WO2010095969A1

WO2010095969A1 - Method for producing dihydroquercetin

Info

Publication number: WO2010095969A1
Application number: PCT/RU2009/000081
Authority: WO
Inventors: Виктор Семенович KAPACEB; Сергей Михайлович СТАРОВЕРОВ; Юрий Арсеньевич КОЛЕСНИК; Алексей Юрьевич КОЛЕСНИК
Original assignee: Закрытое Акционерное Общество "Биoxиmmak Ct"
Priority date: 2009-02-17
Filing date: 2009-02-17
Publication date: 2010-08-26

Abstract

The invention relates to the chromatographic isolation of bioflavonoids. A method is proposed for producing dihydroquercetin, which involves subjecting ground larch wood to extraction with aliphatic alcohol, distilling the alcohol, carrying out at least one cycle of chromatographic isolation of a target product in a column filled with a hydrophobic sorbent while eluting the sorbent with an alcohol-containing mobile phase. An aqueous solution of the same alcohol is used as extractant and mobile phase, the alcohol being selected from the group: methyl, ethyl or isopropyl alcohol. The chromatographic isolation cycle is carried out in two stages: in the first stage, the alcohol concentration in the solution applied on the column and in the mobile phase is maintained at the same level of 20-40 vol.%; in the second stage, the solution is applied on the column when the alcohol concentration in the solution is equal to or less than 20 vol.%, and the sorbent is eluted when the alcohol concentration in the mobile phase is at least 3% greater than the alcohol concentration in the solution from which the solution is applied, but not greater than a concentration of 30 vol.%. The eluate produced during chromatography is evaporated and the precipitate is dried. The method makes it possible to produce a high-purity product at a high yield.

Description

The method of producing dihydroquercetin

The invention relates to the field of isolation and purification of bioflavonoids from larch and can be used to obtain highly purified dihydroquercetin, intended for medical and pharmaceutical purposes.

At the intermediate stages of the process, other components can also be isolated: a bioflavanoid complex, arabinogalactan and essential oils.

A known method for the isolation of anthocyanides from plant materials, by extraction followed by chromatographic purification on brominated polystyrene sorbents using 70% ethyl alcohol.

(US 6,780,442, 2004)

A known method of separation of flavonoids from plant materials by extraction followed by chromatographic purification on a styrene-divinylbenzene sorbent using water-alcohol solutions as eluent.

(US 5912363, 06/15/1999)

However, these methods are designed to isolate a complex of bioflavonoids, and not their individual components.

A known method for producing highly purified dihydroquercetin, including extraction of larch sawdust with an aqueous-acetone mixture, purification of the aqueous-acetone extract by high-performance liquid chromatography on reversed-phase sorbents with a particle size of 5-10 μm using acetone solution with the addition of trifluoroacetic acid as a mobile phase. The method allows to isolate a product with a purity of more than 99% to obtain standard samples.

(RU 2114631, 07/10/1998)

The disadvantage of this method is its complexity and the use of expensive HPLC sorbents, which does not allow it to be effectively scaled for the industrial production of highly purified dihydroquercetin.

Closest to the claimed is a method for producing dihydroquercetin as part of a bioflavonoid complex, by extraction of crushed larch wood with 96.5% ethyl alcohol, followed by reextraction with propylene glycol (propanediol-1,2), mixing propylene glycol extract with a solution of sodium chloride in water, separation of the resin and essential oils, applying a dihydroquercetin-containing raw material to a polyamide sorbent, washing the sorbent and eluting the mixture of bioflavonoids with 96.5% ethyl alcohol, evaporating the alcohol, crystallizing and drying the product.

(RU 2186097, 07.27.2002)

However, this method does not allow to isolate dihydroquercetin as an independent component.

The present invention is to develop a method for producing highly purified monocomponent dihydroquercetin from larch with high yield and purity of the target product.

