stirring and add 10% by weight of ammonium sulfate at pH 8-9 and 25 ° C, 25.0 l of ethyl acetate, discard the heavy bio-phase, extract the organic phase twice with 5.0 liters of 25% phosphoric acid each time, the aqueous phases are combined, adjusted to pH 8-9 with ammonia water and extracted twice with 5.0 liters of ethyl acetate each time, after which the combined organic phases are evaporated at 30 [deg.] C. to 200 ml: n, 400 ml of chloroform are added to the evaporation residue, the reaction mixture is allowed to stand overnight at 4 [deg.] C., the crystals formed are filtered off with suction, dried in a vacuum oven to give 4.20 g or 71.2% of theory of the adduct of ergometrine and chloroform, with a purity of 90.7%. Under the above conditions, the filtrate containing ergotoxin concentrated to a volume of 120 ml is chromatographed using 300 g of activity grade I alumina, eluted with ethyl acetate, the eluate is evaporated to dryness under reduced pressure, the dry residue is dissolved in 30 ml of benzene, the reaction mixture is allowed to stand overnight, , it is dried in a vacuum oven to give 7.95 g, or 73.6%, of a theoretical amount of white crystalline adduct of ergotoxin and benzene with a purity of 91.6%.

Claims (4)

15 5851 4 A process for the simultaneous production of ergocornine, ergocryptine and ergometrine in high yields by fermentation culture and for the isolation and purification of products, characterized in that a new strain of Claviceps purpurea (Fr.) Tul. IMET PA 130 under saprophytic conditions to form conidia, these conidia are used to inoculate a nutrient solution containing sucrose, inorganic nitrogen and other additives known per se, the culture is carried out in a manner known per se from the broth with ethyl acetate or acetone to which a hydrocarbon mixture, especially petroleum ether-xylene mixture has been added, the extract is purified by switching the phase between the acidic aqueous phase and the organic phase, the product thus obtained is concentrated, ergometrine is isolated a very pure epimer-free ergocornine-ergocryptine mixture is crystallized from benzene, toluene or xylene as an aromatic adduct. Process according to Claim 1, characterized in that the conidia are used as inocula in solid or liquid media and at a temperature of 15 to 25 ° C, suitably 18 to 24 ° C. 1. A method for the production of an ergocorn, an ergocryptine and an ergometer with the aid of an enzymatic process for the isolation and processing of a product, the volume of which is obtained by means of a Claviceps purple. IMET PA 130 under saprophytic means, for which the image is concomitant, is shown to be concomitant with the presence of a sachet, an organically occurring condition and and in the case of a pair of means in the case of fermentation processes, the addition of alkaloids to the odorant-tidens slut direkt ur urlingslösningen, ur mycelet eller under med vatten utspädda och tillrenade odlingslösningen med etylacetat eller