EA026930B1 - Method for production of dioscinin from puncture vine - Google Patents

Abstract

The invention is related to pharmaceutical preparations production technology and to a method for producing dioscinin from puncture vine, dioscinin being the main active ingredient of polysponin, a medicinal product that is widely used for treatment and prophylaxis of atherosclerosis. The essence of the invention consists in development of a new process for dioscinin production from puncture vine comprising use of puncture vine Tribulus terrestris as raw material, grinding of dried above-ground parts, degreasing with petroleum ether, removal of less polar glycosides by successive extraction with 95% ethanol, successive extraction of polar glycosides with 50% ethanol, evaporation of ethanol, filtration of water residue, extraction of glycosides from water solution with n-butanol (butanol), washing of n-butanol extract with 10% ammonia solution, complete distilling off of butanol, dilution of the residue with water, addition of acetone, separation of precipitate, boiling of water-acetone solution to dryness, drying, obtaining the target product in the form of off-white, slightly yellowish powder.

Description

The invention relates to a technology for producing pharmaceutical preparations of pharmacy and a method for producing dioscinin from creeping Tribulus, which is the main active ingredient of the drug polysponin, which is widely used for the treatment and prevention of atherosclerosis [1, 2].

Dioscinin is a steroidal glycoside of the spirostane series, obtained for the first time from the plant of Dioscorea Euxogea [3] and which is the main active ingredient of the drug polysponin, also obtained from the said plant and which has an anti-sclerotic effect. By its chemical structure, dioscinin is a tetraoside of the steroid sapogenin diosgenin, the carbohydrate chain of which contains two molecules of L-ramnose and E-glucose.

It should be noted that the depletion of the raw material base of Euxogea due to the destruction of the roots and rhizomes of this plant caused some problems in the production of the aforementioned drug, as well as a complete halt in the production of the first similar drug diosponin [4, 5].

The search for new plant materials containing dioscinin, as well as the development of a method for its production, are an urgent task for pharmacy.

A known method for producing steroidal saponins of a number of spirostane [6] from the roots of the capsicum plant Sar81sit appitite by extraction with 70% methyl alcohol (5 times), evaporation, multiple chromatography on silica gel in three systems (a mixture of chloroform with methanol), acetylation of individual fractions with acetic anhydride in the presence of pyridine, extraction with chloroform, chromatography of acetates on silica gel, elution with a mixture of chloroform and acetone, saponification of peracetates with a 5% solution of potassium hydroxide in methane e and allocation of individual steroidal glycoside.

The disadvantages of the method are the use of methyl alcohol as an extractant, as well as chloroform, acetic anhydride, pyridine, which are highly toxic, inaccessible and expensive solvents, and acetic anhydride is a precursor;

the use of complex, lengthy and time-consuming processes, such as multiple chromatography on silica gel in three systems, elution, acetylation, saponification.

The closest method (prototype) for the production of dioscinin is the method for producing dioscinin from air-dried chopped and fat-free rhizomes of Euxerogero1u51acNua [3], including the extraction of raw materials with 80% methanol in a Soxhlet apparatus for 3 days, distillation of methanol, and the sediment removal is water-insoluble saponins, extraction of saponins from an aqueous ammonia solution with butanol, washing with ammonia water butanol extract, concentration of butanol, separation of the hygroscopic precipitate, followed by washing with a mixture of buta ol with ether (1: 1), dissolving the precipitate in anhydrous alcohol, filtering from an inorganic salt impurity, precipitating water-soluble saponins with ether, which was repeated twice, drying saponins, acetylation with acetic anhydride in pyridine for 48 hours at room temperature, dissolving the acetates in methanol filtration from a paraffin-like substance; distillation of methanol to dryness; treatment with benzene for dehydration; dissolution of acetates in carbon tetrachloride; introduction of magnesium trisilicate in a column; serial eluation first, carbon tetrachloride, then a mixture of it with benzene (1: 1), saponification of eluates, washing with benzene, and then a mixture of benzene and methylene chloride (1: 1), dissolution of individual saponin in anhydrous alcohol, precipitation of the substance with an ether mixture with 10% - acetone, washing the precipitate with petroleum ether and drying the dioscinin product in a gun at 137 ° C for 16 hours.

This method has the following disadvantages:

1) the depletion of raw materials - the roots and rhizomes of the plant Eyubsogea. necessary to obtain dioscinin and the drug polisponin;

2) the method is very complex and multi-stage, since acetylation, saponification, column chromatography, repeated elution, etc. are used, all these operations are very laborious;

3) the method is expensive, dangerous, harmful to humans, since many different solvents are used, such as methyl alcohol, chloroform, ether, acetic anhydride, benzene, carbon tetrachloride, methylene chloride, pyridine, which are poisonous, carcinogenic, dangerous, inaccessible, and acetic anhydride and ether are precursors, the implementation of which is limited.

