RU2078084C1 - Method of ergocrystine preparing - Google Patents

Abstract

FIELD: biotechnology, pharmacy. SUBSTANCE: ergocrystine is prepared from ergot containing low content of ergocrystine and concomitant alkaloids (ergocrystinine and ergotamine). Example: 10 kg milled ergots (content of ergocrystine + ergocrystinine = 0.21%, ergotamine - 0.02%) were extracted three-fold with 100 l 50% aqueous acetone with 100 ml concentrated $$$ (by 1 h at 20 C at stirring). Combined pourings were evaporated, 25% $$$ is added to an aqueous residue to pH = 8-9 and extracted 4 x 5 l $$$. Extract is evaporated up to 400 ml and poured into 4 l petroleum ether (70-100 C). Precipitate (38.1 g) is dissolved in mixture of 300 ml acetone, 150 ml ethanol and 4.5 ml 85% $$$, heated at 60 C for 1 h and ergocrystine phosphate (22.3 g, content is 98.2%) is obtained. Yield is 93%. EFFECT: improved method of preparing, increased yield. 4 cl, 2 tbl

Description

Известно, что для выделения эргоалкалоидов из рожков спорыньи (Clavicers purpurea) используют на головной стадии извлечения суммы алкалоидов из сырья в виде свободных оснований при pH 8-9 с помощью толуола (ксилола) [2] смеси эфир-спирт [3] ацетона [4] дихлорэтана (хлороформа) [5] С другой стороны предложены способы экстракции эргоалкалоидов из сырья при pH 3-4 подкисленными спиртами или 70% ацетоном [6,7] в виде солей (например, в виде тартратов или фосфатов).The invention relates to methods for producing one of the peptide ergot alkaloids of the ergotoxin group, which includes ergocornin, ergocristine and alpha-beta-ergocriptins. Ergocristin 1 attracts the attention of medicine and the pharmaceutical industry in connection with the use of its dihydro derivative [1] as a medication prescribed for cerebral vascular insufficiency, impaired brain function and peripheral circulation disorders (alpha-blocker)

It is known that for the isolation of ergoalkaloids from ergot horns (Clavicers purpurea), the sum of alkaloids from the raw materials in the form of free bases is used at the head stage for extracting pH 8-9 using toluene (xylene) [2] ether-alcohol [3] acetone [4] ] dichloroethane (chloroform) [5] On the other hand, methods are proposed for extracting ergoalkaloids from raw materials at pH 3-4 with acidified alcohols or 70% acetone [6,7] in the form of salts (for example, in the form of tartrates or phosphates).

Known methods [8,9,10] for the reverse transfer of the epimer of ergocristine to a natural compound by treatment with acids and at pH 2-3 (phosphoric, sulfuric) in an environment of organic and aqueous-organic solvents, since ergocristine is compared with other alkaloids of the toxin group it has an increased tendency to epimerization at C ₈ with the transition to ergocristinin in the epimer; this epimerization is especially easy at pH 8-10.

A known method of producing ergoalkaloids [4] in which 10 kg of crushed horns is treated with 12 l of a saturated aqueous solution of sodium bicarbonate and 24 l of acetone, the extraction is repeated 2 more times and the water-acetone extracts are acidified with H ₃ PO ₄ to pH 4. Acidic water-acetone extract treated sequentially with petroleum ether and ether to remove acetone and isolated from the lower layer of the alkaloids in the form of tartrate by a known method, yield 88%, purity of ergoalkaloid 87%. This method is adopted by us as a prototype.

 We propose a method for producing ergocristine from the ergot horns of Claviceps purpurea strain VKM F3410D of VILAR selection with a high content of concomitant alkaloids (ergocristinin and ergotamine), in which water-acetone extractant acidified with sulfuric acid is used at the head stage of extraction, and the ergocrist is used to isolate the main crystallization. bases and / or conversion to phosphate with simultaneous epimerization of ergocristinin.

 The conditions selected by us in the proposed method allow us to use not only high-quality raw materials, but also to work with horns that have a low content of the target alkaloid and / or a high level of impurities of the epimer and other alkaloids of the non-ergotoxin group (for example, ergotamine and its epimer).

Due to differences in the solubility of sulfates and phosphates of these impurities and the target alkaloid, it is possible to get rid of them almost completely without resorting to the laborious chromatographic purification methods proposed for this purpose in other patents [5]
The choice of 50% (by volume) aqueous acetone acidified with sulfuric acid 1: 1000, we justified in a number of experiments, the results of which are presented in table. 1. The dynamics of changes during the allocation of ergocristine qualitative and quantitative composition of raw materials, technical amount and the target substance can be traced according to the table. 2. The above information confirms that when working with raw materials with a reduced content of the target alkaloid, the proposed method allows to achieve good yields (93-96%) and high alkaloid purity (96-98%) of ergocristine substances. At the same time, the task is further solved by reducing the consumption of chlorinated or aromatic and hydrocarbon solvents and increasing the efficiency of the extraction of alkaloids from raw materials, thereby improving the environmental performance of the process. Along with this, the extraction of alkaloids from raw materials with aqueous acetone can reduce the content in the head extract of the main ballast of lipids associated with alkaloids in the raw materials, and in an amount an order of magnitude higher than the content of the target substance.

 The proposed method, thus, reduces the consumption of toxic solvents, increases the yield and quality of recoverable ergocristine, reduces the complexity of cleaning technical amounts and reduces emissions of environmentally harmful substances into the atmosphere and waste streams, allowing the use of raw materials with a high content of impurities and epimer.

