RU2706119C1 - Method for extracting sum of furocoumarins from hemlock herb (conium maculatum l., family apiaceae) - Google Patents

Abstract

FIELD: pharmaceutics.

SUBSTANCE: invention refers to pharmaceutical industry, namely to a method for extracting sum of furocoumarins from hemlock herb. Method for extracting sum of furocoumarins from hemlock herb (Conium maculatum L., fam. Apiaceae), in which raw material is represented by hemlock herb in amount of 100 g, which is dried at temperature of 25 °C in a dark place protected from light, ground, treated with 300 ml of n-octane with constant agitation for 2 hours, then 500 ml of 70 % aqueous acetone solution is added to the rest of the raw material and maintained for 4 hours, filtered and to the filtrate is added 30 ml of 10 % alcohol solution of sodium hydroxide and stirred for 15 minutes, then 100 ml of 10 % aqueous sodium hydroxide solution is added, mixed and left in a closed container for 30 minutes; then, an aqueous layer is separated from the obtained extract, to which is added 100 ml of 10 % hydrochloric acid solution and stirred for 15 minutes; sum of furocoumarins is extracted by adding 250 ml of diethyl ether with constant stirring and drying in a fume cupboard at room temperature.

EFFECT: method described above enables to separate furocoumarins from hemlock with high output.

1 cl, 1 ex

Description

A method of isolating the sum of furocoumarins from the herb hemlock spotted (Conium maculatum L., fam. Apiaceae)

The invention relates to the pharmaceutical industry, in particular to a method for the isolation of pure substances from plant materials, and can be used in the pharmaceutical industry to obtain preparations of furocoumarins from the hemlock sapiens herb.

Furocoumarins - a class of compounds of predominantly natural origin, have a photosensitizing, vasodilating, antitumor, bacteriostatic and antifungal effect, etc. [1]. The following furocoumarins are found in the composition of the hemlock: bergapten, xanthotoxin, and isopimpinellin [2].

A known method of obtaining a dry extract of dental ammonia (patent No. 2171686, RU), including extraction with aqueous ethanol from the fruits of dental ammonia. The extract after washing the raw materials in the first stage of extraction is used as the extractant of the first discharge in the second stage. Then the extracts of the two stages are combined, concentrated and dried. The ratio of the mass of the feedstock and the volume of pure extractant at the stages of the extraction operation is 1: 2.4-1: 2.6. The method allows to increase the content of the sum of chromones in the dry extract.

Dental Ammonia, like spotted hemlock, belongs to the family Umbrella (lat. Apiaceae) and is the producer of furocoumarins. The disadvantage of this method is to obtain ultimately an extract from ammonia with a high content of chromones. Also, this method does not make it possible to obtain a purified amount of furocoumarins.

A known method of producing psoberan (patent No. 419221, SU) by extraction of crushed plant material (fig leaves - Ficus carica) with an organic solvent, evaporation, elution with benzene after passing through a column with an adsorbent, and purification followed by crystallization of the target product.

Psoberan is a mixture of furocoumarins - psoralen and bergapten, which is obtained from the leaves of Central Asian varieties of fig fruits (Ficus carica L.). The disadvantages of this method are the use of a foreign source of raw materials (fig leaves) to obtain furocoumarins and a long extraction time, which leads to an increase in the cost of the final product. In addition, the disadvantage of this method is the use of highly toxic reagents.

A known method of obtaining the drug ammifurin, consisting of a mixture of furocoumarins. Ammifurin is obtained by extraction of ammonium fruits with large ethanol in a ratio of 1: 6 (at a temperature of 50-60 ° C), evaporation, precipitation of furocoumarins with water, treatment with an aqueous solution of caustic soda in a ratio of 1: 2, followed by extraction of ballast substances with a chlorine-containing solvent and acidification with phosphoric acid acid, the precipitate of ammifurin is filtered off and dried in vacuum at 60-70 ° C for 2-3 hours, in addition, part of the furocoumarins passes into the mother liquor, from which these substances are isolated by keeping the solution in the day at 4-6 ° C. Then, the resulting precipitates are combined. A similar method allows you to allocate 58.3% of the total amount of furocoumarins in the feed (copyright certificate No. 1176477, USSR).

