RU2349330C1 - Method of obtaining highly purified dihydroquercetin - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: present invention pertains to chromatographic separation of bioflavonoids. A solution of dihydroquercetin-containing raw material in an aqueous solution of aliphatic alcohol C₁-C₃ is obtained. At least one chromatography cycle is carried out with depositing a sample of the obtained solution on a column, filled with hydrophobic sorbent. The mobile phase is eluted. The sample is deposited at 1-20% vol. concentration of alcohol in the solution. The mobile phase used is an aqueous solution of the same alcohol with 3% more concentration than concentration of alcohol in the solution from which the sample is deposited, but not exceeding 30% vol. The eluate obtained from chromatography is evaporated and the residue is dried.

EFFECT: obtaining high output of product with high degree of purity.

3 cl, 3 ex

Description

The invention relates to the field of chromatographic isolation of bioflavonoids and can be used to obtain highly purified dihydroquercetin, intended for medical and pharmaceutical purposes, the production of biologically active additives, in the food and cosmetic industry.

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 for the 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 of isolating a bioflavonoid complex from larch wood, in which a solution of dihydroquercetin-containing raw materials is passed through a polyamide sorbent, is washed with diluted ethyl alcohol, the sorbent is dried and eluted with ethyl alcohol.

(RU 2186097, 07.27.2002)

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

There is a known method in which a raw extract obtained in a wood processing process is dissolved in an ethyl acetate / water mixture, the resulting solution is applied to a column filled with a urea sorbent, then the column is eluted with an ester or ketone column and the eluent is evaporated to obtain a crystalline product, the crystals are washed and dried.

(RU 2258225, 08.20.2005).

The disadvantage of this method is the use of esters and ketones, which are strictly normalized in substances, as eluents, which requires the introduction of additional purification steps. In addition, the method allows to obtain dihydroquercetin only in a mixture with dihydrocemferol. The need to refill the column with a sorbent after each chromatographic cycle and the low load characteristics of the sorbent (the ratio of the amount of raw material to the mass of the sorbent 1: 100) due to its low specific surface makes it difficult to use on an industrial scale.

Closest to the proposed method is a method for producing highly purified dihydroquercetin, including purification of the water-acetone extract by liquid chromatography on reverse phase sorbents with a particle size of 5-10 μm using an acetone solution with the addition of trifluoroacetic acid as the 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.

The present invention is to develop a method for the separation of highly purified monocomponent dihydroquercetin from dihydroquercetin-containing raw materials with high yield and purity of the target product with the possibility of its scaling.

The problem is solved by the described method for producing highly purified dihydroquercetin, including obtaining a solution of dihydroquercetin-containing raw materials in a polar solvent, at least one chromatography cycle, applying a sample of the resulting solution to a column filled with a hydrophobic sorbent, and eluting the sorbent with a mobile liquid phase, evaporating the eluate, and drying moreover, an aqueous solution of aliphatic alcohol C ₁ -C _{3 is} used as a polar solvent; when the alcohol concentration in the solution is not higher than 20% vol., while an aqueous solution of the same alcohol is used as the mobile phase when its concentration in the solution is at least 3% higher than the alcohol concentration in the solution from which the sample was applied, but not higher than 30% vol.

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.

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

The selected alcohol concentrations are due to the following.

When the alcohol concentration is less than 1%, the solubility of dihydroquercetin decreases and causes 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 breakthrough already at the application stage.

The concentration of alcohol during elution does not exceed 30%, since in this case there is a simultaneous elution of bioflavonoids associated with 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 30 ° C, the solubility of dihydroquercetin is insufficient for high loads. At temperatures above 50 ° 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

6 g of lavitol containing 4.2 g of dihydroquercetin are dissolved in 12 ml of ethanol on a stirrer at room temperature for 20-30 minutes. diluted with distilled water to 190 ml, centrifuged for 10 min at 4000 g, the supernatant was separated from the resin residue and thermostated at 30 ° C.

