DE3104215C2 - - Google Patents

Description

The invention relates to a process for the preparation of alkaloids at a controlled rate and controlled amount ratio by fermentation or fermentation of under saprophytic conditions especially ergocornine, α - and β-ergocryptine generating variant strains of Claviceps purpurea.

It is known that the dihydroergotoxin methanesulfonate or dihydroergotoxin ethane sulfonate in the Medicine as a regulator of the central metabolism and the central and peripheral circulation is used. These Compounds increase protein synthesis in the central nervous system and inhibit those of the pyrocatecholamines stimulated or stimulated adenyl cyclase. Further they have a weak sedative effect, inhibit reflex tachycardia, improve blood flow to the brain and lower it blood pressure. In medicine, they are mainly used for Treatment of diseases due to peripheral circulatory disorders or brain circulatory disorders.

The dihydroergotoxin is produced by hydrogenating the ergotoxin. The product obtained is a mixture of dihydroergocristine, dihydroergocornine and dihydroergocryptine (Hungarian patent 1,229,061). The dihydroergocryptin is available in two different modifications ( α and β form) (Experientia 23 [1967], 991) and, according to the latest studies, the pharmacological effects of the two forms are not entirely identical. The α- form proved to be more active in a contraceptive attempt in rats, while the β- form had a stronger vasopressor effect on decinned cats (Experientia 33 [1977], 1552). For this reason, the quantity ratio of α - and β- dihydroergocryptine is specified in the regulations for dihydroergotoxin-containing preparations. The ratio of the α form to the β form of 2: 1 is considered ideal and the dihydroergotoxin of optimal composition contains the constituents dihydroergocristine, dihydroergocornine, α- dihydroergocryptine and β- dihydroergocryptine in a ratio of 3: 3: 2: 1 Tolerances regarding the quantity ratio of α- dihydroergocryptin to β- dihydroergocryptin differ in the individual countries. In the USA and several European countries, the permissible limit values for the content of b -dihydroergocryptin are 26.7 to 44% by weight of the total content of dihydroergocryptin, while in other European countries and Japan the tolerance is 28.6 to 40%. -% lies.

The initial stage in the production of dihydroergotoxin methanesulfonate or dihydroergotoxin ethanesulfonate is the production of the non-hydrogenated ergot alkaloids (ergocristin, ergocornin, α - and β -ergocryptin).

These alkaloids are produced organically. One way to do this is parasitic biosynthesis, after which rye infected and the alkaloid produced is obtained through drug extraction. This method is mainly used in the production of  Ergocristine applied [strains used: that used in the American Type Culture Collection under number ATCC 20103 filed, described in British Patent 11 92 912 Stamm and that of judge Gedeon Vegy´szeti Gyár RT, Budapest, Hungary under the number MNG 00163 in the National collection of the Hungarian State Institute for health care (Országos Közeg´szs´gügyi Int´zet) deposited trunk].

Furthermore, the biosynthesis can also be carried out saprophytically, the alkaloids being isolated from the fermentation broth. The best known strains for this method are the strain producing ergocryptin and ergotamine, deposited with the American Type Culture Collection under the number ATCC 20102 (British patent specification 11 58 380), the strain producing ergocornin and ergosin with the American Type Culture Collection under the number ATCC 20106 filed strain (British patent 11 84 039), the ergocryptine-producing strain, deposited with the American Type Culture Collection under the number ATCC 20019 (US patent 34 85 772), the β -ergocryptin and ergosin-producing strain of Soc. Farmaceutici Italia (deposited in their own collection under No. FI 7374 and described in Belgian patent 8 24 987).

From this it can be seen that these selected strains not only those important for the production of the ergotoxin Produce alkaloids but also other ergot alkaloids, that is, their fermentation is not selective Ergotoxin oriented.