The disadvantages are the complexity of the method, the long extraction time, the use of temperature conditions, namely heating to 60-70 ° C and cooling to 4-6 ° C, as well as incomplete isolation of furocoumarins from the source of raw materials. This method is designed to isolate ammifurin (the sum of furocoumarins) from large ammonium seeds, while the proposed method is aimed at isolating the amount of furocoumarins from the grass hemlock spotted and can be used to isolate the amount of furocoumarins from other plant sources containing this group of biologically active substances .

In the analyzed patent and medical literature, an adequate prototype was not found.

Spotted hemlock (Conium maculatum L.) - a plant whose toxic properties are determined by alkaloids. It also contains fatty oil, which contains glycerides of petrozelinic and petrozelidic acids, furocoumarins, flavonoids, etc. In folk medicine, hemlock is used as a sedative, anti-inflammatory and analgesic in painful conditions accompanied by seizures or spasm of internal organs - chorea, epilepsy, migraines [3].

The objective of the present invention is to develop a method for the allocation of the sum of furocoumarins from hemlock spotted, providing the highest possible yield.

The problem is solved by including drying of the raw material, extraction of furocoumarins from the raw material with treatment with sodium hydroxide solutions, followed by extraction with diethyl ether, and drying, characterized in that the grass (100 g) of spotted hemlock is used as raw material, which is dried at a temperature of 25 ° С dark place protected from the action of light, crushed, treated with n-octane with constant shaking for 2 hours, extracted with 70% aqueous solution of acetone and gradually treated with 30 ml of 10% alcohols solution of sodium hydroxide, 100 ml of 10% aqueous sodium hydroxide solution, 100 ml of 10% hydrochloric acid solution, followed by extraction with diethyl ether and drying hood at room temperature.

New in the invention is that the raw material is grass (100 g) of hemlock, which is dried at a temperature of 25 ° C in a dark place protected from light, crushed, treated with n-octane with constant shaking for 2 hours, extracted 70 ml of an aqueous solution of acetone and stepwise treated with 30 ml of a 10% alcohol solution of sodium hydroxide, 100 ml of a 10% aqueous solution of sodium hydroxide, 100 ml of a 10% solution of hydrochloric acid, followed by extraction with diethyl ether and dried by fume hood at room temperature.

New features showed in the claimed combination of new properties that are not explicitly derived from the prior art in this field and are not obvious to a specialist. An identical set of features was not found in the patent and medical literature.

Proposed as an invention, the method can be used to obtain the sum of furocoumarins from the herb hemlock spotted, as well as other plant sources containing this group of biologically active substances.

Based on the foregoing, the present invention should be considered appropriate to the conditions of patentability "Novelty", "Inventive step", "Industrial applicability".

The invention will be clear from the following description. The method of isolating the sum of furocoumarins from the hemlock spotted (Conium maculatum L.), includes drying the raw material, extracting the furocoumarins from the raw material with treatment with sodium hydroxide solutions, followed by extraction with diethyl ether, and drying, characterized in that grass (100 g) hemlock is used as the raw material spotted, which is dried at a temperature of 25 ° C in a dark place protected from light, crushed, treated with n-octane with constant shaking for 2 hours, extracted with 70% aqueous solution of acetone and 30 ml of a 10% alcoholic solution of sodium hydroxide, 100 ml of a 10% aqueous solution of sodium hydroxide, 100 ml of a 10% solution of hydrochloric acid are then taped, followed by extraction with diethyl ether and drying in a fume hood at room temperature.

The method is as follows. Spotted hemlock grass is dried in the open air at a temperature of 25 ° C in a dark place protected from the action of light. Grind 100 g in a porcelain mortar until smooth, the resulting mass is treated with n-octane with constant shaking for 2 hours, the supernatant is drained and infused with a 70% aqueous solution of acetone. Then the extraction is filtered and 30 ml of a 10% alcohol solution of sodium hydroxide are successively added to the filtrate, mix thoroughly, 100 ml of a 10% aqueous solution of sodium hydroxide are added. Next, an aqueous layer is separated, to which 100 ml of a 10% solution of hydrochloric acid are added. Then the amount of furocoumarins is recovered by adding 250 ml of diethyl ether. The ether layer is separated and dried in a fume hood at room temperature.

