RU2545799C2 - Method for preparing lappaconitine hydrobromide (versions) - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmaceutical industry, namely to a method for preparing lappaconitine hydrobromide (versions) A method for preparing lappaconitine hydrobromide is implemented by extraction of Aconitum leucostomum root and herb or Aconitum septentrionale root and herb in methylene chloride in a continuous extraction apparatus that is followed by decontamination by means of flash chromatography (version 1), or extraction of a herbal raw material in a polar organic solvent followed by extract removal from the organic solvent (version 2), alkalinisation and extraction of the prepared extract in methylene chloride followed by decontamination of the extract by flash chromatography.

EFFECT: method for preparing lappaconitine hydrobromide provides simplifying the technological process, reducing its length and improving higher yield of the end product of officinal purity.

7 cl, 1 tbl, 9 ex

Description

The invention relates to the pharmaceutical industry, and in particular to a method for producing (and a variant thereof) lappaconitine hydrobromide (allapinin) used as an antiarrhythmic drug.

Lappaconitine hydrobromide is obtained by extraction from the roots or grass of white aconite (Aconitum leucostomum) or the roots or grass of northern aconite (Aconitum septentrionale).

Known methods for producing lappaconitine are based on the extraction of the specified plant material with various solvents: methanol (Marion L., Fonzer L., Wilkins CK, Boca Jr and JP, Sandberg F., Thorsen R., Linden E. // Can J. Chem. 1967 vol. 45, pp. 969-973 [1]); 80% ethanol (Ross A., Pelletier SW // Tetrahedron. 1992, vol. 48, No. 7, pp. 1183-1192 [2]; dichloroethane (Platonova T.F., Kuzovkov A.D., Massagetov P. .S.// JOKH. - 1958, p. 258-261 [3]); chloroform (Usmanova S.K., Telnov V.A., Yunusov M.S., Abdullaev N.D., Schreter A.I. ., Filippova G.B. // KhPS. - 1987, p. 879-883 [4]).

With slight variations in the extraction conditions and extractants, the generally described sequence for preparing lappaconitine described in the prior art comprises the following steps:

- extraction of crushed dry plant material with an organic solvent,

- evaporation of the obtained extract to complete removal of the solvent,

- acidification of the residue to acidic reaction,

- extraction of the resulting acidic solution with a water-immiscible organic solvent to remove non-basic impurities,

- alkalization of the aqueous solution to an alkaline reaction,

- separation of secreted lappaconitine (by filtration or extraction),

- the conversion of the base of lappaconitine to hydrobromide by the action of hydrobromic acid,

- purification to pharmacopoeial purity of lappaconitine hydrobromide by crystallization from alcohol.

In some cases, column chromatography on alumina was additionally used to purify the bulk of the lappaconitine base [1] or the combined mother liquors [2].

The structure of lappaconitine contains an ester fragment of N-acetyl anthranilic (o-aminobenzoic) acid. Due to this, this molecule is very labile; therefore, hydrolysis of both the ester and N-acetyl moieties can occur rather quickly in acidic and basic media. Thus, the process of obtaining lappaconitin from plant materials according to known methods requires great care in observing the pH of the solution and the temperature regime at the stage of acidification of the stripped extract, at the stage of alkalization after extraction of non-basic impurities, as well as in the preparation of hydrobromide by acidification of lappaconitin base with hydrobromic acid. In addition, to avoid hydrolysis, all of these methods should be carried out quickly and not take much time to minimize side hydrolysis processes. Failure to comply with the above conditions can easily lead to a significant decrease in the yield of the target product due to the hydrolysis process at one or more stages of the method.