The objective of the invention is the targeted selection of a new, more suitable plant material having a rich raw material base to obtain dioscinin, simplify, reduce the cost, reduce the duration of the process, eliminate toxicity, harmfulness and danger of the method, increase the purity of the target product.

The problem is solved in that in a method for producing dioscinin, including degreasing chloroform of ground raw materials, removing less polar glycosides by sequential extraction with 80% methanol, distilling off methanol, removing the precipitate, washing with ammonia water in a butanol extract, concentrating butanol, dissolving the precipitate in anhydrous alcohol, precipitation of water-soluble saponins with ether, the following differences are provided: as a source of raw materials, a different kind of Eugenius Euxogee

- 1 026930 a new genus and species - Tribulus terrestris Trpu1i8 1sggs51g15. the dried aboveground parts of which are crushed, degreased with petroleum ether, the less polar glycosides are removed by sequential extraction with 95% ethanol, the polar glycosides are sequentially extracted with 50% ethanol, all the extracts obtained with 50% ethanol are combined, the ethanol is evaporated, the aqueous residue is filtered, the aqueous residue is filtered , glycosides are extracted from the aqueous solution with n-butanol (butanol), the n-butanol extract is washed with 10% ammonia solution, n-butanol is completely distilled off, the residue is diluted with water, acetone is added, the precipitate is separated off, the bottom-acetone solution is evaporated to dryness and dried. A chromatographically homogeneous target product is obtained in the form of a white powder with a slightly yellowish tint, which is dioscinin C ₅₁ N ₈₂ O2 ₁ mol. mass 1030, melting point 208-210 ° C, | α | ² ° υ - 86.2 (with 0.5; 50% ethanol). The yield of the target product is 2.08 g (0.42%). The purity of the target product and the absence of extraneous impurities was established by chromatography on thin layers of sorbent.

The advantage of this method is that, as a starting material for the production of dioscinin, in contrast to the peculiar kind of the plant Euxogea, whose raw material base is depleted, a completely different new genus and species of the plant is proposed - Tribulus creeping Trn1 and 8 Yugger with broad distribution areas and rich raw materials. Sequential extraction of the feed with petroleum ether and 95% ethanol, as well as extraction of glycosides from the aqueous solution with n-butanol and washing of n-butanol extraction with 10% ammonia solution, completely free from fatty, coloring and pectin substances, less polar glycosides (water-insoluble saponins) and other related impurities, thereby ensuring a high degree of purity of the target substance. The precipitation of more polar glycosides (water-soluble saponins) from an aqueous solution with acetone significantly accelerates the process of obtaining dioscinin in an individual pure form without the use of complex, labor-intensive and lengthy processes, such as column chromatographic separation followed by elution, the corresponding chemical conversion of the substance followed by decomposition of the product, etc. D., which may cause partial or significant loss of the target product.

Another advantage of the method is that it does not use toxic, carcinogenic, dangerous, expensive and difficult to reach solvents.

There is a causal relationship between the totality of the essential features and the technical result achieved: the use of creeping TnBi1i8 1egge81g18 as raw materials is of ecological importance and does not deplete natural resources, since the claimed raw materials have wide distribution areas and rich raw materials; sequential extraction of raw materials with petroleum ether and 95% ethanol, as well as extraction of glycosides from an aqueous solution with n-butanol and washing of n-butanol extraction with 10% ammonia solution, completely free from fatty, coloring and pectin substances, less polar glycosides (water-insoluble saponins) and other related impurities, which ensures a high degree of purity of the target substance. The precipitation of more polar glycosides (water-soluble saponins) from an aqueous solution of acetone significantly accelerates the method of obtaining dioscinin in an individual pure form without the use of complex, time-consuming and lengthy processes.

The possibility of carrying out the claimed invention is shown in the following example.

Example.

500 g of dried and ground aerial parts of creeping tributaries were placed in glassware (jar) with a capacity of 3 l. For degreasing, petroleum ether was added until the raw material was completely covered and left for a day. A day later, in addition to the previous level, petroleum ether was added (to supplement the solvent fraction absorbed by the raw materials) and left for a day. The next day, the organic solvent was poured off, the raw material was poured with a new portion of petroleum ether until the raw material was completely covered and left for a day. Degreasing with petroleum ether was carried out only 4 times.

After separation of the organic solvent, the remaining raw materials were dried at ordinary room temperature in a distributed form until the solvent was completely removed, and then dried in an oven at a temperature of 80 ° С for 10 h.