The problem is solved by the described method for the preparation of highly purified dihydroquercetin, including the extraction of crushed larch wood with aliphatic alcohol to obtain an alcohol solution of dihydroquercetin-containing raw materials, distillation of alcohol, at least one chromatographic separation of the target product on a column filled with a hydrophobic sorbent during the elution of the adsorbent eluate, drying the resulting product, moreover, as an extractant and mobile fa PS use an aqueous solution of the same alcohol selected from the group: methyl, ethyl, isopropyl, the chromatographic isolation cycle is carried out in two stages, and in the first stage, the alcohol concentration in the dihydroquercetin-containing solution applied to the column and in the mobile phase are kept the same, equal to 20-40% vol., And in the second stage, the application of the dihydroquercetin-containing solution on the column is carried out at an alcohol concentration of not higher than 20% vol., And the sorbent is eluted at an alcohol concentration of the mobile phase, at least 3% higher than the concentration of alcohol in the solution from which the solution was applied, but not higher than the concentration equal to 30% vol.

At the stage of extraction and distillation of alcohol

Preferably, the distillation of the alcohol is carried out after adding to the extract propylene glycol in an amount of about 1% vol., From its volume, which prevents clumping and sticking of the raw materials with insufficient control over the process of evaporation of the alcohol and allows you to get the raw material in the form of a concentrated solution. Preferably, the propylene glycol concentrate is treated with a 5% solution of sodium chloride, which allows you to remove part of the resinous substances.

At the first stage:

Preferably, the application and elution of the sample on the column is carried out at a concentration of alcohol in a solution of 20-40% vol. In the implementation of the chromatography cycle after elution, the column is washed with a concentrated alcohol solution and balanced with an aqueous alcohol solution with a concentration of the corresponding alcohol concentration in the sample solution and the next chromatography cycle is carried out.

Preferably, the chromatography is carried out in a thermostatic column, at a temperature of 30-50 ⁰ C.

The selected alcohol concentrations are due to the following.

When the alcohol concentration in the solution is less than 20%, the process time increases and the concentration of the target component in the solution decreases.

When the alcohol concentration in the solution is more than 40%, there is insufficient purification from impurities.

In the second stage:

Preferably, the application of the sample to the column is carried out at a concentration of alcohol in a solution of 1-20% vol., And the elution is carried out at a concentration of alcohol in a solution of 12-30% vol.

In the implementation of the chromatography cycle after elution, the column is washed with alcohol and balanced with an aqueous solution of alcohol with a concentration corresponding to the concentration of alcohol in the sample solution and the next chromatography cycle is carried out.

The selected alcohol concentrations are due to the following.

At an alcohol concentration of less than 1%, the solubility of dihydroquercetin is insufficient, which leads to a decrease in the load capacity of the column. With an increase in the alcohol concentration above 20%, dihydroquercetin is poorly retained on the sorbent, which reduces the load capacity of the column or leads to a breakthrough already at the application stage. The alcohol concentration during elution does not exceed 30%, since in this case there is a simultaneous elution of impurities accompanying dihydroquercetin.

Reducing the difference in alcohol concentration between the solution for application and the solution for elution of less than 3% significantly increases the time of elution and reduces the productivity of the process.

The selected temperature range is due to the following.

At temperatures below 3O ⁰ C, the solubility of dihydroquercetin is insufficient for high loads. Above 5O ⁰ C, the load characteristics and selectivity also decrease due to a decrease in sorption binding.

The method of obtaining highly purified dihydroquercetin is illustrated by the following examples:

Example 1

In a column with a jacket measuring 50x1000 mm, 400 g of larch chips are loaded. 2 liters of ethyl 96% alcohol are passed cyclically through the column from the bottom up at a speed of 500 ml / min for 1 hour at a temperature of 6O ⁰ C. The pump is turned off and the pump is connected to the top of the column. From top to bottom, 500 ml of ethanol is passed and combined with the extract. An extract of 2.2 liters is obtained.

The solution is placed in a vacuum evaporator and evaporated until the alcohol is completely removed, and then dissolved in 250 ml of 20% ethyl alcohol (20% by volume based on 96% ethyl alcohol).

