GB2092570A - Ergot alkaloids - Google Patents

Abstract

The invention relates to a new method for controlling the level and distribution of alkaloids produced by Claviceps purpurea variant strains capable of producing primarily ergo-cornine, alpha -ergocryptine and beta -ergocryptine under saprophytic conditions. According to the invention a compound of the bio-synthetic path of isoleucine or a compound which promotes the formation of isoleucine by biochemical control is added to the fermentation broth. In other respects fermentation is performed under conditions known per se. By the method of the invention the ratio of alpha and beta -ergocryptine formed in the fermentation broth can be adjusted to the optimum value.

Description

SPECIFICATION Method for controlling alkaloid formation in a fermentation process The invention relates to a method of controlling the level and distribution of alkaloids produced by Claviceps purpurea variant strains capable of producing primarily ergo-corine, a-ergocryptine and fi-ergocryptine under saprophytic conditions.

It is known that dihydroorgotoxine methanesulfonate or ethanesulfonate has an important role in therapy as a central metabolism controlling and central and peripheral circulation controlling agent. The substance promotes protein synthesis of the central nervous system and inhibits catechol-amine-stimulated adenylcyclase. Moreover it has moderate sedative effects, it inhibits reflex tachycardia, improves cerebral circulation and lowers blood pressure. The substance is applied to therapy primarily for the treatment of diseases connected with cerebral or peripheral circulation disorders.

Dihydroergotoxine is prepared by the hydrogenation of ergotoxine. The hydrogenated product is a mixture of dihydro-ergocrystine, dihydroergocornine and dihydroergocryptine (Hungarian patent specification No. 129,061). Dihydroergo-cryptine, one of the three components, may exist in two modifications [a and ss forms, see Experientia 23, 991 (1967)1, with slightly different pharmacological effects according to the most recent investigations. The a form is more active in conception inhibiting tests performed on rats, whereas the ss form exerts spinal effect, i.e. stronger vasopressor activity on decerebrated cats [Experientia 33, 1 552 (1977)1. Therefore, dihydroergotoxine-containing compositions always have prescribed ratios of a- and P-dihydroergocryptine.The composition of dihydroergotoxine considered as ideal corresponds to a dihydro ergocrystine : dihydroergocornine : a-dihydroergocryptine : fi-dihydroergocryptine ratio of 3:3:2:1, and thus a 2:1 ratio of the a and ss forms of dihydroergocryptine is regarded as optimum. The officially permitted deviation from this ideal ratio of 2:1 varies from country to country. According to the US Pharmacopoeia and some European prescriptions the ratio of ss- ergocryptine may vary within 26.7 to 44% calculated for the total ergocryptine content, whereas according to the regulations of some other European countries and Japan the permitted range is 28.6 to 40%.

The first step of the production of dihydroergotoxine methanesulfonate or ethanesulfonate is the preparation of the non-hydrogenated ergot alkaloid components, i.e. ergo-crystine, ergocornine, ss-ergocryptine and a-ergocryptine.

The components of the mixture are prepared by biological methods.

Biosynthesis may be performed parasitically on rye, whereupon the alkaloids produced are separated by plant extraction. This method is applied primarily for the preparation of ergocrystine. Strains utilized for this purpose include e.g. the strain deposited at the American Type Culture Collection under No. ATCC 20103 by Societa Farmaceutici Italia ( see British Patent Specification No. 1,192,912) and the strain deposited at the Hungarian National Collection under No. MNG 00163 by Richter Gedeon Pharmaceutical Co. Ltd.

Biosynthesis may also be performed in a sapro-phytic way, whereupon the alkaloids produced are separated from fermentation broths. The major methods and strains applied for this purpose are as follows: the method of British Patent Specification No. 1,158, 380 utilizing an ergocryptine-and ergotamine-producing strain deposited under No. ATCC 20102; the method of British Patent Specification No. 1,184,039 utilizing an ergocornine- and ergosine-producing strain deposited under No. ATCC 20106; the method of US Patent Specification No.

