HU188191B - Process for preparing parasitically an alkaloid with ergolene skeleton occurring in nature, ergotamine - Google Patents

Abstract

The present invention relates to the production of ergotamine in a parasitic manner. Ergotamine and its hydrate derivative are dihydroergotamine, widely used in medicine. According to the method of the present invention, the production of a strain begins with the collection of wild-type germs, which are accustomed to rye, and then the additional variant of the "starting" population thus obtained is selected by an analytical method and finally propagated in an irreversible manner. The new strain deposited at 00215 at OKI produces substantially more alkaloids than those known so far. -1-

Description

The present invention relates to a novel Claviceps purpura virulent to rye, which is a naturally occurring ergolene-based alkaloid, a drug containing ergotamine; spore culture.

Various ® formulations containing the ergotamine active ingredient are common and have long been used in obstetrics, gynecology, and internal medicine and neurology. Dihydroergotamine produced by the hydrogenation of ergotamine is a similarly important drug. ¹⁰

It is also known that naturally occurring ergolene skeletal alkaloids can be produced in two different ways:

- sterile fermentation, so-called saprophytic way, or ¹⁵

- by parasitic infestation of rye.

Apart from these two methods, their total synthesis is known, but the latter is not practicable at present due to its high cost.

'The saprophite or the. competition between the production of parasitic alkaloid 20 alkaloids has not yet been decided in favor of either method, so further development of both paths is a must.

In the development of the parasitic method according to the invention, the following objectives have been pursued by us:

- breeding a new strain of Claviceps purpurea containing significantly more alkaloids than previously known.

- can be produced by a fermentation propagate oltóspórát strain capable THEM ^30, továbbmenőleg oltóspóra that provides for the production of stable use and containing higher drug ergotamine rye fertőzé-.

Parasitic strains of ergotamine have been known for a long time (eg Hungarian Patent No. 155,148). The drugs produced by spore culture of these strains contained about 4-5 mg / g of alkaloid.

Important steps in the preparation of the new strain used in the process of the invention are:

1. breeding of Claviceps purpurea, 40

2. production of spores suitable for rye infection by propagation of the strain by fermentation and their preservation until use,

SUMMARY OF THE INVENTION It is an object of the present invention to provide a strain having a higher yield of ergotamine with a higher yield, which is 45

- spores well on artificial media on agar-agar surfaces and in submerged aeration;

- the spores are sufficiently resistant so that they can be well preserved;

- the spores are viable and virulent, ie when re-introduced into 50 media, they germinate at 98-100% and when infused with rye;

- high levels of drug development per unit area;

- the drug has a high ergotamine content and contains only insignificant amounts of secondary alkaloid (s) other than the main alkaloid.

The new strain was prepared as follows:

Selection was started from Claviceps populations collected from wild gramineas (Agropyron repens, Dactylus θθ glomerata, Lohlium sp.) And accustomed to rye. Productivity of the starting population:

3.5 mg / g ergotamine. The home population identifier of the starting population is KGy-19, which is determined by the National Public Health θ ₅ '

was deposited with the Bank of Hungary (OKI) under number 00222.

The initial population provided a broad spectrum of genotypes and balanced viability due to sexual processes. During selection, the plus variants were highlighted by a large-scale analytical method. Further selection was made among the plus variants for microbiological manageability.

The maintenance of the biological material thus highlighted is non-sexed. propagation followed for parasite infection. Based on the parasite life cycle, the strains received the last practical rating.

The new strain, referred to as KGy-1000 and deposited with the National Institute of Public Health (OKI) under number 00215, has beneficial properties.

The new strain in fermentation and cultivation processes gives the following result:

OKI 00215 surface spore production capacity fermentation spore number germination capacity of sprout preparation weight of one thousand drug yield (in experimental plant) ergotamine content secondary alkaloids: ergometrin

1.8-10 ⁰ spores / tube

5.5-610 ⁸ spores / ml 95-98%

180-200 g 250-300 kg / ha

9-10 mg / g 0.1-0.3 mg / g other impurities e.g. greasy 20-25% oil content

Morphological characteristics of the new strain (OKI 00215):

(a) On medium containing malt agar (6% Dutch malt extract, 2% Difco agar), after 12-16 days, formed white, short mycelial colonies with a diameter of 2.0-2.5 μ. Colonies protrude 3-4 mm from the medium. After 16-20 days, conidiospores of 2.5-4 μl are mass-formed on the aerial mycelia.

b) Creates a bulk, unbranched nicellic mass on trypsin, glucose, citric acid-containing submersible medium. After 50-60 hours, the surface of mycelia is wavy, and then the conidium support begins to form on which the conidiospores attach. Their average size is 2.5-5.0 μ.

c) Initially, in a submerged culture containing citrate, mannitol (see example), it forms a bundle with little branching. 38-40 hours of age begins to conidia spore-formation, which is 5-6 days to reach the value 5T0 ⁸ spores / ml. The size of the spores formed is 3-6 μ.

The process of the present invention is illustrated in more detail by the following example.

Example

Strain deposited with OKI under number 00215 24-day malt agar (6% Dutch malt) -21

188,191 were inoculated in a 5 liter glass fermenter with the following medium:

glucose 8.0 s% trypsin 1.5 s% citric acid 0.4 s%

The pH of the medium is 5.8.

Incubation was carried out at 24 ° C with stirring at 440 rpm with aeration of 0.5 l / min for 3 days.

The culture was inoculated with 10 liters of medium of the following composition:

mannitol 5.0 s% citric acid 0.7 s% maize marmalade (based on 50% dry to 0.1 s%) potassium dihydrogen phosphate 0.025 s% magnesium sulphate 0.025 s%

Incubation was carried out at 24 ° C with 230 rpm, aeration of 1.0 rpm at pH 4.5-5.0 for 6 days. The pH was maintained by addition of ammonium hydroxide solution ²⁰ within the specified range. Spore number of fermentation broth: 5.5T0 ⁸ spores / ml.

The biomass was separated and then enriched with malt extract and glucose, bottled and stored at ²⁵ deep cooled.

The spores of the inoculum spore preparation have a germination of 97%. Diluted with the preparation to a concentration of 30,000 spores / mm ^3, cereal stage rye was inoculated on a 30 m ² open field plot. Infection was performed with hand infected sheets. The ter ⁵ MESTO harvested by hand, without loss.

Comparison of the new material and the starting material gave the following results:

Strain deposit number

OKI OKI 00 222 00 215

The weight of a thousand eyes g 160 180 ergotamine mg / g 3.5 9.5 crop yield kg / ha 150 . 280 ergotamine g / ha 525 2660

A patent claim

Claims (1)

A process for the preparation of a naturally occurring ergolene skeletal alkaloid, ergotamine-containing drug, by spore culture of a virulent strain of Claviceps purpurea in rye, wherein the microorganism strain is Claviceps purpurea strain deposited with the National Institute of Public Health under number 00215. Without illustration