LT6013B - Process for preparing dihydroquercetin of a particularly high degree of purity - Google Patents

Abstract

The present invention relates to isolation by chromatography of (2R, 3R)-dihydroquercetin for production of biologically active supplements for medicine and pharmacy and food and cosmetics industry as well. The highly effective process for preparing (2R, 3R)-dihydroquercetin claimed enables to obtain the extremely pure product. The raw material containing dihydroquercetinis subjected to dissolution in aqueous ethanol solutionat 2-29 oC until the dihydroquercetin amount in solution of 50-250 g/l. Then the solution is subjected to chromatographic separation in a column under pressure 100-500 kg/cm2, the column being filled with reverse-phase sorbent. The sorbent is subjected to eluation with degassed modified ethyl alkohol aqueous solution. The portions of eluate are controlled by refractometry and mass spectrometry methods; then the product is subjected to crystallization, filtration and lyophylization. The product obtained contains at least 99,9 % of (2R,3R)-dihydroquercetin.

Description

Technical field

The present invention relates to the field of chromatographic isolation of (2R, 3R) -dihydro-quercetin, to the manufacture of biologically active supplements for medical and pharmaceutical purposes, and to the food and cosmetics industry.

BACKGROUND OF THE INVENTION (2R, 3R) -Dihydroquercetin ((+) - (2R, 3R) -transdihydrocvercetin), also known as "taxifoline", belongs to bioflavonoids and is a reference antioxidant of natural origin.

There is a known method of isolating flavonoid compounds from vegetable raw materials, namely, proanthocyanidins (U.S. Pat. No. 5,912,363, C01D 311/62, 1999). In this way, heating of a mixture of solid plant materials and water at high pressure and reduced oxygen content, solution separation, filtration and adsorption followed by elution of the adsorbed proanthocyanidin with the aid of a polar solvent. Although this method allows the isolation of flavonoid compounds, which include dihydrocvercetin, from a variety of plant-rich antioxidant substances such as grapes, nut shells, bark and many others, the purity of the resulting products does not exceed 88 - 92%.

Several methods are known for the preparation of dihydro-quercetin with a purity not exceeding 97%.

In one way, dihydro-quercetin is extracted from pulverized hardwood by organic solvent extraction immediately after treatment of the raw material with water, further evaporation of the extract, and crystallization from hot water (Patent ΡΦ N 2038094, A 61 K 35/78, 1995).

Alternatively, dihydro-quercetin is obtained by the water extraction of hardwood sawdust, cooling, filtration and purification with a polyamide sorbent, eluting with ethyl acetate and crystallization from hot water. (Patent ΡΦ N 2000797, A 61 K 35/78, 1993).

In yet another embodiment, dihydrocvercetin is obtained by treating hardwood with 96.6% ethyl alcohol 5 times. Before processing, the wood is chopped to a size of 1-5 mm. Further, for 30-60 minutes, with constant stirring, extraction is carried out at 50-60 ° C until the desired product is obtained. After extracting, the filtrate is subjected to filtration and addition of propylene glycol. After removal of the ethyl alcohol from the resulting mixture, a 5% sodium chloride solution is added to the resulting compound. After mixing and filtration, the resulting filtrate is passed through a polyamide sorbent to precipitate the bioflavonoid complex. After washing the sorbent with distilled water and dilute aqueous ethanol solution, the sorbent is dried and the bioflavonoid complex is washed out. The invention allows the quality of the bioflavonoid complex to be improved by removing unwanted materials (Patent ΡΦ 2186097, C11B9 / 00, A23L1 / 30, A23L1 / 29, 2001).

A common disadvantage of the presented methods is that the resulting product contains foreign substances of various origins, including flavonoid compounds.

There is a known process for the preparation of high purity dihydroquercetin, which involves pretreating hardwood with boiling water. The following steps involve the extraction of acetone with aqueous solution and purification of the water-acetone extract by liquid chromatography using reversible phase Sverisorb C2 - C18 sorbents having a particle size of 5 to 10 μιτι and using 30% 50% acetone in 0.1% trifluoro as mobile phase. acetic acid solution and evaporation of the acetone. The technical result is the production of dihydro-quercetin of greater than 99.5% purity (Patent ΡΦ Ns 2114631 A61K35 / 78, C07D311 / 32, 1997).

The main disadvantages of this process are the complexity of the process and the consequent inefficiency of product production; moreover, there is an unknown proportion of the stereoisomers of dihydro-quercetin in the resulting product.