Closest to the claimed methods is a method for producing lappaconitine hydrobromide by 3-5 times extraction of roots or grass of northern aconite or roots or grass of white aconite with 50-80% by volume aqueous solution of acetone, followed by distillation of acetone from the combined water-acetone extract by removing impurities of a non-alkaloid nature from the aqueous residue acidified with sulfuric acid by a single treatment with dichloroethane, isolation of alkaloids from the alkalized solution by 2-3-fold extraction with dichloroethane or ethyl acetate, treatment of alkaloids with an alcoholic solution of hydrobromic acid and a single recrystallization of lappaconitine hydrobromide from aqueous alcohol (RU 2123347, 1998 [5]).

The disadvantages of this method include the duration of the production cycle (about 60 hours: 21-28 hours are spent on the extraction of plant materials by 3-5 times extraction, a certain time is spent on evaporation of the extractant, 4 extractions are carried out with dichloroethane, about 24 hours are spent on crystallization of the base lappaconitine and about 10 hours on crystallization of lappaconitine hydrobromide), significant amounts of resources spent on the production cycle, the possibility of adverse reactions idrolysis, insufficiently high yield of the product (the average yield of the target product is about 0.595% based on the initial dry raw material).

The objective of the present invention is to simplify the process, reduce its duration and increase the yield of the target product of pharmacopeia purity.

The goal is achieved by the proposed method for producing lappaconitine hydrobromide, which is carried out as follows.

The roots or grass of northern aconite (Aconitum septentrioale) or white aconite (Aconitum leucostomum) is subjected to extraction with methylene chloride in a continuous extraction apparatus, the obtained extract is directly subjected to flash chromatography.

As the sorbent for flash chromatography, alumina or silica gel is used. In this case, the adsorbed alkaloids are eluted with an organic solvent, such as acetone or ethyl acetate. The resulting ethyl acetate solution of lappaconitine base is evaporated, dissolved in acetone, and the acetone solution of lappaconitine base is treated with a solution of hydrobromic acid in acetone, the crude lapapconitin hydrobromide is filtered, dried, and lapapconitin pharmacopoeial purity hydrobromide is obtained by recrystallization from ethanol.

Silica gel is preferably used as flash chromatography sorbent using ethyl acetate as eluent.

Alumina using acetone as eluent is preferably used as a flash chromatography sorbent.

As a device for continuous extraction using conventional devices for extraction in the system "solid substance - liquid", such as Soxhlet apparatus, percolators.

The proposed method using continuous extraction of the original plant material, in addition, can significantly reduce the amount of solvent used. The extractant used is methylene chloride, which is a cheap low-boiling solvent. Extraction with boiling methylene chloride does not lead to tarring, since its boiling point is 42 ° C.

It should be noted that the proposed method using continuous extraction of the initial plant material with methylene chloride eliminates the acidification and subsequent alkalization of the extracts, that is, exclude the possibility of side hydrolysis reactions. For purification, the obtained extract is passed through a thin layer of sorbent (flash chromatography), which results in the sorption of the sum of alkaloids and more polar impurities on the sorbent and the removal of low-polar colored impurities.

The goal is also achieved by a variant of the proposed method for producing lappaconitine hydrobromide, as follows:

The roots or grass of northern aconite (Aconitum septentrioale) or white aconite (Aconitum leucostomum) is subjected to extraction with a polar organic solvent selected from the group consisting of ethanol, acetone and their aqueous solutions, followed by removal of the polar organic solvent from the extract, alkalization and extraction of the resulting chloride with chlorine methylene, followed by purification of the obtained extract flash chromatography.

As the sorbent for flash chromatography, alumina or silica gel is used. In this case, the adsorbed alkaloids are eluted with an organic solvent, such as acetone or ethyl acetate. The resulting ethyl acetate solution of lappaconitine base is evaporated, dissolved in acetone and the acetone solution of lappaconitine base is treated with a solution of hydrobromic acid in acetone, the crude lapapconitin hydrobromide is filtered, dried and pharmacopoeial purity lapapconitin hydrobromide is obtained by recrystallization from ethanol

Silica gel is preferably used as flash chromatography sorbent using ethyl acetate as eluent.

