WO2013172693A1

WO2013172693A1 - Process for preparing of highly pure dihydroquercetin

Info

Publication number: WO2013172693A1
Application number: PCT/LT2012/000008
Authority: WO
Inventors: Natalia KISELEVA; Yury GARIPOV; Sergey PONKRATOV; Viačeslavas MASLOVAS
Original assignee: UAB "Rokiskio Pragiedruliai"
Current assignee: UAB "Rokiskio Pragiedruliai"
Priority date: 2012-05-18
Filing date: 2012-11-21
Publication date: 2013-11-21
Anticipated expiration: 2014-11-18
Also published as: LT6013B; LT2012035A; EP2850069A1

Description

PROCESS FOR PREPARING

OF HIGHLY PURE DIHYDROQUERCETIN

Field of the Invention

The present invention relates to chromatographic extraction of (2R, 3R) - dihydroquer- cetin for production of biologically active additives for medical and pharmaceutical purposes as well as for cosmetics and food industry.

Background of the Invention

(2R, 3R)- dihydroquercetin ((+)-(2R,3R)-trans- dihydroquercetin), also known as„Taxi- folin", is attributed to bioflavonoids and is a reference antioxidant of natural origin.

US No. 5912363 disclose a method for extracting flavonoid compounds from vegetable raw materials, in particular - for extracting proanthocyanidins. The method includes heating a mixture of water and hard vegetable raw materials under high pressure and in low-oxygen conditions; the separation, filtration and absorption of the solution, followed by the elution of adsorbed proanthocyanidin with a polar solvent. Although said method allows the extraction of flavonoid compounds, including dihydroquercetin, from various vegetable materials rich in antioxidants, such as grape seeds, nut shell, bark etc., the target products, acquired through the use of this method are only 88-92% pure at best.

There is a variety of ways to acquire up to 97% pure dihydroquercetin.

RU No. 2038094 discloses another method for acquiring dihydroquercetin from shredded deciduous wood by means of extraction, using an organic solvent immediately after the material is treated with water and later evaporating the extract and crystallizing from hot water.

RU No. 2000797 discloses yet another method for acquiring dihydroquercetin by extraction of deciduous wood sawdust with water, including heating, cooling, filtering and purification by using polyamide sorbent, elution of the target product with ethyl acetate and crystallizing from hot water.

RU No. 2186097 discloses one more method for acquiring dihydroquercetin by treat- ing the deciduous wood five times with 96.6% ethanol. Before treatment, the lumber is shredded to 1-5 mm size pieces. Afterwards extraction is done at 50-60°C temperature for 30-60 minutes, under constant stirring until the target product has been achieved. The resulting ex- tract is filtered and propylene glycol is added. Ethanol is then removed from the resulting compound, and a 5% solution of sodium chloride is added to the compound, which is then stirred and filtered. The resulting filtrate is percolated through polyamide sorbent in order to precipitate the bioflavonoid complex. The sorbent is rinsed with distilled water and a diluted water solution of ethanol, and then dried, after what the bioflavonoid complex is washed out. The invention allows increasing the quality of bioflavonoid complex by removing from it all extraneous substances.

General drawback of the aforementioned methods is that the target product contains extraneous substances of different origins, including flavonoid compounds.

RU No. 2114631 discloses a method for acquiring highly pure dihydroquercetin, which method consists of advance treatment of deciduous wood with boiling water, followed by extraction of acetone using a solution of water and purifying the water-acetone extract with fluid chromatography by using a reverse-phase Spherisorb C2 - CI 8 type sorbents having particle size from 5 to 10 microns, using a 30-50% solution of acetone in 0.1% water solution of trifluoroacetic acid as the mobile phase, and removing the acetone by evaporation. The technical result - acquired dihydroquercetin having purity over 99.5%.

The essential drawbacks of said method are the complexity of the process and related insufficient capacity regarding the target product, as well as the unknown proportion of dihydroquercetin stereoisomers in the target product.

RU No. 2114631 discloses closest method, that consists of acquiring highly purified dihydroquercetin, by obtaining a solution of raw material containing dihydroquercetin in a water solution of aliphatic alcohol C₁-C₃ wherein the concentration of alcohol is 1 - 20%; executing one or several chromatography cycles in a thermostatic column with a sorbent, the sorbent being eluted by a water solution of aliphatic alcohol CrC₃, with concentration not ex- ceeding 12 -30%.; vaporising the eluat and drying the resulting product. The advantage of this method is purity of the resulting target product, which is about 98.5-99.5%.