The particular process for producing the pure pure dihydrocvercetin disclosed in this application is the process whereby a solution of the raw material containing dihydrocvercetin in an aqueous solution of aliphatic alcohol C1-C3 is obtained at a concentration of 1-20% vol. Also, the thermostatic column is subjected to one or more chromatography cycles with a sorbent, eluting with an aqueous solution of aliphatic alcohol C1-C3 in a concentration of not more than 12-30% by volume. At the end of the process, after evaporation of the eluate, the resulting product is dried. The advantage of this method is the high purity of the product obtained: 98.5 - 99.5%. (ΠβτθΗΤ ΡΦ Ns

2349330 A61K36 / 00, B01D15 / 08, B01D15 / 42, June 2007)

Among its major disadvantages are the complexity of the process, the insufficiently high yield of the product of 89-93%, the antioxidant activity associated with the presence of all optical dihydro-quercetin in the resulting product, and the low yield of the resulting solution of 20 ml / min.

In addition, the use of in-stream ultraviolet detection, which results in partial degradation of the product, must be considered as a major disadvantage of all described chromatographic recovery methods for (2R, 3R) -dihydrocvercetin.

It is an object of the present invention to provide a high-throughput process for the production of (2R, 3R) -dihydro-quercetin, which provides a highly pure product.

Description of the Invention

The production of highly effective, highly pure (2R, 3R) -dihydrocercercetin is achieved in an aqueous solution of ethyl alcohol at a concentration of 20-95% by volume at 20-29 ° C by dissolving the solid dihydrocercercetin-containing raw material until the dihydrocercercetin composition reaches 50-250. g / l followed by chromatographic separation on a column with a dynamic axial pressure of 100 - 500 kg / cm ² , wherein the column is filled with a reversible phase sorbent with increased polar bond retention, with a sorbent particle size of 10-20 µm and a pore size 120-200 A, further eluting the sorbent with pre-degassed and modified ethyl alcohol in water, collecting the eluate fractions by refractometric and mass spectrometric analysis, and then crystallizing the product by cooling, filtration and lyophilization at the end of the process.

Modification of the eluent is preferably carried out with methanol, isopropanol, acetic acid, formic acid when the composition in the eluate is 0.001-5% by volume.

The product obtained in this way contains at least 99.9% ((+) - (2R, 3R) -transdihydroquercetin).

During initial laboratory testing of this process, the present inventors unexpectedly found that the resulting product contained only (2R, 3R) -dihydroco-quercetin and was completely free of optical stereoisomers; was subjected to chromatographic separation of the dihydrocvercetin degassed water-alcohol solution at a temperature of 20-29 ° C and a pressure of 100-500 kg / cm ^{2 on a} column with dynamic axial pressure, wherein the column was filled with a reversible phase sorbent with increased polar bond retention size was 10-20 μιτι and pore size was 120-200 A, where the sorbent was eluted with a modified, pre-degassed aqueous alcoholic solution and the eluate fractions were collected by refractometric and mass spectrometric analysis followed by crystallization refrigeration, for filtration and lyophilization steps.

The ranges of values for ethyl alcohol of 20 - 95% vol and 50 - 250 g / l dihydrocvercetin volume in the resulting solution are selected based on the process maximum performance condition due to the different initial technical solubility of dihydrocvercetin and impurities.

The pre-degassing of the resulting water-alcohol dihydrocercercetin and eluate solution prevents oxidation of the product due to dissolved oxygen and significantly improves the accuracy of (2R, 3R) -dihydrocercercetin refractometric and mass spectrometric determination of the eluate, thereby substantially increasing production efficiency, product purity and yield.

The pressure range of the chromatographic separation process, 100-500 kg / cm ² , was determined on a YMC DAU 100-700 preparative column with dynamic axial pressure using a reversible-phase sorbent with increased YMC ODC-AQ-HG particle size of 10 -20 μιτι and a pore size of 120200 A. The latter were determined by the nature of the product, the maximum feed rate in the range 150-250 ml / min, and related to the possible presence of three dihydro-quercetin stereoisomers other than (2R, 3R). ) in the presence of dihydro-quercetin.

It is of particular importance that the resulting dihydro-quercetin is dissolved and chromatographically isolated in a temperature range of 20 ° C to 29 ° C. If the process temperature is below 20 ° C, a significant decrease in process performance will be noticeable, and if the temperature is above 29 ° C, the likelihood of stereoisomerization of the product will increase.