To remove less polar glycosides, fat-free raw materials were placed in the same glass dish, poured with 95% ethanol until completely covered, and left for a day. The next day, the alcoholic extract was drained and the raw material was poured with a new portion of 95% ethanol. Extraction with 95% alcohol was performed 3 times. To isolate polar glycosides, the feed was extracted four times with 50% ethanol under the indicated conditions. All extracts obtained with 50% ethanol were combined, ethanol was distilled off in a water bath using a water jet pump to a water residue. The aqueous solution was left for a day. The precipitate was separated through a paper filter. The aqueous filtrate was placed in a separatory funnel, 150 ml of n-butanol was added, the mixture was shaken for 15 minutes and left to separate phases. The n-butanol phase was separated from the aqueous. A new portion of nbutanol (150 ml) was added to the aqueous phase, shaken, left and the n-butanol phase was separated. Thus, extraction with n-butanol was carried out 4 times. All n-butanol extracts were combined and washed three times with 10% ammonia solution (150 ml each). From the organic extract, n-butanol was completely distilled off. The aqueous 2,229,930 residue was diluted with water to 100 ml. 600 ml of acetone was added to the aqueous solution and left for 3

h. The precipitate was separated through a paper filter. The water-acetone filtrate was evaporated to dryness and dried in an oven at 70-80 ° C to constant weight. Yield: 2.08 g (0.42%). Received a white slightly yellowish chromatographically homogeneous powder, which is a dioscinin With ₅₁ N ₈₂ About ₂₁ , mol. mass 1030, melting point 208-210 ° C, [α] ² % - 86.2 (with 0.5; 50% ethanol). The purity of the target product and the absence of extraneous impurities was established by chromatography on thin layers of sorbent.

Literature

1. Mashkovsky M.D. Medicines, 15th edition, Moscow 2005, p. 474.

2. Sokolova L.N., Turova A.D., Schreter A.I. Nippon dioscorea is a source of raw materials for producing polysponin - an anti-sclerotic drug. // Plant resources, 1968, No. 1, p. 4345.

3. Madaev O.S., Ryzhkova V.K., Panina V.V. Saponins Iuksogea ro1u81asyuya XIV. Dioscinin. // Chemistry of natural compounds, 1967, No. 3, p. 155-158.

4. Sokolova L.N., Kichenko V.I., Rostotsky B.K., Gubina G.P. Diosponin is a new drug for the treatment of patients with atherosclerosis. // Medical industry of the USSR, 1961, No. 7, p. 4345.

5. Schroeter A.I., Zadorozhny A.M. The results of the VILR to create new herbal medicines. // Plant resources, 1982, No. 4, p. 539-547.

6. Gutsu E.V., Kintya P.K., Shvets S.A., Lazurevsky G.V. Steroid glycosides of the roots of sarkuit appit. // Chemistry of natural compounds, 1986, No. 6, p. 708-712.

Used materials and devices

1. Flasks of various capacities: 2 l (1a ^^ ae 8 & H ΌΙΝ | Ι8Ο | Α | Βοτο 3.3, 29/32), 1 l (OO-17, 29/32).

2. Glass funnel.

3. Dividing funnel.

4. Spray gun.

5. Glass bottle (with a capacity of 3 l).

6. Porcelain cups.

7. Oven (OBOBITERM R-40).

8. Plates for thin-layer chromatography ZyiToG '(Czech Republic), ZotLy1 (Ki881a, Kgakpobag 350072).

9. Water bath (NN-8).

10. Installation for distillation.

11. Petroleum ether (1 _bale = 70-100 ° C).

12. The aerial parts of the Tribulus terrestris Tg1i1i8 Yugge Chr (0.5 kg).

Claims (1)

A method of producing dioscinin from creeping Tribulus, including the degreasing of crushed raw materials, removal of less polar glycosides by sequential extraction, distillation, sediment removal, washing with butanol extract with ammonia water, concentration of butanol, dissolution of the precipitate, precipitation of water-soluble saponins, characterized in that Tribulus Tg1i1i8 1egge81g18 is used, the dried aboveground parts of which are crushed, degreased with petroleum ether, and less polar glycosides are removed. by solvent extraction with 95% ethanol, polar glycosides are sequentially extracted with 50% ethanol, all extracts obtained with 50% ethanol are combined, ethanol is evaporated, the aqueous residue is filtered, the glycosides are extracted from the aqueous solution with n-butanol, washed with n-butanol extract with 10% ammonia solution, n-butanol is completely distilled off, the residue is diluted with water, acetone is added, the precipitate is separated, the aqueous-acetone solution is evaporated to dryness and dried, the desired product is obtained in the form of a white powder with a slightly yellowish tint .