The essential drawbacks consists of complexity of the process, related insufficient output - only 89-93%, the antioxidant activity, which related to the presence of all the optical dihydroquercetin isomers in the final product, and low output rate of the obtainable solution - 20 ml/min.

Besides essential drawbacks of the aforementioned methods for chromatographic extraction of (2R, 3R) - dihydroquercetin, another one is the use of streaming ultraviolet detection, which leads to partial disintegration of the target product. The objective of this invention is to develop a highly productive method for acquiring (2R, 3R) - dihydroquercetin, resulting in high purity and high output of the target product.

Detailed Description of the Invention

The objective of acquiring highly pure dihydroquercetin is achieved by dissolving hard raw material containing dihydroquercetin in water solution of ethanol, having concentration of 20-95% at temperature of 20-29°C, wherein the content of dihydroquercetin reaches 50-250 g/1, followed by chromatographic separation under pressure of 100-500 kg/cm² on a column with dynamic axial compression filled with reverse-phase sorbent, having increased retention of polar bonds, particle size from 10 to 20 microns, pore size from 120 to 200 A. The sorbent is eluted with previously degassed and modified water solution of ethanol, the eluat fractions are gathered by means of refractometric and mass spectrometric analysis, which is followed by crystallization by freezing, filtering and lyophilisation of the target product.

The eluat is modified preferably with methanol, isopropanol, acetic acid, formic acid, until the presence of these elements in the eluat reaches 0,001 - 5 % by volume.

The product acquired by the use of such method contains no less than 99,9 % ((+)- (2R,3R)-trans- dihydroquercetin.

During the primary tests in the laboratory, the authors have unexpectedly discovered that the product produced by means of chromatographic separation of degassed water-alcohol solution of dihydroquercetin at a temperature of 20-29°C and pressure of 100-500 kg/cm² on a column with dynamic axial compression filled with reversed-phase sorbent with increased retention of polar bonds, wherein sorbent particle size is from 10 to 20 microns, size of pores is from 120 to 200 A, sorbent being eluted with previously degassed and modified water solution of ethanol, the eluat fractions being gathered by means of simultaneous refractometric and mass spectrometric analysis, followed by crystallization by freezing, filtering and lyophilisation, contains only the (2R,3R)- dihydroquercetin with the full absence of its optical stereoisomers.

The range of concentration values of ethanol and range of volume values of dihydroquercetin in the obtained solution, respectively being 20 - 95% and 50 - 250 g/1, were cho- sen in accordance with the condition of the maximum capacity of the process, taking into consideration the different solubility of the primary technical dihydroquercetin and its admixtures. The advance degassing of the initial water and alcohol solution of dihydroquercetin and the eluat, allows to avoid the oxidation, caused by dissolved oxygen, of the target product and considerably increase the precision of refractometric and mass spectrometric determination of (2R,3R)- dihydroquercetin in the eluat, which in turn allows to increase production capacity, purity and output of the product.

The range of pressure values, 100-500 kg/cm², during the chromatographic separation is determined by the preparative column YMC DAU 100-700 with dynamic axial compression using a reverse-phase sorbent having increased retention of polar bonds YMC ODC-AQ-HG, sorbent having particle size from 10 to 20 microns, size of pores from 120 to 200 A. The lat- ter were determined in accordance with the nature of the target product and related to the possible presence of three dihydroquercetin stereoisomers having different polarity then (2R,3R)- dihydroquercetin, as well as in compliance with the maximum rate of 150 - 250 ml/min at which eluat is supplied.

It is crucial to maintain the temperature within the range of 20-29°C during the process of dissolving the initial dihydroquercetin and its chromatographic separation. The capacity of the process decreases substantially if the temperature falls below 20°Cand the rise of temperature above 29°C increases the risk of stereo isomerization of the target product.

The gathering of eluat fractions by means of simultaneous refractometric, spectrometric and mass spectrometric analysis allows reusing the solvent from the eluat without a thorough distillation of the column; ensures more precise analysis of fractions with the molecular weight within the range of 100 - 1000 units; and also allows isolating the admixtures separately as pure target products. These procedures allow to increase the output of the target product up to 99,5 - 99,9 %.