3,485,722 utilizing an ergocryptine-producing strain deposited under No. ATCC 20019; and the method of Belgian Patent Specification No. 824,987 utilizing a fi-ergocryptine; and ergosine-producing strain deposited in the home collection of Society Farmaceutici Italia under No. Fl 7374.

It appears from the above that these known strains produce not only the alkaloid(s) applied in the production of ergotoxine, but ergot alkaloids other than ergotoxine type, too. Thus, with these strains, fermentation is not selective to ergotoxine.

Hungarian research workers were the first in selecting Claviceps purpurea strains which produce primarily ergocornine, a-ergocryptine and ssergocryptine ( see, for example strain No.

MNG 0022, see Hungarian Patent Specification No. 152,238, and strain No. MNG 0088, see Hungarian Patent Specification No. 164,816). It has remained, however, an unsolved problem to control the ratio of the alkaloids produced by these strains during the fermentation process.

The ratio of the three alkaloids produced could be adjusted to the required value only after fermentation by separating the individual components of the alkaloid mixture and mixing them again in the proper ratio. However, the individual ergot alkaloid components are difficult to separate from each other.

Swiss Patent Specification No. 577,556 describes a process based on the simultaneous utilization of two strains. According to this method a strain which produces ergocornine, ergocryptine and isomers thereof is cultivated on a culture medium together with an ergocrys tine-producing strain, and the alkaloids are produced simultaneously in a non-specified ratio.

Thus, the alkaloid ratio cannot be adjusted by this method, either. Further problems arise from the fact that when two strains are applied simultaneously, fermentation is much more difficult to control than with the cultivation of a single strain.

H. Kobel (lecture held on the 1977 session of the Federation of the European Microbiological Societies) recognized that the biosynthesis of peptide alkaloids can be influenced by certain amino acids. More particularly, he presented that the biosynthesis of ergocornine, which contains valine in the peptide moiety, can be promoted by adding valine to the mixture, whereas the biosynthesis of a-ergocryptine, which contains leucine in the peptide chain, can be promoted by introducing leucine. In contrast, the bio-synthesis of ss-ergocryptine, which contains isoleucine in the peptide rnoie.y, cannot be influenced favourably by introducing isoleucine. As a probable explanation of this strange phenomenon it was presumed that isoleucine inhibits the growth of fungus strains capable of producing fi-ergocryptine.

Since, generally the fermentation broths of the known strains which produce ergocryptine or ergocornine and ergocryptine contain just ss-ergocryptine in a ratio less than required, the problem of adjusting the proper ratio has still remained unsolved. Therefore, we attempted to find a way of influencing the fermentation of Claviceps purpurea variant strains in a favourable direction.

We have unexpectedly found that when, instead of isoleucine, a compound in the biosynthetic path of isoleucine (further on: a bioprecursor of isoleucine) is applied to influence the alkaloid production of known Claviceps purpurea variant strains, the ratio of fi-ergocrytine increases in the fermentation broth. Consequently, the bioprecursors of isoleucine do not inhibit the growth of the fungus strains.

It has also been found that the alkaloid production can also be controlled appropriately by compounds which promote the formation of isoleucine via biochemical control (further on: biocontrolling compounds). Both the bioprecursors and the biocontrolling compounds are ketoacids, hydroxyacids and amino acids of 4 to 6 carbon atoms, the biosynthesis of which starts from aspartic acid or aspartic acid phosphate.

We have also attempted to influence the fermentation in the required direction by a new variant strain obtained by selective cultivation. In these tests we succeeded in isolating a new variant of Claviceps purpurea with a yeast-like morphology, deposited at the Hungarian National Collection on 9th May, 1979 under No. MNG 00186. This strain produces primarily ergocornine and ss-ergocryptins, thus it can be applied to enrich the proportion of fi-ergocryptine in fermentation broths.

Furthermore, it has been found that if the new variant strain is used for alkaloid production, and a bio-precursor of isoleucine or a biocontrolling compound which promotes the fermentation of isoleucine is added to the fermentation broth, the total alkaloid level of the broth increases, without influencing significantly the ratio of the individual alkaloids produced.