Alumina using acetone as eluent is preferably used as a flash chromatography sorbent.

In this method, the removal of the polar organic solvent from the extract is carried out under reduced pressure, then the still bottoms are made alkaline to pH 8-10 and extracted with methylene chloride. The resulting solution in methylene chloride is then used in the same sequence for flash chromatography as in the case of the proposed method using continuous extraction.

The extraction of plant materials using a polar solvent in one of the embodiments of the present method is carried out in an apparatus for continuous extraction. This embodiment of the claimed method allows to obtain an additional reduction in the duration of the process.

In the proposed methods, flash chromatography is used to purify the methylene chloride extract by passing the extract through a thin layer of sorbent, which results in the sorption of the sum of alkaloids and more polar impurities on the sorbent, and the removal of low-polar colored impurities.

As an adsorbent for flash chromatography, alumina or silica gel is used. In this case, the adsorbed alkaloids are eluted with an organic solvent, for example ethyl acetate or acetone. Evaporation of an ethyl acetate or acetone solution gives a crude base of lappaconitine, the content of the target alkaloid in which reaches 80%. Thus, the eluent used in the method (ethyl acetate or acetone) is almost completely regenerated and can be used again for the next elution.

The resulting crude base of lappaconitine is dissolved in a minimum amount of acetone, cooled and carefully acidified with a 48% solution of hydrobromic acid in acetone. The precipitate of lappaconitine hydrobromide (allapinin) is filtered off, washed with a minimum amount of cold acetone, methylene chloride and dried.

In the case of using alumina, acetone is used to elute the crude base of lappaconitine. In this case, the resulting solution of lappaconitine base is concentrated by evaporation of the solvent in vacuo and acidification is carried out further, and lappaconitine hydrobromide is isolated and purified just like ethyl acetate is used as an eluent.

The purity obtained by the proposed methods of the preparation of lappaconitine hydrobromide is more than 90%. Post-treatment to pharmacopoeial purity is achieved by a single recrystallization from ethanol.

After a single crystallization from ethanol, the purity of the obtained lappaconitine hydrobromide exceeds 97%.

In the proposed method using continuous extraction of plant materials, the process time is about 30 hours, and in the case of using the extraction of plant materials with a polar organic solvent, it is about 12 hours. The average yield of the product based on the initial dry raw material in the proposed methods is 0.73% (which is 20% higher than the average yield of the product according to the known method).

Thus, the goal, which is to simplify the process, reduce its duration and increase the yield of the target product of pharmacopeia purity, is achieved by the proposed methods for producing lappaconitine hydrobromide (options) based on a fundamentally new approach to the purification of lappaconitin, which is based on the use of flash chromatography for purification of the obtained alkaloid extract by passing it through a thin layer of sorbent, which achieves the sorption of the amount of alkaloids and more Yarnykh contaminants on the sorbent, and removing colored impurities of low polarity. This highly effective method has never been used to obtain pharmacopoeial purity lappaconitine. This method does not require the use of special equipment and devices, allows the use of significantly smaller amounts of sorbent and eluent, and, in addition, the chromatography process takes less time, and this method is also easily scalable.

Thus, the proposed methods are a very flexible and effective approach to obtaining lappaconitine hydrobromide. Depending on the requirements for the process, it is possible to use various equipment, as well as a wide range of solvents. Regardless of the sequence used, the number of process steps is significantly reduced and a high yield and purity of allapinin is achieved using just one recrystallization.

The invention is further illustrated by the following specific embodiments of the invention.

The results of the isolation of lappaconitine hydrobromide from plant materials under the conditions of the proposed methods are summarized in the Table.