For the first time, Hungarian researchers selected strains of Claviceps purpurea, which mainly produce α - and β- ergorcryptin [Hungarian patent specification 1 52 238, deposit number MNG 0022 at the National Collection of Strains of the Hungarian State Institute for Health Care (Országos Közeg´szs´gügyi Int ´zet); Hungarian patent specification 1 64 816, deposit number MNG 0088 at the national trunk collection of the Hungarian State Institute for Health Care (Országos Közeg´szs´gügyi Int´zet)]. Even with these strains, however, the adjustment of the quantitative ratio of the alkaloids to one another during fermentation is not regulated. The ratio of the 3 alkaloids produced to one another can only be adjusted retrospectively by dividing the alkaloid mixture obtained into its constituent parts using the laborious methods available and mixing them again in the appropriate ratio.

Furthermore, a method in which 2 strains of those of the one Ergocornin, Ergocryptin and their isomers and the other Ergocrist creates, fermented at the same time become known (Swiss patent 5 77 556). From the common culture fluid of the two tribes the alkaloids listed are not specified in one designated ratio won. A regulation of this Ratio is not achieved with this method either. Another problem is that the fermentation at the same time Use of 2 trunks is much more difficult to control can be as in a process in which only the breeding optimum of a single tribe must be taken into account.

H. Kobel (in his lecture at the Federation of the European Microbiological Societies in 1977 in Basel) found that certain amino acids can influence the biosynthesis of peptide alkaloids. The biosynthesis of the ergocornin contained in the peptide part valine can be promoted by adding valine and the biosynthesis of the leucine-containing α- ergocryptine can be promoted by adding leucine. In contrast, the biosynthesis of the isoleucine-containing β- ergocryptine cannot be influenced by the addition of isoleucine. As a probable reason for this strange appearance, Kobel stated that the growth of the strains capable of producing β- ergocryptin is inhibited by isoleucine.

Since the proportion of β- ergocryptine in the culture broths of the known ergocryptin or ergocornin and ergocryptin-producing strains is smaller than required, the appropriate ratio could still not be set.

Also in terms of the amount produced the desired Overall, alkaloids allow the known processes wish left.

The invention is based on the object under remedy the disadvantages of the prior art, a method for the production of alkaloids from the fermentation of under saprophytic conditions especially ergocornine α - and β-ergocryptine generating variant strains purpurea of Claviceps, wherein this fermentation in a favorable direction , that is to say in the sense of the higher production of the β- ergocryptine and / or the production of the individual alkaloids in the desired ratio for the production of ergotoxins and / or the higher production of the alkaloids used for the ergotoxin as a whole is influenced.

This object was surprisingly achieved by the invention solved.

It was surprisingly found that the too low proportion of β- ergocryptin in the fermentation broth increases when the alkaloid production of the variant strains of Claviceps purpurea is influenced instead of with isoleucine with 1 or more compounds (s) acting as precursors in the biosynthesis of isoleucine. The precursors of isoleucine therefore do not inhibit the growth of the strains mentioned.

It was also surprisingly found that the Alkaloid production also with compounds through biochemical Regulation to stimulate the formation of isoleucine respectively stimulate, can be regulated.

Furthermore, it was surprisingly found that by the precursor (s) acting in the biosynthesis of isoleucine Compound (s) or by biochemical Regulation stimulating the formation of isoleucine Compound (s) also produce higher levels of ergotoxin used alkaloids can be achieved in total can.

The invention therefore provides a process for the preparation of alkaloids at a controlled rate and controlled quantity ratio from the fermentation of under saprophytic conditions especially ergocornine, α - and β-ergocryptine purpurea generating variant strains of Claviceps, which is characterized in that during the fermentation as a controller 1 or more compound (s) acting as a precursor in the biosynthesis of isoleucine and / or 1 or more compound (s) stimulating or stimulating the formation of the isoleucine by biochemical control.

Acting as a precursor in the biosynthesis of isoleucine Compounds are in Lehninger, Biochemistry, Worth Publ. Inc. N. Y. [1975], page 704.

As a precursor in the biosynthesis of isoleucine acting compounds and through biochemical regulation Formation of isoleucine-stimulating compounds are pre all with keto acids, hydroxy acids and amino acids 4 to 6 carbon atoms, whose biosynthesis from aspartic acid or whose phosphate runs out.