Accordingly, we provide a method for controlling the level and distribution of alkaloids produced by Claviceps purpurea variant strains capable of producing primarily ergocornine, aergocryptine and ssergocryptine under saprophytic fermentation conditions. According to the invention a compound of the biosynthetic path of isoleucine or a compound which promotes the formation of isoleucine by biochemical control is introduced to the fermentation broth as controlling substance for part or all of the fermentation process.

In the process of the invention a Claviceps purpurea strain which produces primarily ergocornine, a-ergocryptine and fi-ergocryptine, preferably a variant strain deposited under No.

MNG 0022, MNG 0088 or MNG 00186, is used as alkaloid-producing strain.

Fermentation is preferably performed in a submerged culture under aerobic conditions, utilizing a liquid culture broth which contains carbon and nitrogen sources, mineral salts and optionally other additives as well.

The strain is generally fermented at 20 to 26 C for 4 to 8 days; the pH of the culture medium is generally maintained between 5.2 and 6.8.

Fermentation is performed, generally in a manner known per se, in the presence of an additive which controls the ratio of the alkaloids produced. As additive controlling the ratio of the alkaloids a bioprecursor of isoleucine [Lehninger Biochemistry; Worth Publ. Inc. N.Y.

(1975), p. 704] or a biocontrolling agent which promotes the formation of isoleucine is applied.

The bioprecursors of isoleucine and the sequence of their formation is as follows: aketobutyric acid [formed through homoserine and threonine or 3-methylaspartic acid (alternatively 2-amino-3-methylsuccinic acid) and methyloxalic acid (alternataly 2-methyl-3-oxosuccinic acid)], which converts in turn to a-acetyl-a-hydroxybutyric acid, a,ss-dihydroxy-ss-methylvaleric acid and a-keto-ss-methylvaleric acid.

From biochemical aspects, the biocontrolling agents which promotes the formation of isoleucine belong to the so-cailed aspartate family, i.e. their biosynthesis starts from aspartic acid or aspartic acid phosphate. These compounds are primarily ketoacids, hydroxyacids or amino acids or 4 to 6 carbon atoms, of which homocysteine and/or methionine are the mosx preTerred.

The compound which regulates alkaloid production is preferably applied in an amount of 0.01 to 10 kg/m3 of fermentation broth, most preferably 0.05 to 5.0 kg/m3 of fermentation broth.

The regulating additive is desirably applied as an aqueous or slightly acidic aqueous solution, which is introduced into the fermentation broth after sterilization. The solution may be added either at onece or in portions; alternataly the solution can be introduced continuously during a certain period of fermentation.

The total alkaloid content of the fermentation broth was determined by photometry on the basis of colour reactions. We do not consider this value as a specific characteritic of the process, although previous patent specifications frequently characterize the production levels in terms of the total alkaloid content determined by spectrophotometry.

To characterize the fermentation process, the individual ergotoxine components were determined separately by elution chromatography or quantitative liquid chromatography.

The invention is elucidated in detail by the aid of the following non-limiting Examples.

Example 1 A culture of Claviceps purpurea MNG 0022 variant strain grown on AIC agar culture medium is inoculated onto 200 ml of a GK culture medium filled into a conic flask of 750 ml capacity, and is incubated for 3 days at 24"C on a rotary shaker (300 r.p.m.). The resulting broth is applied to inoculate 5 litres of an St culture medium filled into a laboratory fermenter of 10 1 capacity, and the mixture is fermented for 7 days at 24"C under stirring at 240 r.p.m. and aerating at a rate of 0.5 litres of air/litre of fermentation broth/min. In the 40th, 64th, 88th and 112th hours of fermentation a 10% aqueous solution of methyl-oxalacetic acid is aded to the broth as bioprecursor in an amount of 0.5 g of precursor/1 litre of fermentation broth.The ergocornine : ergocryptine ratio reaches the optimum level in the fermentation broth on the 7th day. At this stage fermentation is stopped. The total alkaloid level, determined by colour reaction, is 11 50 y/ml. According to thin layer of chromatography [J. Chrom. 87, 433 (1 973)] the fermentation broth contains 300y/ml of ergocornine and 180y/ml of ergocryptine, and the dextrorotatory epimers thereof amount to 1107/ml. Based on high pressure liquid chromatography [J. Parm. Sci. 67, 98 (1 978)], the ratio of a- and fi-argocryptine is 66:34.