EXAMPLES

Example 1

The use of continuous extraction (methylene chloride), the sorbent is silica gel

In a Soxhlet apparatus for continuous extraction with a volume of 500 ml, 250 g of ground to a state of fine powder of dry plant material containing lappaconitine (white aconite roots) is ground. Extraction of 250 ml of methylene chloride is carried out for 24 hours (in about 18 hours, about 90% of the target substance is extracted). The obtained extract is passed by gravity through a layer of silica gel on a filter with a porous plate (D = 3 cm, H layer = 4 cm, silica gel weight ~ 12 g). The remainder of methylene chloride, not merged by gravity, is pumped to dryness in vacuo. The eluent (methylene chloride) is yellow-brown in color, containing slightly polar colored impurities discarded, with further regeneration of methylene chloride on a rotary evaporator.

25 ml of ethyl acetate are applied to the filter and allowed to drain by gravity. This portion of the eluent is discarded, sent for the regeneration of ethyl acetate.

The target lappaconitin was eluted with silica gel ~ 150 ml ethyl acetate (control - TLC, silufol, acetone, R _f lappaconitine ~ 0.65), and the resulting lappaconitine solution was evaporated to dryness on a rotary evaporator, obtaining about 1.5 g of a yellow, viscous, crystallized upon standing oil . According to HPLC, the content of the target lappaconitine in this residue reaches 80%.

The residue was dissolved in 3 ml of acetone and cooled to -10 ° C. A solution of ~ 0.3 ml of 48% hydrobromic acid in 2 ml of acetone prepared in advance is added dropwise to the resulting solution with stirring from a dropping funnel (control - universal indicator paper, pH ~ 5). The precipitated white crystalline precipitate of lappaconitine hydrobromide is triturated with a spatula, left in the freezer at -18 ° C for 30 minutes, and then filtered on a filter with a porous plate. The filter cake was washed with a minimum amount of cold acetone (3 ml), and then washed with methylene chloride (5 ml) and dried. Obtain about 1.5 g of lappaconitine hydrobromide, purity according to HPLC more than 90%.

For the final purification of lappaconitine hydrobromide, it is crystallized from alcohol, at the rate of 2 ml of alcohol per 850 mg of hydrobromide. About 1.61 g of lappaconitine hydrobromide are obtained with a purity of more than 97%.

The total yield of extraction of lapapconitin from plant materials in the case of using this approach is 0.645%.

Example 2

Use of continuous extraction (methylene chloride), sorbent - alumina

In a Soxhlet apparatus for continuous extraction with a volume of 500 ml, 250 g of ground to a state of fine powder of dry plant material containing lappaconitin (roots of northern aconite) is ground to a state of fine. Extraction of 250 ml of methylene chloride is carried out for 24 hours (in about 18 hours, about 90% of the target substance is extracted). The obtained extract is passed by gravity through a layer of aluminum oxide on a filter with a porous plate (D = 3 cm, H layer = 3 cm, weight of aluminum oxide ~ 15 g). The remainder of methylene chloride, not merged by gravity, is pumped to dryness in vacuo. The eluent (methylene chloride) is yellow-brown in color, containing slightly polar colored impurities discarded, with further regeneration of methylene chloride on a rotary evaporator.

The target lappaconitin is eluted with alumina using ~ 200 ml of acetone (control - TLC, silufol, acetone, R _{f of} lappaconitin ~ 0.65). The resulting solution of lappaconitine is concentrated on a rotary evaporator to a volume of 3-4 ml. The resulting residue was cooled to -10 ° C and, with stirring, a solution of ~ 0.3 ml of 48% hydrobromic acid in 2 ml of acetone prepared in advance was added dropwise from a dropping funnel (control - universal indicator paper, pH ~ 5). The precipitated white crystalline precipitate of lappaconitine hydrobromide is triturated with a spatula, left in the freezer at -18 ° C for 30 minutes and then filtered on a filter with a porous plate. The filter cake was washed with a minimum amount of cold acetone (3 ml), and then washed with methylene chloride (5 ml) and dried. Obtain about 1.3 g of lappaconitine hydrobromide, purity according to HPLC more than 90%.