Ethanol and water is removed from eluat fraction, acquired by chromatographic isola- tion, by using Hei-VAP Value HB/G3 Heidolph rotor of the plate-rotor pump in the vacuum at a temperature of 30°C. The solvent from remaining fractions is distilled by using Laborota 20 (LR 20 eco) rotor.

The obtained (2R, 3R) - dihydroquercetin is transferred to the lyophilizer, wherein special containers with (2R, 3R) - dihydroquercetin are automatically sealed after drying in deep vacuum at a temperature of -55°C.

The chemical purity of the target product is controlled directly during the chromatographic analysis when the flow of eluat is split and directed to fraction collector and detectors by means of a guide. The refractometric, spectrometric and mass spectrometric detectors are used simultaneously. The product is additionally checked for the content of polymers (bound dihydroquercetin monomers), using a gas-vapour osmometer K-7000 KNAUER.

The antioxidant activity is assessed by spectrofluorometer FLUOstar Optima in accordance to the known method ORAC, disclosed in US No. 7132296.

The (2R,3R)- dihydroquercetin obtained by said method is at least 99.9% pure, not taking into account the bound water, and is kept at a temperature of -30° C.

The eluat is modified with methanol, isopropanol, acetic acid, formic acid or other volatile compounds that are compatible with mass spectrometric detectors, until the presence of these elements in the eluat reaches 0,001 - 5 %, thus reducing the time, necessary for dihy- droquercetin to be kept in the reverse-phase sorbent which significantly boosts the capacity of such method.

The target product, obtained by said method, contains at least 99.9% ((+)-(2R,3R)-trans- dihydroquercetin, not taking into account the bound water. Detailed Description of Preferred Embodiments

Example 1

62 grams of semi-finished product, containing 70,07 % (2R,3R)- dihydroquercetin, 3,14 % aromadendrene, 0,96 % eriodictyol, 0,39 % quercetin, 0,15 % naringenin, 0,01 % kaempferol and 0,24 % pinocembrin are dissolved in 400 ml of 50% alcohol solution.

The prepared sample is loaded into 100x350 mm column with dynamic axial compression, which is filled with sorbent YMC ODC-AQ-HG, having particle size 20 microns, pore size200 A, at 200 ml/min rate. Afterwards, eluat (ethanol: methanol: water in respective ratio of 50: 1 :49) is passed through the column at the rate of 200 ml/min. The main stream from the column is directed to the fraction collector, and the auxiliary one - to the refractometric and mass spectrometric detectors. The target fraction, containing (2R, 3R) - dihydroquercetin, is collected according to the readings from these detectors.

The target fraction is evaporated in vacuum until the alcohol has been fully removed, then it is cooled down to the temperature of 4°C, the sediment is filtered and dried in lyophi- lizer at a temperature of - 55°C.

Thus, 43.1 grams of the target product are acquired, containing 99,60% (2R,3R)- dihydroquercetin and 0,35 % (2R,3S)- dihydroquercetin. The output degree of (2R, 3R) - dihydroquercetin is 98,5%. Example 2

82 grams of semi-finished product, containing 88,09 % (2R,3R)- dihydroquercetin, 2,49 % aromadendrene, 0,86 % eriodictyol, 0,59 % quercetin, 0,19 % naringenin, 0,03 % kaempferol and 0,07 % pinocembrin are dissolved in 300 ml of 70% alcohol solution.

The prepared sample is loaded into 100x350 mm column with dynamic axial compression, filled with sorbent YMC ODC-AQ-HG, having particle size 10 microns, pore size 120 A, at the rate of 150 ml/min. Afterwards, eluat (ethanol : isopropanol : water in respective ratio of 70 : 5 : 25) is passed through the column at the rate of 150 ml/min. The main stream from the column is directed to the fraction collector, and the auxiliary one - to the refracto- metric and mass spectrometric detectors. The target fraction, containing (2R, 3R) - dihydroquercetin, is collected according to the readings from these detectors.

Thus, 71.6 grams of the target product are acquired, containing 99,81% (2R,3R)- dihydroquercetin and 0,014 % (2R,3S)- dihydroquercetin. The output degree of (2R, 3R) - dihydroquercetin is 99,0%.

