HU188190B - Process for preparing parasitically a drug containing an alkaloid with ergolene skeleton occurrung in nature, ergochristine - Google Patents

Abstract

According to the method of the present invention, the production of the strain begins by collecting from the gramines, which is used for rye, and then the additional variant of the "starting" population thus obtained is selected by an analytical method and finally necropsied up / down. significantly more alkaloids than those known in the art.

Description

The present invention relates to a process for the production of a drug containing a naturally occurring ergolene skeletal alkaloid, ergocristine, by spore culture of a novel strain of Claviceps purpurea that is virulent to rye.

The three components of ergotoxine hydrogenated derivatives ^5, more accurately and mixtures thereof: the dihydroergocornine, dihydroergocryptine, and dihydroergocristine in medicine still an indispensable compounds adrenolytic activity, etc. serotonin activity. (Straub., ¹⁰ FB Biological Lexicon, Vol. 1, pp. 171-172). She. (1978) Akadémiai Kiadó, Budapest].

Furthermore, it is also known to occur in nature ergolene skeleton alkaloids may be prepared in two different ways: ¹⁵

- by sterile fermentation saproptata, or

- by parasitic infestation of rye.

In addition to these two well known method of total synthesis is the latter, however, is currently not a viable option due to the high cost of ²⁰ practical way.

Parasitic strains of ergocristine are already known (Hungarian Patent Nos. 154,035 and 176,003). However, these strains produced spóratenyészetével drugs contained only 3-6 mg / ²⁵ g of an alkaloid.

The saprophite or the. competition between the production of parasitic alkaloids by parasites has not yet been decided in favor of either method, so the development of both paths is a must. ³⁰

SUMMARY OF THE INVENTION It is an object of the present invention to provide a strain having higher ergocristine content in a higher yield which

- spores well on artificial medium on agar-agar surfaces and in submerged aeration,

- the spores are resistant properly, so well-preserved ^35,

- the spores are viable and virulent, ie when re-introduced into culture medium they sprout from 98% to 100% and, when applied to rye, infect them,

- developing the drug quantity per unit area of ⁴⁰ high-ge,

- the drug contains high levels of major alkaloids and contains only insignificant amounts of minor alkaloids other than the main alkaloids.

Important steps in the preparation of the new strain 45 used in the method of the invention are:

1. Breeding of Claviceps purpurea.

2. Production of spores suitable for rye infection by propagation of the strain by fermentation and their preservation until use. 50

The new strain was prepared as follows:

Selection was started from Claviceps populations harvested from wild gramineas (Agropyron repens, Dactylus glomerata, Lohlium sp.) And cultured in rye. Productivity of the starting population:

5.0 mg / g ergocristine. The home population of the starting population is KGy-2, deposited with the National Institute of Public Health (OKI) under number 00 224. 60

The initial population provided a wide range of genotypes and balanced viability due to sexual processes. During selection, the plus variants were highlighted by a large-scale analytical method. More Tented ₅ g historically selection among the variants plus a microbiological point of view of handling. The material highlighted in this way is maintained and / or maintained. propagation followed for parasite infection. Based on the parasite life cycle, these strains have been awarded the last practical rating.

The new strain, referred to as KGy-300 and deposited with the National Institute of Public Health (OKI) under number 00 216, has beneficial properties.

The new strain gives the following results in fermentation and natural processes:

OKI 00 216 surface spore production capacity fermentation juice spore number inoculum sprout preparation germination thousand grain weight drug yield (in experimental plant) ergocristine content secondary alkaloids: ergometrine ergotamine other impurities eg. greasy oil content

210 ⁹ spores / tube

5-6 · 10 ⁸ spores / ml 95-98%

170-190 g 200-250 kg / ha

10-12 mg / g 0.1-0.3 mg / g

0.1 mg / g 15-25%

Morphological characteristics of the new strain (OKI 00 216):

(a) After 14 to 18 days on white agar medium (6 s% Dutch malt extract, 2 s% Bacto agar), it produces white short aerial mycelium colonies with a diameter of 1.5 to 2.5 cm. The colonies protrude 1-2 mm from the surface of the medium. After 10 to 12 days, conidia spores are formed which are 2.5 to 4 μ in size.

b) On medium containing malt extract, trypsin and citric acid (see example), it generates abundant, unbranched, bundled filamentous fungi which are 2-4 μm thick. After 3-4 days, the conidia holders and the conidiospores attached to them appear.

c) In citrate mannitol submersible cultures a slightly branched mass of mycelium is formed, on which conidia spores are deposited after 38-44 hours. Spore formation is complete on day 5 with 5-6 to ^{10 8} spores / ml. The resulting spore mass is heterogeneous with an average size of 4 μ.

It should be noted that in the case of Claviceps fungi, unlike other fungi, in our experience morphological features are scarce and this fact explains the close similarity to the hitherto known strains.

The following example illustrates the process according to the invention.

Example

The strain deposited with OKI at 00 216 was inoculated with 24-day-old malt agar in a 5-liter glass fermenter with the following medium

188,190 in: malt extract (Dutch) 3% by weight, trypsin 1.5% by weight, citric acid 0.4% by weight, glucose 1.0% by weight

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 containing: mannitol 5.0%, citric acid 0.7%, corn jam (50% on a dry basis) 0.1%, potassium dihydrogen phosphate 0.025%, magnesium sulfate 0.025% ¹

Incubation was carried out at 24 ° C with 230 rpm, with aeration of 1.0 rpm at pH 4.5-5.0 for 6 days. The pH was maintained by the addition of ammonium hydroxide solution within the stated range. Spore number of fermentation broth: ¹ 5.5T0 ⁸ / ml. The biomass was separated, then enriched with malt extract, bottled and stored frozen.

The germination capacity of the spores of the inoculum spore preparation is 98-100%. ²

The composition, diluted to a concentration of 30,000 spores / mm ³ , was infested with rye on 30 m ² open field plots. Infection was performed with hand infected sheets. The fruit was harvested by hand without loss. ²

Comparative study of the new strain and the parent strain gave the following result:

Strain deposit number WHO IS HE 00224 WHO IS HE 00 216 One thousand eyes weight g 160 180 ergocristine mg / g 5.5 11.5 crop yield kg / ha 150 250 ergocristine - g / ha 825, 2875

A patent claim

Claims (1)

A method for producing a drug containing a naturally occurring ergolene skeletal alkaloid, ergocristine, by spore culture of a virulent strain of Claviceps purpurea on rye, wherein the microorganism strain is Claviceps purpurea strain deposited with the National Institute of Public Health under number 00 216.