For the final purification of lappaconitine hydrobromide, it is crystallized from alcohol, at the rate of 2 ml of alcohol per 850 mg of hydrobromide. About 1.25 g of lappaconitine hydrobromide are obtained with a purity of more than 97%.

The total yield of extraction of lappaconitine from plant materials in the case of using this approach is 0.5%.

Example 3

The method is carried out analogously to example 2, but using as a plant raw material grass aconite white. The conditions and results of the isolation of lappaconitine hydrobromide are presented in the Table below.

Example 4

The method is carried out analogously to example 2, but using the northern aconite grass as plant material. The conditions and results of the isolation of lappaconitine hydrobromide are presented in the Table below.

Example 5

The use of continuous extraction (acetone), the sorbent is silica gel.

In a Soxhlet apparatus for continuous extraction with a volume of 500 ml, 250 g of ground to a state of fine powder of dry plant material containing lappaconitin (roots of northern aconite) is ground to a state of fine. Extraction is carried out with 250 ml of acetone for 4 hours. The extract obtained is evaporated to dryness on a rotary evaporator, the residue is dissolved in 250 methylene chloride and passed by gravity through a layer of silica gel on a filter with a porous plate (D = 3 cm, H layer = 4 cm, silica gel weight ~ 12 g). The remainder of methylene chloride, not merged by gravity, is pumped to dryness in vacuo. The eluent (methylene chloride) is yellow-brown in color, containing slightly polar colored impurities, discarded, with further regeneration of methylene chloride on a rotary evaporator.

25 ml of ethyl acetate are applied to the filter and allowed to drain by gravity. This portion of the eluent is discarded, sent for the regeneration of ethyl acetate.

The target lappaconitin is eluted with silica gel ~ 150 ml ethyl acetate (control - TLC, silufol, acetone, R _f lappaconitine ~ 0.65), and the resulting lappaconitine solution is evaporated to dryness on a rotary evaporator, obtaining about 2.5 g of a yellow, viscous, crystallized upon standing oil . According to HPLC, the content of the target lappaconitine in this residue reaches 80%.

The residue was dissolved in 4 ml of acetone and cooled to -10 ° C. A solution of ~ 0.3 ml of 48% hydrobromic acid in 2 ml of acetone prepared in advance is added dropwise to the resulting solution with stirring from a dropping funnel (control - universal indicator paper, pH ~ 5). The precipitated white crystalline precipitate of lappaconitine hydrobromide is triturated with a spatula, left in the freezer at -18 ° C for 30 minutes and then filtered on a filter with a porous plate. The filter cake was washed with a minimum amount of cold acetone (3 ml) and then washed with methylene chloride (5 ml), and dried. Receive about 1.8 g of lappaconitine hydrobromide, purity according to HPLC more than 90%.

For the final purification of lappaconitine hydrobromide, it is crystallized from alcohol, at the rate of 2 ml of alcohol per 850 mg of hydrobromide. About 2.0 g of lappaconitine hydrobromide are obtained with a purity of more than 97%.

The total yield of extraction of lapapconitin from plant materials in the case of using this approach is 0.8%.

Example 6

The use of conventional extraction (80% ethanol), the sorbent is silica gel.

In a 1 L round-bottom flask, 100 g of milled to a fine powder of dry plant material containing lappaconitin (white aconite roots) are ground. 900 ml of 80% aqueous ethanol are added and stirred for 2 hours. Decant the solvent through a paper filter. An additional 900 ml of 80% aqueous ethanol was added to the residue in the flask and stirred for 2 hours. The solvent was decanted through a paper filter, combined with the previous extract and ethanol was evaporated in vacuo (50 mbar). To the residue was added 20 ml of a 10% aqueous solution of sodium carbonate (pH ~ 9) and the resulting solution was extracted with methylene chloride (3 times 50 ml). The combined extract is dried with anhydrous sodium sulfate and passed by gravity through a layer of silica gel on a filter with a porous plate (D = 3 cm, H layer = 3 cm, silica gel weight ~ 9 g). The remainder of methylene chloride, not merged by gravity, is pumped to dryness in vacuo. The eluent (methylene chloride) is yellow-brown in color, containing slightly polar colored impurities, discarded, with further regeneration of methylene chloride on a rotary evaporator.