The alkaloids are separated from the fermentation broth in a manner known per se.

The compositions of the culture media utilized in this Example are as follows: AIC agar medium: mannitol 40.0 9 citric acid 7.0 g corn steep liquor 2.0 9 potassium dihydrogen phosphate 1.0 g magnesium sulfate 0.3 g agar powder (Difco-Registered Trade Mark) 25.0 g ammonium hydroxide to pH 5.2-5.3 water to 1000 ml The pH of the culture medium is adjusted to the required value (5.2-5.3) during boiling.

Thereafter the culture medium is filled into test tubes in portions of 6 ml each, sterlized, and slant cultures are prepared.

GK culture medium: trypcasine 7.0 g citric acid 4.1 g potassium dihydrogen phosphate 0.3 g magnesium sulfate 0.3 g ammonium hydroxide to pH 5.7-5.8 water to 840 ml 84 ml or 1 68 ml portions of the above mixture are filled into flasks, and 1 6 ml or 32 ml, respectively, of a 50% glucose solution are added under sterile conditions.

St culture medium: sucrose 100.0 g succinic acid 10.0 g potassium dihydrogen phosphate 0.25 g magnesium sulfate 0.25 g ammonium nitrate 1.0 g calcium chloride 1.0 g ammonium hydroxide to pH 5.2-5.3 water to 1 000 ml The culture medium is sterilized in portions of 0.1, 5 or 100 litres.

Example 2 A culture of Claviceps purpurea MNG 0088 variant strain grown on AIC agar culture medium is inocculated onto 200 ml of a GK culture medium filled into a conic flask of 750 ml capacity, and is incubated for 3 days at 24"C on a rotary shaker (300 r.p.m.). The resulting broth is applied to inoculate 5 litres of a TC 54 culture medium filled into a laboratory fermenter of 10 1 capacity, and the mixture is fermented for 3 days at 24"C under stirring at 240 r.p.m. and aerating at a rate of t).5 litres of air/litre of fermentation broth/min.The resulting broth is applied to inoculate 100 litres of an St culture medium filled into an acid-fast fermenter equipped with a stirrer, and the mixture is fermented for 6 days at 24"C under stirring at 1 20 r.p.m. and aerating at a rate of 0.3 litres of air/litre of fermentation broth/min. In the first 5 days of fermentation a 5 % aqueous solution of a-ketobutyric acid is added to the broth as precursor at a rate of 20 ml/hour.

The total alkaloid level of the broth obtained after stopping fermentation is 920 y/ml. The concentrations of the individual alkaloids were determined by the chromatographic methods described in Example 1. According to this measurement the broth contains 260 y/ml of ergocornine, 155y/ml of a-ergocryptine and 95 y/ml of ss-ergocryptine, furthermore 80 y/ml of ergocorninine-cryptinine.

The compositions of the AIC, GK and St culture media are the same as given in Example 1.

The TC-54 culture medium has 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 ammonium hydroxide to pH 5.7-5.8 water to 1000 ml The culture medium is sterilized in a laboratory fermenter in portions of 5 litres.

Example 3 A culture of Claviceps purpurea MNG 0088 variant strain grown on St agar culture medium is inoculated onto 100 ml of a GK culture medium filled into a conic flask of 500 ml capacity, and is incubated for 3 days at 24"C on a rotary shaker (300 r.p.m.). 10 ml of the resulting culture are applied to inoculate 100 ml of a T 25 culture medium, and the mixture is incubated for 5 days at 20"C under shaking as described above. In the 20th hour of fermentation 0.5 g of threonine are added to the broth as precursor. On the 5th day of fermentation the total alkaloid level of the broth amounts to 1 200 y/ml. The concentrations of ergocornine and ergocryptine were determined as described in Example 1. According to this measurement the broth contains 260 y/ml of ergocornine, 1 40 y/ml of a-ergocryptine and 80y/ml of (3-ergocryptine. The total amount of ergocorninine and -cryptinine is 1 30m y/ml.

The composition of the GK culture medium is the same as described in Example 1.