The compounds which act as precursors in the biosynthesis of isoleucine are the following in the order in which they are formed: via homoserine and threonine or 3-methylaspartic acid (2-amino-3-methylsuccinic acid) and methyloxaloacetic acid (2-methyl-3-oxosuccinic acid) α- ketobutyric acid , subsequently α-acetyl-α-hydroxybutyric acid, α, β-dihydroxy β -methyl valeric acid and α-keto-β -methyl valeric acid. Methyl oxaloacetic acid, α- keto butyric acid, threonine and / or homoserine are preferably used in the process according to the invention.

The formation of isoleucine by biochemical regulation stimulating or stimulating compounds belong biochemically to the so-called aspartates, that is, compounds whose biosynthesis is different from that of  Aspartic acid or its phosphate starts. First Line are keto acids, hydroxy acids and Amino acids with 4 to 6 carbon atoms. Is preferred or according to the invention become homocysteine and / or methionine used.

The or is preferably the regulators in an amount of 0.01 to 10 kg / m³ fermentation broth, in particular 0.05 to 5.0 kg / m³ fermentation broth used. It is also preferred, the regulator in the form of an aqueous, optionally weakly acidic solution after sterilization to add the nutrient medium, all at once, in several Servings or in one of the periods of fermentation continuously.

In the method according to the invention, the the following variant strains of Claviceps purpurea are used:

Claviceps purpurea, deposited on April 24, 1963
under the designation MNG 0022,
Claviceps purpurea, deposited on January 25, 1972
under the designation MNG 0088 and
Claviceps purpurea, deposited on May 9, 1979
under the designation MNG 00186,

each at the Hungarian National Collection State Institute for Health Care (Országos Közeg´szs´gügyi Int´zet).  

If the variant strain Claviceps purpurea MNG 00186 is used, the β- ergocryptin is obtained in large quantities even without a regulator. According to the invention, it has now surprisingly been found that in this case, when using the fixed regulators according to the invention, the ratio of the alkaloids produced hardly changes, but the total amount of alkaloids and thus also the alkaloids used for the ergotoxin in the fermentation broth increases.

The fermentation is expediently submerged in culture aerobic conditions at a carbon and nitrogen source and mineral salts and, if appropriate, other additives containing liquid nutrient medium, preferably at 20 to 26 ° C and a pH of 5.2 to 6.8 4 to 8 days long, done.

In the literature, the fermentation broth contains alkaloids often indicated by spectrophotometric values. The total alkaloid content was also present here Trap measured spectrophotometrically, the measurement results however, were not specific.

The individual ergotoxin components were eluted chromatography or quantitative liquid Chromatography determined separately.

The invention is illustrated by the following examples explained in more detail.  

Example 1

200 cm 3 of a nutrient medium GK described below were inoculated in a 500 cm 3 Erlenmeyer flask with a culture of the strain Claviceps purpurea MNG 0022 grown on an agar nutrient medium AIC described below. The Erlenmeyer flask was incubated at 24 ° C on a shaking table with a frequency of 300 minutes ^{-1 for} 3 days. The inoculum or inoculum obtained was inoculated into 5 l of a nutrient medium St described below in a 10 l fermenter. The culture was grown at 24 ° C and a stirring speed of 240 revolutions / minute with aeration with 0.5 l air per 1 fermentation broth and minute for 7 days. In the 40th, 64th, 88th and 112th hour, a 10% aqueous solution of methyloxalacetic acid in a quantity corresponding to 0.5 g of methyloxalacetic acid (dry substance) / l of fermentation broth was added to the fermentation broth as a compound which acts as a precursor in the biosynthesis of isoleucine. The quantitative ratio of the alkaloids ergocornin and ergocryptin reached the desired value on the 7th day of fermentation. The total alkaloids content, measurable by color reaction, was 1150 γ / cm³. The thin-layer chromatographic determination (J. of Chrom. 87 [1973], 433) gave 300 γ / cm³ of their right-rotating epimer. The ratio of α- ergocryptin to β- ergocryptin was 66:34 according to the investigation by means of high pressure liquid chromatography (J. Pharm. Sci. 67 [1978], 98).

The alkaloids were known from the fermentation broth Way isolated.