Example

100 g of dried and ground raw hemlock grass was treated with 300 ml of n-octane with constant shaking for 2 hours. Then the supernatant was discarded and 500 ml of a 70% aqueous acetone solution was added to the residue of the feed and insisted for 4 hours. Next, the obtained water-acetone extract was filtered through a paper filter and 30 ml of a 10% alcohol solution of sodium hydroxide was added to the filtrate, stirred for 15 minutes, and then 100 ml of a 10% aqueous solution of sodium hydroxide was added, stirred and left in a closed container for 30 minutes. Then, the aqueous layer was separated from the obtained extract using a separatory funnel, to which 100 ml of a 10% aqueous solution of hydrochloric acid was added, and stirred for 15 minutes. The amount of furocoumarins was recovered by adding 250 ml of diethyl ether with constant stirring. The ether layer was separated using a separatory funnel and dried in a fume hood at room temperature. The resulting sum of furocoumarins was a crystalline powder of light beige color, odorless. The output of the sum of furocoumarins was 0.2 g (0.2%). Furocoumarins were separated by thin layer chromatography (TLC) on Sorbifill plates (Sorbpolymer CJSC, Krasnodar). As reference samples, standard substances of furocoumarins were used: bergapten, xanthoxin, isopimpinellin, and also a model mixture based on them (1: 1: 1). Identification was carried out by the results of chromatographic mobility and color shades of fluorescence of the isolated substances before and after treatment with reagents (10% solution of NH ₄ OH; Br ₂ ), comparing with the model mixture and standard samples of furocoumarins. According to TLC, the Rf value and color shades of fluorescence of the isolated substances before and after treatment with reagents completely coincided with the Rf value and color shades of fluorescence of the substances of the model mixture and standard samples of furocoumarins: for isopimpineline - 0.42, bergapten - 0.55, xanthotoxin - 0, 85.

Quantitative analysis of the sum of furocoumarins was carried out by high performance liquid chromatography on an Alliance system instrument (Waters) with a detector with a photodiode array on an Aliance C18 column (4.6 × 150), 5 mkm in elution mode: 0.1% phosphoric acid / acetonitrile (78:22).

List of references

1. Lozhkin A.V., Sakanyan E.I. Natural Coumarins: Methods of Isolation and Analysis / Chemical-Pharmaceutical Journal, 2006. - Volume 40., No. 6. - p. 47-56 (p. 48).

2. Meier P., Hotti N., Rischer H. Elicitation of furanocoumarins in poison hemlock (Conium maculatum L.) cell culture / Plant Cell Tiss Organ Cult (PCTOC), 2015. - 123., No. 3. - P. 443-453 (p. 444).

3. Maznev H.I. Encyclopedia of medicinal plants. - 3rd ed., Rev. and add. - M: Martin, 2004 .-- S. 496 (p. 102-103).

Claims (1)

The method of isolating the sum of furocoumarins from the grass of the hemlock spotted (Conium maculatum L., family Apiaceae), characterized in that the raw material used is the grass of the hemlock spotted in the amount of 100 g, which is dried at a temperature of 25 ° C in a dark place protected from light, crushed, treated with 300 ml of n-octane with constant shaking for 2 hours, then 500 ml of 70% aqueous acetone solution was added to the residue of the raw material and insisted for 4 hours, filtered and 30 ml of 10% alcohol solution was added to the filtrate sodium hydroxide and stirred for 15 minutes, then added 100 ml of a 10% aqueous solution of sodium hydroxide, stirred and left in a closed container for 30 minutes, then the aqueous layer was separated from the resulting extract, to which 100 ml of a 10% solution of hydrochloric acid was added and mixed for 15 minutes; the amount of furocoumarins is recovered by adding 250 ml of diethyl ether with constant stirring and dried in a fume hood at room temperature.