 To determine the quantitative and qualitative content of ergoalkaloids in the feedstock, technical amount and preparations of ergocristine and its phosphate, the reverse phase HPLC method was used. The analysis was performed on a Cilson Gradient HPLC System (Gilson) liquid chromatograph (France) with a Mobel 116 spectrophotometric detector controlled by an IBM PCAT personal computer using the 714 HPLC System Controller Software program. The chromatograph is equipped with a Rheodine 7125 loop injector with a 20 µl loop. Column (4 • 250 mm) with sorbent Diasorb C16, 10 μm (Elsiko, Russia), mobile phase: a mixture of acetonitrile and 0.01 M phosphate buffer pH 7.0 (38:62 and 48: 52).

 The proposed method for producing ergocristine can improve the yield (93-96% instead of 88%) and the alkaloid purity (96-98% instead of 87%) compared to the prototype [4], use raw materials with a reduced content of the target alkaloid and associated alkaloids (ergocristinin and ergotamine ) (0.20% instead of 0.40%), and also reduce the consumption of acetone (50 l per 10 kg of raw material instead of 72 l of the prototype).

 The inventive step of the proposed method is determined by the fact that the increase in yield and pure ergocristine is achieved through the use, on the one hand, of extraction with acidic aqueous 50% acetone containing sulfuric acid (1: 1000), on the other hand, use at the stage of separation of the conversion to phosphate with epimerization ergocristinin in the target alkaloid. Such a combination of head extraction techniques with epimerization during the isolation of ergot alkaloids has not been used before. The epimerization conditions (composition of the reaction medium acetone-alcohol-phosphoric acid) were selected by us for ergocristine and do not repeat the data given in the patents [8,9,10].

Example 1. 5 kg of crushed horns (the content of ergocristine and its epimer 0.33%) is poured into 40 l of 50% aqueous acetone containing 40 ml of concentrated sulfuric acid, and extracted for 1 h at 20 ^o With stirring, the solvent is drained and repeated extraction twice more, each time using 30 l of extractant of the same composition. Acetone was removed in vacuo, 25% aqueous ammonia was added to the residue to pH 8-9, and 4 times 5 L of chloroform was recovered. The chloroform extract is dried, evaporated to a volume of 400 ml and poured into 4 l of petroleum ether. The evaporated precipitate was separated (19.6 g, the content of ergocristine and its epimer 82.4%). Yield 98.0%. The precipitate was recrystallized from ethyl acetate to obtain 15.8 g of ergocristine as a solvate with ethyl acetate.

Alkaloid composition of the substance,
Ergocristine 96.6
Ergocristinin 0.9
Ergocristins 1.0
Ergocornin 0.2
Ergotamine 0.3
The residue after removal of ethyl acetate from the mother liquor (5.7 g) was dissolved in 40 ml of acetone and 20 ml of ethanol containing 0.5 ml of concentrated phosphoric acid was added. Heated for 60 minutes at 60 ^{° C.} and left for 12 hours. The precipitate formed was filtered, dried, and 2.53 g of ergocristine phosphate were obtained (ergocristine content 97.5%).

Total feed yield 96.0%
Example 2. 10 kg of crushed horns (the content of ergocristine and its epimer 0.21%) is extracted in three doses with 100 l of 50% aqueous acetone, acidified with 100 ml of concentrated sulfuric acid for 1 h at 20 ^o C and with stirring. Further processing is carried out, as in example 1, and obtained after precipitation with petroleum ether 38.1 g of sediment (ergocristine and its epimer content 54.72%, 99.2% yield). The resulting precipitate was dissolved in 300 ml of acetone and 150 ml of ethanol containing 4.5 ml of conc. phosphoric acid. Heated for 60 minutes at 60 ^° C and left for 12 hours. The precipitate was filtered, dried, and 22.3 g of ergocristine phosphate were obtained (ergocristine content 98.2% ergocristinin traces). The yield of 95.5% of the initial amount of ergocristin and ergocristinin.

Total feed yield 93.0%
Literature
1. Application for a patent of Russia N 5033362/14/013626 from 03.20.92.

 2. USSR patent N 1801006, 1993.

 3. Patent of Czechoslovakia N 264880, 1989.

 4. The patent of Germany N 1240871, 1967.

 5. Application for a patent of Russia N 93007749 from 09.02.93.

 6. Switzerland patent N 79879, 1918.

 7. R. Brunner, P. L. Stutz, H. Tscherter, P. A. Stadler, J. Canad. Cnem. 57 (N 5), 1638-41, 1979.

 8. Patent GDR N 285357, 1990.

 9. Patent GNP N 180286, 1987.

 10. Copyright certificate of Czechoslovakia N 227996.1986.

Claims (4)

 1. The method of producing ergocristine by extraction of crushed ergot horns with aqueous acetone followed by its removal, extraction of the target product from the aqueous residue and using phosphoric acid in the process, characterized in that crushed ergot horns with a reduced content of ergocristine and associated alkaloids are used for extraction and the extraction is carried out 50% aqueous acetone containing sulfuric acid, with a ratio of 1000: 1, respectively, the aqueous residue after removal of acetone at pH 8 9 is extracted with chloroform, the amount of ergocristin and ergocristinin is precipitated with petroleum ether and epimerization is carried out using phosphoric acid in a mixture of acetone-alcohol with heating and ergocristine is isolated in the form of phosphate or base. 2. The method according to claim 1, characterized in that the extraction with 50% aqueous acetone is carried out for 1 h at 20 ^o C, with stirring and with a ratio of raw material extractant = 1:10 20 when triple extraction. 3. The method according to claim 1, characterized in that the epimerization is carried out in an environment of acetone ethanol 85% phosphoric acid in a ratio of 2: 1: 0.025, respectively, at 60 ^o C for 1 hour  4. The method according to claim 3, characterized in that the epimerization is carried out in an environment of acetone ethanol 85% phosphoric acid in a ratio of 2: 1: 0.03.

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