The target lappaconitin is eluted with silica gel ~ 150 ml ethyl acetate (control - TLC, silufol, acetone, R _f lappaconitine ~ 0.65) and the resulting solution of lappaconitine base is evaporated to dryness on a rotary evaporator, obtaining about 1.3 g of a yellow, viscous, crystallized upon standing oil .

The residue was dissolved in 3 ml of acetone and cooled to -10 ° C. A solution of ~ 0.3 ml of 48% hydrobromic acid in 2 ml of acetone prepared in advance is added dropwise to the resulting solution with stirring from a dropping funnel (control - universal indicator paper, pH ~ 5). The precipitated white crystalline precipitate of lappaconitine hydrobromide is triturated with a spatula, left in the freezer at -18 ° C for 30 minutes and then filtered on a filter with a porous plate. The filter cake was washed with a minimum amount of cold acetone (3 ml) and then washed with methylene chloride (5 ml) and dried. Receive about 1.1 g of lappaconitine hydrobromide, purity according to HPLC more than 90%.

For the final purification of lappaconitine hydrobromide, it is crystallized from alcohol, at the rate of 2 ml of alcohol per 850 mg of hydrobromide. Get about 0.95 g of lappaconitine hydrobromide with a purity of more than 97%.

The yield of extraction of lappaconitine from plant materials in the case of using this approach is 0.95%.

Example 7

The use of conventional extraction (80% ethanol), the sorbent is aluminum oxide.

In a 1 L round-bottom flask, 100 g of milled to a state of fine powder of dry plant materials containing lappaconitin (roots of northern aconite) are ground to a state. 900 ml of 80% aqueous ethanol are added and stirred for 2 hours. Decant the solvent through a paper filter. An additional 900 ml of 80% aqueous ethanol was added to the residue in the flask and stirred for 2 hours. The solvent was decanted through a paper filter, combined with the previous extract and ethanol was evaporated in vacuo (50 mbar). To the residue was added 20 ml of a 10% aqueous solution of sodium carbonate (pH ~ 9) and the resulting solution was extracted with methylene chloride (3 times 50 ml). The combined extract is dried with anhydrous sodium sulfate and passed by gravity through a layer of aluminum oxide on a filter with a porous plate (D = 3 cm, H layer = 3 cm, weight of aluminum oxide ~ 15 g). The remainder of methylene chloride, not merged by gravity, is pumped to dryness in vacuo. The eluent (methylene chloride) is yellow-brown in color, containing slightly polar colored impurities, discarded, with further regeneration of methylene chloride on a rotary evaporator.

The target lappaconitin is eluted with alumina ~ 200 ml of acetone (control - TLC, silufol, acetone, R _{f of} lappaconitin ~ 0.65), and the resulting solution of lappaconitine base is evaporated on a rotary evaporator to a volume of 3-4 ml. The resulting residue was cooled to -10 ° C and, with stirring, a solution of ~ 0.3 ml of 48% hydrobromic acid in 2 ml of acetone prepared in advance was added dropwise from a dropping funnel (control universal indicator paper, pH ~ 5). The precipitated white crystalline precipitate of lappaconitine hydrobromide is triturated with a spatula, left in the freezer at -18 ° C for 30 minutes and then filtered on a filter with a porous plate. The filter cake was washed with a minimum amount of cold acetone (3 ml) and then washed with methylene chloride (5 ml), and dried. Get about 0.9 g of lappaconitine hydrobromide, purity according to HPLC more than 90%.