On a column with dimensions 22 × 180 mm, volume 70 ml, filled with a hydrophobic sorbent Diasorb-130-Phenyl based on silica gel with a specific surface area of 280-350 m ² / g with grafted phenyl groups with a particle size of 40-60 microns, thermostatically controlled at 30 ° C and balanced with 6.3% aqueous ethanol (6.3% by volume based on 96% ethyl alcohol), a sample is applied at a rate of 20 ml / min. Elution is carried out with a 10% solution of ethyl alcohol. The volume of the eluted sample is 500 ml.

The eluate is evaporated, then cooled to 4-7 ° C, the precipitated precipitate is discarded on a glass filter and dried in a vacuum oven. 3.75 g of product is obtained with a purity of 98.5% according to HPLC. The yield is 89%.

The column is washed with 96% ethanol (2-3 column volumes) and equilibrated with a 10% solution of ethanol in water before applying the next sample.

Example 2

3 g of the Sibel bioflavonoid complex containing 2.7 g of dihydroquercetin is dissolved in 10 ml of methanol on a stirrer at room temperature for 20-30 minutes, diluted with distilled water to 1000 ml, centrifuged for 10 minutes at 4000 g, the supernatant is separated from the resin residue and thermostat at 50 ° C.

On a column with dimensions 22 × 180 mm, volume 70 ml, filled with a hydrophobic sorbent Diasfer-ST-500 based on styrene-divinylbenzene with a specific surface of 600-1000 m ² / g with a particle size of 150-300 microns, thermostated at 50 ° C and balanced 1% water methanol, apply the sample at a speed of 20 ml / min. Elution is carried out with an 11% solution of methyl alcohol. The volume of the eluted sample is 400 ml.

The eluate is evaporated, cooled to 4-7 ° C, the precipitated precipitate is discarded on a glass filter and dried in a vacuum oven. Obtain 2.4 g of product with a purity of 99.5% according to HPLC. The yield is 90%.

The column is washed with methanol (2-3 column volumes) and equilibrated with a 1% solution of methanol in water before applying the next sample.

Example 3

6 g of Lavitol containing 4.2 g of dihydroquercetin are dissolved in 20 ml of isopropyl alcohol on a stirrer at room temperature for 20-30 minutes, diluted with distilled water to 100 ml, centrifuged for 10 minutes at 4000 g, the supernatant is separated from the resin residue and thermostated at 40 ° C.

For a column 22 × 180 mm in size, 70 ml in volume, filled with a Diasfer-130-C8 reversed-phase sorbent based on silica gel with a specific surface area of 280-350 m ² / g, particle size 60-100 μm, temperature-controlled at 40 ° С and balanced 20 % aqueous isopropanol, apply the sample at a speed of 20 ml / min. Elution is carried out with a 30% solution of isopropyl alcohol. The volume of the eluted sample is 300 ml.

The eluate is evaporated, cooled to 4-7 ° C, the precipitated precipitate is discarded on a glass filter and dried in a vacuum oven. 3.9 g of product are obtained with a purity of 97.1% according to HPLC. The yield is 93%.

The column is washed with pure isopropyl alcohol (2-3 column volumes) and equilibrated with a 20% solution of isopropyl alcohol in water before applying the next sample.

Claims (3)

1. A method of obtaining highly purified dihydroquercetin, including obtaining a solution of dihydroquercetin-containing raw materials in a polar solvent, at least one chromatography cycle, applying the obtained sample to a column filled with a hydrophobic sorbent, and eluting the sorbent with the mobile phase, evaporating the eluate, drying the resulting product, characterized in that as a polar solvent use an aqueous solution of aliphatic alcohol C ₁ -C ₃ , chromatography is carried out in a thermostated column at 30-50 ° C, applied Samples are carried out at an alcohol concentration in the solution of 1-20 vol.%, while an aqueous solution of the same alcohol is used as the mobile phase when its concentration in the solution is at least 3% higher than the concentration of alcohol in the solution from which the application is made samples, but not higher than 30 vol.%. 2. The method according to claim 1, characterized in that the elution is carried out at a concentration of alcohol in a solution of 12-30 vol.%. 3. The method according to claim 1, characterized in that after elution, the column is washed with alcohol and balanced with an aqueous solution of alcohol with a concentration equal to its concentration in the solution for applying the sample, and the next chromatography cycle is carried out.