The culture media used had the following Compositions:

Culture medium GK

Trypcasin 7.0 g citric acid 4.1 g potassium dihydrogen phosphate 0.3 g magnesium sulfate 0.3 g ammonia until reached
pH from 5.7 to 5.8 water to 840 cm³

84 cm³ or 168 cm³ of the nutrient medium were in Piston filled. In each flask were 16 cc respectively 32 cm³ of a 50% glucose solution brought in.

Agar medium AIC

Mannitol 40.0 g citric acid 7.0 g corn steep liquor 2.0 g potassium hydrogen phosphate 1.0 g magnesium sulfate 0.3 g agar powder (Difco) 25.0 g ammonia until reached
pH from 5.2 to 5.3 water to 1000 cm³

The desired pH of the nutrient medium was maintained during of boiling. Then that became Nutrient medium in 6 cm³ portions in test tubes filled and by tilting the glasses solidified as a slant agar.

St

Sucrose 100.0 g succinic acid 10.0 g potassium hydrogen phosphate 0.25 g magnesium sulfate 0.25 g ammonium nitrate 1.0 g calcium chloride 1.0 g ammonia until reached
pH from 5.2 to 5.3 water to 1000 cm³

The nutrient medium was in portions of 0.1 l or 5 l or 100 l sterilized.

Example 2

200 cm 3 of a nutrient medium GK described in Example 1 were inoculated in a 750 cm 3 Erlenmeyer flask with the culture of the variant strain Claviceps purpurea MNG 0088 grown on an agar nutrient medium AIC described in Example 1. The culture was incubated at 24 ° C on a shaking table with a frequency of 300 minutes ^{-1 for} 3 days. The culture obtained was inoculated onto 5 l of a TC 54 nutrient medium described below in a 10 l laboratory fermenter. The culture was incubated at 24 ° C. and a stirring speed of 240 revolutions / minute with aeration with 0.5 l air per l fermentation broth and minute for 3 days. With the inoculum or inoculum obtained, 100 l of a nutrient medium St described in Example 1 were inoculated in an acid-proof fermenter provided with a stirrer and the culture was at 24 ° C. and a stirring speed of 120 revolutions / minute with aeration of 0.3 l of air each Fermentation broth and minute fermented for 6 days. In the first 5 days of fermentation, a 5% aqueous solution of α- keto butter acid was added to the culture as a precursor in the biosynthesis of isoleucine at a rate of 20 cc / hour.

After the fermentation was stopped, the total alkaloid content was 920 γ / cm³. Using the high-pressure liquid chromatography procedure given in Example 1, 260 γ / cm³ ergocornin, 155 γ / cm³ α -ergocryptin and 95 γ / cm³ β -ergocryptin were detected; a content of 80 γ / cm³ ergocorcorninin and ergocryptinin (ergocorninincryptinin) was also measurable.

The nutrient medium TC 54 had the following composition:

Sucrose 100.0 g citric acid 10.0 g sodium chloride 10.0 g potassium dihydrogen phosphate 0.5 g magnesium sulfate 0.5 g ammonia until reached
pH from 5.7 to 5.8 water to 1000 cm³

The nutrient medium was in 5 l portions sterilized in a laboratory fermenter.  

Example 3

100 cm 3 of a nutrient medium GK described in Example 1 were inoculated in a 500 cm 3 Erlenmeyer flask with the culture of the variant strain Calviceps purpurea MNG 0088 grown on an agar nutrient medium St described below. The culture was incubated at 24 ° C on a shaking table with a frequency of 300 minutes ^{-1 for} 3 days. 10 cm 3 of the culture obtained were inoculated onto 100 cm 3 of a nutrient medium T 25 described below. This culture was also incubated at 20 ° C on the shaker table for 5 days. In the 20th hour, 0.5 g of threonine was added to the fermentation broth as a compound which acts as a precursor in the biosynthesis of isoleucine. On the 5th day the total alkaloid concentration was 1200 γ / cm³. Using the high-pressure liquid-chromatographic procedure given in Example 1, 260 γ / cm³ ergocornin, 140 γ / cm³ α -ergocryptin and 80 γ / cm³ β -ergocryptin were detected; the total content of ergocorninin and ergocryptinin was 130 γ / cm³.