For the final purification of lappaconitine hydrobromide, it is crystallized from alcohol, at the rate of 2 ml of alcohol per 850 mg of hydrobromide. Get about 0.75 g of lappaconitine hydrobromide with a purity of more than 97%.

The total yield of extraction of lappaconitine from plant materials in the case of using this approach is 0.75%.

Example 8

The use of conventional extraction (80% aqueous acetone), the sorbent is silica gel.

A 100 g round-bottom flask was charged with 100 g ground to a fine powder of dry plant material containing lappaconitin (white aconite roots). 900 ml of 80% aqueous acetone are added and stirred for 2 hours. Decant the solvent through a paper filter. To the residue in the flask was added another 900 ml of 80% aqueous acetone and stirred for another 2 hours. The solvent is decanted through a paper filter and combined with the previous extract. Evaporate acetone in vacuo (60 mbar). To the residue was added 20 ml of a 10% aqueous solution of sodium carbonate (pH ~ 9) and the resulting solution was extracted with methylene chloride (3 times 50 ml). The combined extract is dried with anhydrous sodium sulfate and passed by gravity through a layer of silica gel on a filter with a porous plate (D = 3 cm, H layer = 3 cm, silica gel weight ~ 9 g). The remainder of methylene chloride, not merged by gravity, is pumped to dryness in vacuo. The eluent (methylene chloride) is yellow-brown in color, containing slightly polar colored impurities, discarded, with further regeneration of methylene chloride on a rotary evaporator.

The target lappaconitin is eluted with silica gel ~ 150 ml ethyl acetate (control - TLC, silufol, acetone, R _f lappaconitine ~ 0.65) and the resulting solution of lappaconitine base is evaporated to dryness on a rotary evaporator, obtaining about 1.2 g of a yellow, viscous, crystallized upon standing oil .

The residue was dissolved in 3 ml of acetone and cooled to -10 ° C. To the resulting solution, a solution prepared in advance of ~ 0.3 ml of 48% hydrobromic acid in 2 ml of acetone (control is a universal indicator paper, pH ~ 5) is added dropwise with stirring from a dropping funnel. The precipitated white crystalline precipitate of lappaconitine hydrobromide is triturated with a spatula, left in the freezer at -18 ° C for 30 minutes and then filtered on a filter with a porous plate. The filter cake was washed with a minimum amount of cold acetone (3 ml) and then washed with methylene chloride (5 ml), and dried. Receive about 1.0 g of lappaconitine hydrobromide, purity according to HPLC more than 90%.

For the final purification of lappaconitine hydrobromide, it is crystallized from alcohol, at the rate of 2 ml of alcohol per 850 mg of hydrobromide. Get about 0.85 g of lappaconitine hydrobromide with a purity of more than 97%.

The total yield of extraction of lappaconitine from raw materials in the case of using this approach is 0.85%.

Example 9

The use of conventional extraction (80% aqueous acetone), the sorbent is aluminum oxide.

In a 1 L round-bottom flask, 100 g of milled to a state of fine powder of dry plant materials containing lappaconitin (roots of northern aconite) are ground to a state. 900 ml of 80% aqueous acetone are added and stirred for 2 hours. Decant the solvent through a paper filter. To the residue in the flask was added another 900 ml of 80% aqueous acetone and stirred for another 2 hours. The solvent is decanted through a paper filter and combined with the previous extract. Evaporate acetone in vacuo (60 mbar). To the residue was added 20 ml of a 10% aqueous solution of sodium carbonate (pH ~ 9) and the resulting solution was extracted with methylene chloride (3 times 50 ml). The combined extract is dried with anhydrous sodium sulfate and passed by gravity through a layer of aluminum oxide on a filter with a porous plate (D = 3 cm, H layer = 3 cm, weight of aluminum oxide ~ 15 g). The remainder of methylene chloride, not merged by gravity, is pumped to dryness in vacuo. The eluent (methylene chloride) is yellow-brown in color, containing slightly polar colored impurities, discarded, with further regeneration of methylene chloride on a rotary evaporator.