Collecting the eluate fractions by refractometric, spectrometric and mass spectrometric analysis without the need for careful column distillation allows the second use of the eluate solution and significantly increases the analytical accuracy of the fraction with molecular weight in the range 100-1000 units, as well as separate impurities as isolation of clean products. The following procedures increase the yield of the product to 99.5 - 99.9%.

From the eluate fraction obtained after chromatographic separation, ethyl alcohol and water are removed in a plate-rotor pump under vacuum with a Hei-VAP Value HB / G3 Heidolph at 30-30 ° C. From the remaining fractions, the solvent is distilled on a Laborota 20 rotor (LR 20 eco).

The collected (2R, 3R) -dihydro-quercetin, packaged in special containers, is transferred to a lyophilizer, where the special containers are automatically sealed after drying in a deep vacuum at -55 ° C.

The chemical purity of the product is directly controlled by chromatographic analysis, in which the eluate flow guide is split by directing it to the fraction collector and detectors. Refractometric, spectrometric and mass spectrometric detectors are used simultaneously. The product is additionally tested on a K-7000 KNAUER gas-vapor osmometer for the presence of polymers (dihydrocvercetin bound monomers).

Antioxidant activity is evaluated by a FLUOstar Optima spectrofluorimeter according to the known procedure ORAC (US Patent No. 7132296, G01N 33/00, 2006).

The (2R, 3R) -dihydro-quercetin, which is at least 99.9% pure, irrespective of the binder water, obtained in the proposed manner is stored at -30 ° C.

Modification of the eluate is carried out with methanol, isopropanol, acetic acid, formic acid or other volatile compounds compatible with the mass spectrometric detector until the amount of eluate reaches 0.001 to 5% by volume, which reduces the retention time of the dihydrocvercetin in the sorbent and significantly increases performance.

The product thus obtained contains at least 99.9% of ((+) - (2R, 3R) -transdihydro-quercetin, regardless of binding water.

Possible embodiments of the invention

Embodiments of the invention that demonstrate its industrial applicability are illustrated by the following examples.

example

Dissolve 62 grams of a semi-product containing 70.07% (2R, 3R) -dihydrocvercetin, 3.14% aromadendrin, 0.96% eriodicitol, 0.39% quercetin, 0.15% naringenin in 400 ml of 50% alcohol solution. 01% Kempferol and 0.24% Pinocembrin.

Into a 100x350 mm column with dynamic axial pressure filled with YMC ODC-AQ-HG sorbent having a particle size of 20 µm and a pore diameter of 200 A at 200 ml / min. at the speed the sample produced above is allowed. The eluate (50: 1: 49 ethanol: methanol: water) is then passed through the column at 200 ml / min. speed. From the column, the main channel is directed to the fraction collector and the additional channel to the refractometric and mass spectrometric detector. The (2R, 3R) -dihydro-quercetin-containing fraction is collected from the detector readings.

The collected fraction is evaporated in vacuo to complete removal of the alcohol, then cooled to 4 ° C and the precipitate is filtered and dried in a lyophilizer at -55 ° C.

43.1 g of the target product are thus obtained, containing 99.60% (2R, 3R) -dihydro-quercetin and 0.35% (2S, 3S) -dihydro-quercetin. The yield of (2R, 3R) -dihydro-quercetin is 98.5%.

example

In 300 ml of a 70% alcohol solution, dissolve 82 grams of a semi-product containing 88.09% (2R, 3R) -dihydro-quercetin, 2.49% aromadendrin, 0.86% eriodicitol, 0.59% quartzetin, 0.19% naringenin, 0, 03% Kempferol and 0.07% Pinocembrin.

Into a 100 x 350 mm column with dynamic axial pressure filled with a YMC ODC-AQ-HG sorbent having a particle size of 10 µm and a pore diameter of 120 A at 150 ml / min.

at the speed the sample produced above is allowed. The eluate (70: 5: 25 ethanol: isopropanol: water) was then passed through the column at 150ml / min. speed. From the column, the main channel is directed to the fraction collector and the secondary channel to the refractometric and mass spectrometric detectors. The (2R, 3R) -dihydro-quercetin containing fraction is collected on the basis of the detector readings.

The collected fraction is evaporated in vacuo until complete removal of the alcohol, then cooled to 4 ° C and the precipitate is filtered and dried in a lyophilizer at -55 ° C.