A column with a 24x45 mm jacket filled with a hydrophobic sorbent Diafer-CT-500 based on styrene-divinylbenzene with a specific surface area of 600-1000 m / g with a particle size of 150-300 microns, balance with 20% ethyl alcohol at a temperature of 4O ⁰ C and apply prepared higher sample at a rate of 20 ml / min. Then 250 ml of 20% alcohol are passed through the column at a rate of 20 ml / min.

The first 250 ml is collected in the form of the target fraction, diluted to 10% vol. and applied to a 24x70 mm column filled with a hydrophobic sorbent Diacopb-130-Phenyl based on silica gel with a specific surface area of 280-350 m / g with grafted phenyl groups with a particle size of 40-60 microns, thermostated at 3O ⁰ C and balanced 10% - aqueous ethanol (10% volume based on 96% ethyl alcohol) apply the sample at a rate of 20 ml / min. Elution is carried out with a 13% solution of ethyl alcohol. The volume of the eluted sample is 500 ml.

The eluate is evaporated to 125 ml, then cooled to 4-7 ^° C, the precipitate is discarded on a glass filter and dried in a vacuum oven.

Receive 6.2 grams of dihydroquercetin with a purity of 98.2% according to HPLC.

Before the next chromatographic cycle, the columns are washed with a concentrated solution of ethyl alcohol and equilibrated with a solution of ethyl alcohol with a concentration of 20% (first column) and 10% (second column).

Example 2

In a column with a jacket measuring 50x1000 mm, 400 g of larch chips are loaded. 2 liters of methyl alcohol are passed cyclically through the column from the bottom up at a speed of 500 ml / min for 1 hour at a temperature of 6O ⁰ C. The pump is turned off and the pump is connected to the top of the column. 500 ml of methyl alcohol are passed from top to bottom and combined with the extract. An extract of 2.2 liters is obtained.

20 ml of propylene glycol are added to the obtained extract and vacuum evaporation is carried out until the removal of alcohol and water. 35 ml of syrupy concentrate are obtained. 60 ml of 5% sodium chloride were added to the concentrate and stirred for 30 minutes until a gummy precipitate formed. The resulting emulsion is decanted, dissolved in 150 ml of 30% methanol and subjected to chromatographic purification.

A column with a 24x45 mm jacket and a jacket, filled with a Diafep-CT-500 hydrophobic sorbent based on styrene-divinylbenzene with a specific surface of 600-1000 m ² / g with a particle size of 150-300 microns, is balanced with 30% methyl alcohol at a temperature of 30 ° C and apply the sample prepared above at a speed of 20 ml / min. Then 150 ml of 30% alcohol are passed through the column at a rate of 20 ml / min.

The first 200 ml is collected as a target fraction, diluted with water to an alcohol content of 1% vol. In water. and applied to a 24x70 mm column filled with a hydrophobic sorbent Diafer-CT-500 based on styrene-divinylbenzene with a specific surface of 600-1000 m ² / g with a particle size of 150-300 μm, thermostated at 3O ⁰ C and balanced with 1% methyl alcohol at a rate of 50 ml / min. Elution is carried out with l l-% methanol. The target fraction is collected in a volume of 600 ml. The solution is evaporated to a volume of 150 ml and cooled to 1-4 ⁰ C and incubated for 4 hours until a precipitate of dihydroquercetin. The precipitate was filtered off and dried in a vacuum oven at 4O ⁰ C over anhydrous granular potassium hydroxide.

Receive 7.8 grams of dihydroquercetin with a purity of 99.5% according to HPLC.

Before the next chromatographic cycle, the columns are washed with a concentrated solution of methyl alcohol and equilibrated with a solution of methyl alcohol with a concentration of 30% (first column) and 1% (second column).