The culture media used had the following Compositions:

Agar nutrient medium St

The agar nutrient medium St differed from that in Example 1 described liquid nutrient medium St in that 25 g agar powder (Difco) was added to it per liter were. After adding the agar, the nutrient medium became boiled, then in 6 cm³ portions Test tubes filled, sterilized and inclined the glasses freeze to slant agar.

Culture medium T 25

Sucrose 300.0 g citric acid 15.0 g yeast extract 1.0 g potassium dihydrogen phosphate 0.5 g magnesium sulfate 0.5 g ammonia until reached
pH from 5.2 to 5.3 water to 1000 cm³

The nutrient medium was in portions of 100 cm³ filled into flasks and sterilized.

Example 4

The procedure was as described in Example 3, except that 0.5 g of homoserine was used as the precursor in the biosynthesis of isoleucine instead of threonine. On the 5th day of fermentation, the total alkaloids content was 880 γ / cm³. The fermentation broth contained 220 γ / cm³ ergocornin, 150 γ / cm³ α -ergocryptin and 80 γ / cm³ β -ergocryptin; Ergocorninin and ergocryptinin together made up 180 γ / cm³.

Example 5

The procedure was as described in Example 3, but 0.05 g of homocysteine was used as the compound which stimulates the formation of the isoleucine instead of the threonine by means of biochemical regulation. On the 5th day the total alkaloid concentration was 700 γ / cm³. The fermentation broth contained 190 γ / cm³ ergocornin, 85 γ / cm³ α -ergocryptin and 90 γ / cm³ β -ergocryptin; Ergocorninin and ergocryptinin together made up 150 γ / cm³.

Example 6

100 cm 3 of a culture medium GK described in Example 1 were inoculated in a 500 cm 3 Erlenmeyer flask with the culture of the strain Claviceps purpurea MNG 00186 grown on an agar culture medium St described in Example 3. The culture was incubated at 24 ° C on a shaking table with a frequency of 300 minutes ^{-1 for} 4 days. 10 cm 3 each of the culture obtained were introduced into 8 flasks, each containing 100 cm 3 of a nutrient medium St described in Example 1. The cultures were also fermented on the shaker table at 24 ° C for 7 days. No compound stimulating the formation of the isoleucine by biochemical regulation was introduced into 4 flasks. In the other four flasks, 0.02 g of methionine in solution in 2 cm 3 of water each was introduced in the 24th and 48th hours of fermentation. The fermentation was stopped on the 7th day.

The fermentation broths obtained without the addition of the compound stimulating the formation of the isoleucine by biochemical regulation were combined and the determination of their alkaloid content gave: 80 γ / cm³ ergocornin, 15 γ / cm³ α -ergocryptin and 30 γ / cm³ β -ergocryptin.

The fermentation broths obtained with the compound which stimulates the formation of the isoleucine by means of biochemical regulation were also combined and investigated. 250 γ / cm³ ergocornin (average content), 50 γ / cm³ α -ergocryptin and 100 γ / cm³ β -ergocryptin were detected.

Claims (5)

1. A process for the preparation of alkaloids in a controlled amount and controlled ratio by fermentation of variant strains of Claviceps purpurea which produce ergocornin, a - and β -ergocryptin under saprophytic conditions, characterized in that one or more in the fermentation as regulator Biosynthesis of the isoleucine as a precursor compound (s) and / or 1 or more by biochemical regulation uses the formation of the isoleucine stimulating compound (s). 2. The method according to claim 1, characterized in that methyloxaloacetic acid, α- keto butyric acid, threonine and / or homoserine is used as the compound (s) acting as a precursor in the biosynthesis of isoleucine. 3. The method according to claim 1 or 2, characterized in that, as a result of biochemical regulation, the formation of the Isoleucine stimulating compound (s) homocysteine and / or Uses methionine. 4. The method according to any one of claims 1 to 3, characterized in that the regulator in an amount of 0.01 up to 10 kg / m³ fermentation broth. 5. The method according to claim 4, characterized in that one the regulator in an amount of 0.05 to 5.0 kg / m³ fermentation broth starts.