This gives 71.6 g of product containing 99.81% (2R, 3R) -dihydro-quercetin and 0.014% (2S, 3S) -dihydro-quercetin. The yield of (2R, 3R) -dihydro-quercetin is 99.0%.

example

Dissolve 80 grams of a semi-product containing 92.43% (2R, 3R) -dihydro-quercetin, 2.38% aromadendrin, 0.87% eriodicyclic, 0.53% quercetin, 0.18% naringenin in 500 ml of 40% alcohol solution. 03% Kempferol and 0.07% Pinocembrin.

a 100 x 350 mm column with dynamic axial pressure filled with a YMC ODC-AQ-HG sorbent having a particle size of 10 µm and a pore diameter of 120 A at 250 ml / min. at the speed the sample produced above is allowed. The eluate (40: 1: 59 ethanol: acetic acid: water) was then passed through the column at 250 ml / min. speed. From the column, the main channel is directed to the fraction collector and the additional channel to the refractometric and mass spectrometric detector. The (2R, 3R) -dihydro-quercetin fraction is collected based on the detector readings.

The collected fraction is evaporated in vacuo until complete removal of the alcohol, then cooled to 4 ° C, the precipitate is filtered and dried in a lyophilizer.

This gives 72.76 g of the target product containing 99.91% (2R, 3R) -dihydro-quercetin, 0.012% (2S, 3S) -dihydro-quercetin. The yield of (2R, 3R) -dihydro-quercetin is 99.0%.

The essential features of the invention are also:

- the mode of preparation of the resulting solution of dihydro-quercetin, in particular the value of ethyl alcohol in aqueous solution is 20-95%, the proportion of dihydrocvercetin in aqueous solution is 50 250 g / l and the dissolution temperature is 20-29 ° C;

- pre-degassing the resulting dihydrocercercetin water-alcohol solution;

- pressure range of the chromatographic separation process -100 - 500 kg / cm ² ;

- use of a reversed-phase sorbent with increased polar bond retention with a particle size of 10-20 μηι and a pore size of 120-200 A;

- pre-degassing the eluate;

- pre-modification of the eluate;

- Modification of the eluate with methanol, isopropanol, acetic acid, formic acid until they form from 0.001 to 5% by volume of the eluate;

- collecting the eluate fractions by simultaneous refractometric, spectrometric and mass spectrometric analyzes with flow separation;

- crystallization by freeze-drying, filtration and freeze-drying.

Thus, the technical result of the present invention is a process for the preparation of a highly pure dihydrocvercetin, which yields only ((+) - (2R, 3R) -transdihydrocvercetin with a content of at least 99.9%, a yield of (2R, 3R) dihydrocvercetin of 99%. -99.5% and the solution yield is in the range of 150-250 ml / min, also the product obtained in the indicated manner.

Sources of information

U.S. Patent No. 5,912,363, C01D 311/62, 1999.

Patent ΡΦ N 2038094, A 61 K 35/78, 1995.

Patent ΡΦ N 2000797, A 61 K 35/78, 1993.

Patent ΡΦ 2186097, C11B9 / 00, A23L1 / 30, A23L1 / 29, 2001. Patent ΡΦ Ns 2114631 A61K35 / 78, C07D311 / 32, 1997. Patent US Ns 7132296, G01N 33/00, 2006.

Claims (3)

A process for the preparation of a highly pure dihydrocercercetin, comprising dissolving a solid crude dihydrocercercetin in an aqueous solution of ethyl alcohol at a concentration of 20-95% by volume at 20-29 ° C to a fraction of dihydrocercercetin in the solution of 50-250 g / l followed by chromatography. separation at 100 - 500 kg / cm ² pressure on a column with dynamic axial pressure, wherein the column is filled with a reversible phase sorbent with increased polar bond retention, with a sorbent particle size of 10 to 20 µm and a pore size of 120 to 200 A, eluting the sorbent. collecting the eluate fractions by means of refractometric and mass spectrometric analysis, followed by crystallization, freeze-drying and lyophilization of the product by means of a pre-degassed and modified aqueous solution of ethyl alcohol. 2. A process according to claim 1, wherein the eluate is modified with methanol, isopropanol, acetic acid until the amount of eluate in the eluate reaches 0.001 to 5% by volume. The product of the process of claims 1 and 2, containing at least 99.9% ((+) - (2R, 3R) -trans-dihydrocercercetin).