Example 3

In a column with a jacket measuring 50x1000 mm, 400 g of larch chips are loaded. 2 liters of isopropyl alcohol are passed cyclically through the column from the bottom up at a speed of 500 ml / min for 1 hour at a temperature of 6O ⁰ C. The pump is turned off and the pump is connected to the top of the column. 500 ml of isopropyl alcohol is passed from top to bottom and combined with the extract. An extract of 2.2 liters is obtained.

To the resulting solution was added 20 ml of propylene glycol and vacuum evaporation was carried out until the removal of alcohol and water. 35 ml of syrupy concentrate are obtained. 60 ml of 5% sodium chloride were added to the concentrate and stirred for 30 minutes until a gummy precipitate formed. The resulting emulsion is decanted, dissolved in 250 ml of 40% isopropyl alcohol and chromatographed.

A column with a 24x45 mm jacket and a jacket, filled with a Diafep-CT-500 hydrophobic sorbent based on styrene-divinylbenzene with a specific surface of 600-1000 m ² / g with a particle size of 150-300 microns, is balanced with 40% isopropyl alcohol at a temperature of 5O ⁰ C and apply the sample prepared above at a speed of 20 ml / min. Next, the column is washed with 250 ml of 40% alcohol at a rate of 20 ml / min.

The first 250 ml is collected as the target fraction, diluted twice to the content of isopropyl alcohol in water of 20% vol. and applied to a 24x70 mm column filled with a hydrophobic reversed-phase sorbent Diafer-130-C8 based on silica gel with a specific surface area of 280-350 m ² / g, a particle size of 60-100 μm, thermostated at 5O ⁰ C and balanced 20% - solution isopropyl alcohol at a rate of 20 ml / min. Elution is carried out with a 30% solution of isopropyl alcohol. The volume of the eluted sample is 400 ml.

The eluate is evaporated to 100 ml, then cooled to 4-7 ^° C, the precipitate is discarded on a glass filter and dried in a vacuum oven.

Receive 6.0 grams of dihydroquercetin with a purity of 98.5% according to HPLC.

Before the next chromatographic cycle, the columns are washed with a concentrated solution of isopropyl alcohol and equilibrated with a solution of isopropyl alcohol with a concentration of 40% (first column) and 20% (second column).

Thus, the proposed method allows to obtain high purity dihydroquercetin (more than 98%) using low pressure chromatography, which provides a cheaper process and allows it to scale.

Claims

Claim

1. A method of producing dihydroquercetin, including the extraction of crushed larch wood with aliphatic alcohol to obtain an alcohol solution of dihydroquercetin-containing raw materials, distillation of alcohol, at least one chromatographic isolation of the target product on a column filled with a hydrophobic sorbent when eluting the sorbent with an alcohol-containing mobile phase, the resulting product, characterized in that as an extractant and a mobile phase using an aqueous solution of the same a pirte selected from the group: methyl, ethyl, isopropyl, the chromatographic separation cycle is carried out in a thermostated column at 30-50 ° C in two stages, and in the first stage, the alcohol concentration in the dihydroquercetin-containing solution applied to the column and in the mobile phase are maintained the same, equal to 20-40% vol., and in the second stage, the application of the dihydroquercetin-containing solution on the column is carried out at an alcohol concentration in the solution of 1-20% vol., and the sorbent is eluted at an alcohol concentration in the mobile se, at least 3% excess of the alcohol concentration in the solution from which the coating solution is carried out, but no higher than a concentration of about 30%.

2. The method according to p. 1, characterized in that before distillation of the aliphatic alcohol from a solution of dihydroquercetin-containing raw materials, propylene glycol is introduced into it, and the resulting concentrate is treated with a solution of sodium chloride.

3. The method according to claim 1, characterized in that in the second stage of chromatography, elution is carried out at an alcohol concentration in the mobile phase of 12-30% vol.

4. The method according to p. characterized in that several cycles of chromatographic isolation are carried out, and after elution, the column is washed with alcohol and equilibrated with an aqueous solution of alcohol with a concentration equal to its concentration in the sample solution and the next chromatography cycle is carried out.