St agar culture medium: 25 9 of powdered agar (Difco) are added to each litre of the St culture medium with the composition given in Example 1, the resulting mixture is boiled, then distributed to flasks in portions of 6 ml each, sterilized, and slant cultures are prepared.

T 25 culture medium: 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 ammonium hydroxide to pH 5.2-5.3 water to 1000 ml The culture medium is distributed to flasks in portions of 100 ml and sterilized.

Example 4 One proceeds as described in Example 3 with the difference that 0.5 g of homoserine are added to the broth as precursor instead of threonine. On the 5th day of fermentation the total alkaloid level of the broth amounts to 880 y/ml. The broth contains 220 y/ml of ergocornine, 1 50 y/ml of a-ergocryptine and 80 y/ml of ssergocryptine. The total amount of ergocorninine and -cryptinine is 180 y/ml Example 5 One proceeds as described in Example 3 with the difference that 0.05 g of homocysteine are added to the broth as precursor, instead of threonine. On the 5th day of fermentation the total alkaloid level of the broth amounts to 700 y/ml.The broth contains 1 90 y/ml of ergocornine, 85 y/ml of a-ergocryptine and 90 y/ml of fi-ergocryptine. The total amount of ergocornine and ergocryptine is 1 50 y/ml.

Example 6 A culture of Claviceps purpurea MNG 00186 variant strain grown on St agar culture medium is inoculated onto 100 ml of a GK culture medium filled into a conic flask of 500 ml capacity, and is incubated for 4 days at 24"C on a rotary shaker (300 r.p.m.). 10 ml portions each of the resulting culture are applied to inoculate 8 flasks containing 100 ml of St culture medium, each.

The cultures are fermented for 7 days at 24"C under shaking as described above. A solution of 0.02 g of methionine in 2 ml of water is added to 4 of the 8 flasks in the 24th and 48th hours of fermentation, whereas the remaining 4 flasks, to which no precursor is added, serve as controls. Fermentation is stopped on the 7th day, the control broths (i.e. the contents of the flasks without precursor) are combined, and the alkaloid content of the broth is determined. The broth contains 80 y/ml of ergocornine, 1 5 y/ml of a-ergocryptine and 30 5/mi of ss- ergocryptine.

The contents of the flasks to which precursor was added are combined as well, and the alkaloid content of the broth is determined. The broth contains 250 y/ml of ergocornine, 50 y/ml of a-ergocryptine and 100 y/ml of ss-ergocryptine.

The compositions of culture media GK. St and St-agar are the same as given in Examples 1 and 3, respctively.

Claims (9)

1. A method for controlling the level and distribution of alkaloids produced by Claviceps purpurea variant strains capable of producing primarily ergocornine, a-ergocryptine and ss- ergocryptine under saprophytic fermentation conditions, wherein a compound of the biosynthetic path of isoleucine or a compound which promotes the formation of isoleucine by biochemical control is introduced to the fermentation broth as a controlling substance for part or all of the fermentation process.

2. A method as claimed in claim 1 wherein the controlling substance is methyloxalacetic acid, a-ketobutyric acid, threonine and/or homoserine, said substance being a compound of the biosynthetic path of isoleucine.

3. A method as claimed in claim 1 wherein the controlling substance is homocysteine and/or methionine, said substance being a compound which promotes the formation of isoleucine by biochemical control.

4. A method as claimed in any of claims 1 to 3, wherein the controlling substance is used in an amount of 0.01 to 10 kg/m3 of fermentation broth.

5. A method as claimed in claim 4 wherein the controlling substance is used in an amount of 0.05 to 5.0 kg/m3 of fermentation broth.

6. A method as claimed in any of claims 1 to 5 wherein the strain of Claviceps purpurea used is selected from strains MNG 0022, MNG 0088 and MNG 00186.

7. A method as claimed in any of claims 1 to 6 wherein fermentation is performed at from 20 to 26"C from 4 to 8 days at a pH between 5.2 and 6.8.

8. A method for producing alkaloids substantially as hereinbefore described.

9. A method for producing alkaloids substantially as hereinbefore described with reference to any of Examples 1 to 6.