RU2308267C1 - Method for isolation of biologically active dihydroquercetin isomers - Google Patents

Abstract

FIELD: chemical and pharmaceutical industry.

SUBSTANCE: ground larch wood is extracted with 90-95 % ethanol at 55-66°C for 1-2 h; extract is filtered; propylene glycol is added; mixture is boiled out; obtained rest is treated with 5 % sodium chloride solution in softened water at 20-30°C; and obtained resin is separated. Obtained flavonoid complex is eluted from column with 90-95 % ethanol; eluate is boiled out, precipitated with water cold to 0-2°C; deposited precipitate is crystallized, filtered, and dried. Obtained product representing bioflavonoid complex is ground followed by dissolving in methanol/water (10:90) mixture and subjected to reverse phase chromatography wherein as mobile phase β-cyclodextrine solution in methanol/water (10:90) mixture is used, and column is cold to 0°C. Collected fractions of individual enantiomeres are boiled out and dried.

EFFECT: complex of increased purity.

1 ex

Description

The invention relates to the field of the pharmaceutical industry and relates to the production of individual biologically active isomers (enantiomers) of dihydroquercetin from larch wood.

Larch wood of various species, for example Siberian larch (Larix sibirica Ledeb.) Or Gmelin larch (Larix gmelinii Rupr.), Serves as a rich source of biologically active substances, including flavonoid compounds. Representatives of almost all classes of flavonoids are present in their composition: flavanones, flavononols, biflavonoids, condensed tannins, arabinogalactan. Flavonoids are one of the most numerous and widespread groups of natural compounds that have attracted attention, especially in recent years, because of their unusually wide spectrum of biological activity. The study of flavonoid compounds of larch wood was started in the 1970s, but now this type of raw material is the main one in the production of dihydroquercetin.

Dihydroquercetin (DHQ) - (3,3 ', 4', 5,7-pentahydroxyflavanone or 3,4 ', 5,7-pentahydroxyflavanonol) is a well-known antioxidant, capillary-protective, anti-inflammatory and antihistamine.

The dihydroquercetin molecule can exist in the form of two optical isomers (enantiomers). In the general case, the enantiomers of various compounds exhibit unequal pharmacological and toxicological activity, so that in the pharmaceutical industry there is currently a tendency to isolate and use enantiomers with lower toxicity and greater target effectiveness.

Numerous patents are known that describe a method for producing mixtures (complexes) of flavonoids from larch and purified dihydroquercetin.

So, patent RU 2206568, 2003.06.20 describes a method that involves preparing wood by grinding, fractioning and pre-soaking, followed by extraction of natural substances from wood in a fluidized state of the components of the extraction mass. As a solvent, a mixture of ethyl alcohol and deionized water is used. The process is carried out at a temperature of 30-40 ° C. After removing the solvent from the obtained aqueous-alcoholic extracts, mixtures of flavonoids are isolated, from which individual components can be isolated by extraction with aqueous-acetone mixtures, which are then recrystallized from water.

A known method (RU 2236863, 2004.09.27), which includes processing when heating sawdust of larch wood with an extractant representing a 60-70% aqueous solution of ethyl alcohol, evaporating the extract, separating resinous substances, heating the extract, crystallization, purification from an aqueous solution of isopropanol or ethanol in the presence of activated carbon while heating the mixture to a boil, followed by isolation of the target product by cooling and separation of the precipitate. The resulting preparation "Flavitex" is a mixture of two main components: dihydroquercetin (94-98 wt.%) And dihydrocampferol (2-6 wt.%).

Patent RU 2183966, 2002.06.27 describes a method for producing a bioflavonoid complex for the production of biologically active additives, medical and chemical-pharmaceutical products, which consists in treating larch wood with a working agent (extraction) to isolate the target product. Before processing, larch wood is crushed to particle sizes of 1-5 mm, ethanol (96.6%) is used as a working agent, and processing is carried out with 5 times the amount of ethanol of the same concentration. The extraction is carried out at a temperature of 50-60 ° C for 30-60 minutes with constant stirring, the obtained extract is filtered, propylene glycol is added to the filtrate, ethanol is completely distilled off from the resulting mixture, then a 5% sodium chloride solution is added to the obtained residue, the resulting mixture is stirred and filtered, the filtrate is passed through a polyamide sorbent to precipitate a bioflavonoid complex, then the sorbent is washed with distilled water and a dilute aqueous solution of ethyl alcohol to remove interfering their substances. After washing, the sorbent is dried and the bioflavonoid complex with the sorbent ethanol is eluted; the resulting eluate is evaporated to dryness to obtain the dry bioflavonoid complex.