The target lappaconitin is eluted with alumina ~ 200 ml of acetone (control - TLC, silufol, acetone, R _{f of} lappaconitin ~ 0.65) and the resulting solution of lappaconitine base is evaporated on a rotary evaporator to a volume of 3-4 ml. The resulting residue was cooled to -10 ° C and, with stirring, a solution of ~ 0.3 ml of 48% hydrobromic acid in 2 ml of acetone prepared in advance was added dropwise from a dropping funnel (control - universal indicator paper, pH ~ 5). The precipitated white crystalline precipitate of lappaconitine hydrobromide is triturated with a spatula, left in the freezer at -18 ° C for 30 minutes and then filtered on a filter with a porous plate. The filter cake was washed with a minimum amount of cold acetone (3 ml) and then washed with methylene chloride (5 ml) and dried. Get about 0.9 g of lappaconitine hydrobromide, purity according to HPLC more than 90%.

For the final purification of lappaconitine hydrobromide, it is crystallized from alcohol, at the rate of 2 ml of alcohol per 850 mg of hydrobromide. Get about 0.7 g of lappaconitine hydrobromide with a purity of more than 97%.

The total yield of extraction of lappaconitine from raw materials using this approach is 0.7%.

Table
Conditions and results of the isolation of lappaconitine hydrobromide from plant materials No. p / p The mass of raw materials, g Type of extraction, extractant The output of the pharmacopeia hydrobromide of lappaconitine (allapinin) Used plant materials g % by weight of dry raw materials one 250 continuous methylene chloride 1,61 0.645 Roots of aconite 2 250 continuous methylene chloride 1.25 0.5 The roots of aconite northern 3 250 continuous methylene chloride 0.875 0.350 Aconite grass four 250 continuous methylene chloride 0.75 0,300 Northern aconite grass 5 250 continuous
acetone 2 0.8 The roots of aconite northern 6 one hundred ordinary
80% ethanol 0.95 0.95 Roots of aconite 7 one hundred ordinary
80% ethanol 0.75 0.75 The roots of aconite northern 8 one hundred plain, 80% aqueous acetone 0.85 0.85 Roots of aconite 9 one hundred plain, 80% aqueous acetone 0.7 0.7 The roots of aconite northern

Claims (7)

1. The method of producing lappaconitine hydrobromide by extraction of the roots or grass of northern aconite (Aconitum septentrioale) or white aconite (Aconitum leucostomum) with an organic solvent, purification of the extract, treatment of lappaconitine with a solution of hydrobromic acid and crystallization of the target product, characterized in that the extraction said plant material is carried out with methylene chloride in a continuous extraction apparatus followed by purification of the obtained extract by flash chromatography. 2. The method according to claim 1, where silica gel is used as a sorbent for flash chromatography, and ethyl acetate is used as an eluent. 3. The method according to claim 1, where alumina is used as a sorbent for flash chromatography, and acetone is used as an eluent. 4. A method of producing lappaconitine hydrobromide by extracting the roots or grass of northern aconite (Aconitum septentrioale) or white aconite (Aconitum leucostomum) with an organic solvent, purifying the extract, treating lappaconitine with a solution of hydrobromic acid and crystallizing the target product, characterized in that the extraction said plant material is carried out with a polar organic solvent selected from the group comprising ethanol, acetone and their aqueous solutions, followed by removal of the polar organic solvent from the extract, alkalization and extraction of the resulting residue with methylene chloride, followed by purification of the obtained extract by flash chromatography. 5. The method according to claim 4, where silica gel is used as a sorbent for flash chromatography, and ethyl acetate is used as an eluent. 6. The method according to claim 4, where alumina is used as the sorbent for flash chromatography, and acetone is used as the eluent. 7. The method according to claim 4, where the extraction carried out in an apparatus for continuous extraction.