Example 3

80 grams of semi-finished product, containing 92,43 % (2R,3R)- dihydroquercetin, 2,38 % aromadendrene, 0,87 % eriodictyol, 0,53 % quercetin, 0,18 % naringenin, 0,03 % kaempferol and 0,07 % pinocembrin are dissolved in 500 ml of 40% alcohol solution.

The prepared sample is loaded into 100x350 mm column with dynamic axial compression, filled with sorbent YMC ODC-AQ-HG, having particle size 10 microns, pore size 120 A, at the rate of 250 ml/min. Afterwards, eluat (ethanol : acetic acid : water in respective ratio of 40 : 1 : 59) is passed through the column at the rate of 250 ml/min. The main stream from the column is directed to the fraction collector, and the auxiliary one - to the refractometric and mass spectrometric detectors. The target fraction, containing (2R, 3R) - dihydroquercetin, is collected according to the readings from these detectors.

The target fraction is evaporated in vacuum until the alcohol has been fully removed, then it is cooled down to the temperature of 4°C, the sediment is filtered and dried in lyophi- lizer. Thus, 72.76 grams of the target product are acquired, containing 99,91% (2R,3R)- dihydroquercetm and 0,012 % (2R,3S)- dihydroquercetin. The output degree of (2R,3R)- dihydroquercetm is 99,0%.

Further essential features of this invention are:

- the preparation conditions of the initial dihydroquercetin solution, in particular the content of ethanol, which equals to 20-95%in a water solution, the content of dihydroquercetin, which equals to 50-250 g/1 in a water solution, and the dissolving temperature, which equals to 20-29° C;

- advance degassing of the initial water-alcohol solution of dihydroquercetin;

- the range of pressure values during the process of chromatographic separation is 100-

500 kg/cm²;

- the application of reverse-phase sorbent with increased retention of polar bonds, sorbent having particle size from 10 to 20 microns, pore size from 120 to 200 A;

- advance degassing of the eluat;

- advance modification of the eluat;

- modification of the eluat with methanol, isopropanol, acetic acid, formic acid, until the presence of these elements in the eluat reaches 0,001 - 5 %.

- the eluat fractions are gathered by means of simultaneous refractometric, spectrometric and mass spectrometric analysis, with the separation of streams;

- crystallization by freezing, filtering and lyophilisation of the target product.

Therefore, the technical result of this invention is the method of acquiring highly pure dihydroquercetin, which contains only no less than 99.9% ((+)-(2R,3R)-trans- dihydroquercetin, whereby the (2R,3R)- dihydroquercetin output degree - 99-99,5%, capacity of the initial solution being within the range of 150 - 250 ml/min, and the target product obtained with the method described herein.

References

1. Patent US No. 5912363, COID 311/62, 1999.

2. Patent RU No. 2038094, A 61 K 35/78, 1995.

3. Patent RU No. 2000797, A 61 K 35/78, 1993.

4. Patent RU No. 2186097, CI 1B9/00, A23L1/30, A23L1/29, 2001.

5. Patent RU No. 2114631 A61K35/78, C07D311/32, 1997.

6. Patent US No. 7132296, G01N 33/00, 2006.

Claims

1. A method for acquiring a highly pure dihydroquercetin by dissolving hard raw material containing dihydroquercetin in water solution of ethanol, having concentration of 20- 95%, at temperature of 20-29°C, until the content of dihydroquercetin in the solution reaches 50-250 g/1, followed by chromatographic separation under pressure of 100-500 kg/cm² on a column with dynamic axial compression filled with reverse-phase sorbent with increased retention of polar compounds, sorbent having particle size from 10 to 20 microns and pore size from 120 to 200 A, eluting sorbent with in advance degassed and modified water solution of ethanol, the eluat fractions are gathered by means of refrac- tometric and mass spectrometric analysis, which is followed by crystallization by freezing, filtering and lyophilisation of the target product.

2. Method according to claim 1, characterised in, that the eluat is modified with methanol, isopropanol, acetic acid, until the content of these elements in the eluat reaches 0,001 - 5 % by volume.

3. The product obtained by the method according to claim 1 and 2, contains at least 99,9 % ((+)-(2R,3R)-trans- dihydroquercetin.