A known method of separation of proanthocyanidins from plant materials by heating it in deoxygenated water, separating the liquid, concentrating it with ultrafiltration or reverse osmosis, followed by drying of the product. The resulting product contains several flavonoid compounds, including the trans isomer of dihydroquercetin (US 5,968,517, October 19, 1999). This source may be indicated as the closest analogue. However, this known method, like all previous ones, does not provide for the isolation of individual enantiomers.

The objective of the invention is to obtain individual optically active isomers (enantiomers) of dihydroquercetin, exhibiting biologically active properties and which may be suitable for use in therapy or for screening drugs.

Isolation of individual (2R, 3R) - and (2S, 3S) enantiomers 3.3 ', 4', 5.7 pentahydroxyflavanone (dihydroquercetin) from the flavonoid complex is achieved using high-performance liquid chromatography in reverse phase under isocratic elution in the presence of a chiral modifier-eluent (β-cyclodextrin), and in order to reduce the concentration of the chiral modifier of the mobile phase, the process is carried out by cooling the chromatographic column to 0 ° C.

It is known that the separation of enantiomers in the presence of a chiral modifier occurs due to the reversible formation of inclusion complexes. Complexation constants depend on the process temperature. We found that if the process is carried out at a low temperature, the separation efficiency of enantiomers is significantly increased, which is the essence of the proposed method. The technique used made it possible for the first time to separate the enantiomers of 3.3 ', 4', 5.7 pentahydroxyflavanone (dihydroquercetin) isolated from natural plant materials. Achieving a similar separation of enantiomers without cooling (at room temperature) is possible only at a concentration of β-cyclodextrin of at least 15 mmol / L and a significantly higher methanol content in the mobile phase (25 vol.%, Instead of 10 vol.%).

The technological process carried out in the installation of periodic action includes the following stages:

- preparation of basic raw materials - larch wood chopping;

- extraction of crushed raw materials with ethyl alcohol (90-95%);

- filtering the resulting extract;

- evaporation of the extract to a constant volume;

- salting out (resin separation);

- saline filtration;

- purification of BFK (bioflavonoid complex) on a preparative chromatographic column;

- evaporation of the alcohol eluate from the column;

- reprecipitation of BFK in softened water;

- crystallization of the crystallizate;

- drying;

- dissolution;

- application to the column;

- elution and collection of fractions containing isomers;

- evaporation and drying;

- packaging of end products.

Larch stock wood is crushed in a special plant to an average particle size of 1-5 mm. The crushed raw materials are extracted with 90-95% ethyl alcohol at a temperature of 55-66 ° C for 1-2 hours. The extract is filtered. Propylene glycol is poured into the alcohol extract in an amount of 1-2 vol.% Of the amount of extractant. The mixture is evaporated, and the residue (propylene glycol solution of BFK) is salted out using a 5% solution of sodium chloride in softened water (at a temperature of 20-30 ° C). The resin is separated. Saline solution is applied to a column with a polyamide sorbent. The column is washed with softened water. The flavonoid complex is eluted from the column with ethyl alcohol of 90-95%. The alcohol eluate is evaporated, the dissolved substances are precipitated with chilled (0 - + 2 ° С) softened water, the precipitate is left in the cold for complete crystallization. The crystallizates are filtered off and dried. After drying, the product is cooled to room temperature and ground.

The resulting product, which is a bioflavonoid complex, is dissolved in a mixture of: methyl alcohol - water (10:90) and chromatographed in reverse phase using a solution of β-cyclodextrin (10 mmol / l) in a mixture of methyl alcohol - water as a mobile phase (10:90) while cooling the column to 0 ° C. Separate eluate fractions containing (2R, 3R) -dihydroquercetin and (2S, 3S) -dihydroquercetin are collected separately. The fractions are evaporated to dryness and the final products are dried.

The invention can be illustrated by the following specific embodiment.

Example 1

The original wood of larch is crushed in a special installation. A measured amount of larch wood (70 kg) is sent to an extractor filled with 350 l of ethyl alcohol 90-95% with the stirrer constantly on. The extraction process proceeds at a temperature of 60 ° C and uniformly mixing the extracted mass. The extraction time after reaching the working temperature in the extractor is 1-2 hours. The extracted mass is discharged through the lower drain valve of the extractor with the mixer constantly switched on and fed to the vacuum filter for filtration. The filtered solution is pumped by vacuum into a vacuum evaporator. Before evaporation, 3.5 L of propylene glycol is poured into the alcohol extract. Evaporation of alcohol is carried out fractionally. One stripped off residue (BFK propylene glycol solution) is collected in a plastic container and fed to a salting out container, in which 70 l of a 5% sodium chloride solution in softened water are preliminarily placed (temperature 20-30 ° С). After completion of the process, the saline solution is fed to separate the resin on the suction filter. The resulting filtrate is pumped by vacuum to a storage tank, from where it is fed to a sorption column. After sorption, the column is washed with 40 l of softened water. The flavonoid complex is eluted from the column with 90-95% ethyl alcohol (50 L) fractionally. The first fraction is the dead volume of the column (about 10 l), discarded in a drain tank. The second fraction is the target product, the intensely colored zone (about 10 l) is collected in a receiving tank and then pumped to the evaporator. The third fraction - washing the columns, is collected in a receiving tank for the target product after pumping the second fraction into the evaporator and evaporated separately. One stripped off residue of the second fraction is poured into a plastic container filled with 5 l of chilled (0 - + 2 ° С) softened water, and then transferred to a refrigeration unit in which it is maintained until the BFC is completely crystallized. One stripped off residue of the third fraction is poured into a plastic container filled with 2 l of chilled (0 - + 2 ° С) softened water, and then transferred to a refrigeration unit, in which it is also kept until complete crystallization. The crystallizates are filtered and dried in a vacuum oven at a temperature of + 40 ° C for 3-4 hours or at 65-70 ° C without evacuation. The final drying of the product is carried out in a vacuum oven at a temperature of + 80 ° C for 60 min or at 105 ° C without evacuation. After drying, the product is cooled to room temperature and ground.

Preparation of the mobile phase solution for HPLC analysis. A portion of β-cyclodextrin is dissolved in water when heated to obtain a solution with a concentration of 10 mmol / L. After cooling to room temperature, the resulting solution was mixed with methyl alcohol in a ratio of 10:90 by volume, filtered and used as the mobile phase.

Analysis conditions. A Beckman high-pressure chromatograph with an UV detector and a Luna C ₁₈ column 4.6 × 150 mm (particle size of the sorbent 5 μm) with a 20 mm pre-column filled with the same sorbent. Used isocratic elution mode with the eluent prepared as described above. For detection, a wavelength of 290 nm was chosen. The eluent flow rate is 1.0 ml / min. The chromatographic column is cooled to 0 ° C. by placing ice in a column thermostat. The separation cycle time under the indicated conditions is about 30 minutes.

Separate eluent fractions containing (2R, 3R) -dihydroquercetin and (2S, 3S) -dihydroquercetin are collected separately. The fractions were evaporated and the resulting products were dried. The specific optical rotation values of the isolated enantiomers [α] _D ²⁰ are in accordance with the literature: + 20 ° (2R, 3R) and -21 ° (2S, 3S) (G. - J. Nonaka, et. Al. Chem. Pharm Bull. 1987. V.35. No. 3. P.1105-1108); (Hyang - GO et. Al. Brain Researsch. 965. (2003). P.130-136); (Wakako Ohmura et. Al. Holzforchung. 56. (2002). 493-497).

In this way, optically pure enantiomers of dihydroquercetin in amounts up to several tenths of a gram can be obtained.

Claims (1)

The method of isolation of (2R, 3R) - and (2S, 3S) enantiomers 3.3 ', 4', 5.7 pentahydroxyflavanone (dihydroquercetin), characterized in that the crushed larch wood is extracted with ethyl alcohol 90-95% at a temperature 55-66 ° C for 1-2 hours, the extract is filtered, propylene glycol is added, the mixture is evaporated, the obtained residue is treated with 5% sodium chloride solution in softened water at a temperature of 20-30 ° C, the resulting resin is separated, the resulting flavonoid complex is eluted 90-95% ethyl alcohol from the column, the eluate is evaporated, cooling is precipitated water to 0 - 2 ° C, the precipitated precipitate is crystallized, filtered and dried, the resulting product, which is a bioflavonoid complex, is crushed, followed by dissolution in a mixture: methyl alcohol - water (10:90) and chromatographed in reverse phase using as a mobile phase, a solution of β-cyclodextrin in a mixture of methyl alcohol - water (10:90) while cooling the column to 0 ° C, the collected fractions of the individual enantiomers are evaporated and dried.