RU2642294C1 - Method for obtaining concentrate of unsaturated alkyl-glycerine ethers from sea hydrobionts - Google Patents

Abstract

FIELD: food industry; pharmaceutical industry.

SUBSTANCE: invention relates to the food and pharmaceutical industry, in particular to a process for the production of alkyl-glycerol esters (AGE) from marine fats. Method consists in that sea fats containing the AGE in their composition are subjected to hydrolysis to recover free fatty acids and release the unsaponifiable fraction, the mixture is neutralized with acid and crystallized from acetone to obtain a precipitate of saturated AGE, the remaining lipid mixture is evaporated, further, by chromatography on a column of silica gel and an eluting system of organic solvents, with increasing polarity, a total fraction of alkyl-glycerol esters is isolated, the solvent is evaporated, the mixture is dissolved in a non-polar organic solvent to a concentration of 0.5 to 2 % of alkyl glycerol esters and mixed with solutions of silver nitrate in 20–60 % aqueous ethanol with a non-polar:polar = 1:1–2.8, ratio, and the ratio of unsaturated alkyl-glycerols: AgNO₃ = 1:3–7, mol/mol, stirred, centrifuged, the lower aqueous-ethanol layer separated, acidified with hydrochloric acid to precipitate the silver salt, filter the solution and evaporate the solvent to isolate the unsaturated alkyl-glycerol esters. Above described method takes into account the processes of interaction of silver salts with unsaturated organic compounds and allows using the advantages of this fact in the development of a simple technology for the concentration of unsaturated alkyl-glycerol esters.

EFFECT: method is effective and economical for the production of unsaturated alkyl-glycerol esters from marine fats, providing not only high quality of the target product, but also the integrated use of valuable marine raw materials.

4 cl, 2 dwg, 1 tbl

Description

The invention relates to the food and pharmaceutical industries and can be used to produce alkyl-glycerol esters with high biological effect from sea fats.

Under the name “marine lipids” a large number of biologically active substances are combined - polyunsaturated fatty acids, phospholipids, hydrocarbons such as squalene and squalane, carotenoids and others. The composition of the total lipid of some aquatic organisms includes unusual lipids - 1-O-alkyl-diacylglycerols - compounds formed by fatty acids and alcohols (butyl, chemical and selachilic, etc. - 1-O-alkyl-glycerol ethers (AGE), see Fig. . 1 and Fig. 2.

Alkyl radicals in the 1st position of glycerol are saturated, both in the case of chymyl and batyl alcohols, and unsaturated in selachyl alcohol. The chemical structure of alkyl glycerol esters (AGE) is shown in FIG. 1, AGE is successfully used in rehabilitation therapy in the treatment of cancer, radiation injuries, immunodeficiency states, disorders of higher nervous activity, etc.

Recent studies have shown that AGEs with an unsaturated radical (primarily selachyl alcohol) have greater biological activity, possibly significantly greater than saturated AGEs (A.-L. Deniau, P. Mosset, D.Le Bot, A.V. Legrand Which alkylglycerols from shark liver oil have anti-tumor activities? / Biochimie. 2010. P. 1-3). Therefore, the search for methods for isolating such compounds and creating drugs with their participation is important in the light of the prevention and treatment of many pathologies.

At the moment, there is no simple way to obtain unsaturated and saturated AGE in one process with a high yield, production efficiency, reduction of the time parameter and simplification of the entire technological scheme that allows the method to be carried out by affordable means in any laboratory.

A known method of obtaining concentrates of unsaturated AGE from the liver of a prickly shark (Katran), a Greenland shark and a chimera (Hallgren B., Larsson SO "Separation and identification of alkoxyglycerols" // Acta Chem. Scand. 1959. Vol.13, N 7. P. 2147-2148). The method is as follows: unsaponifiable substances extracted from hydrolyzed liver oil of fish are chromatographed on an activated alumina column, a mixture of 10% methanol in methylene chloride is used to elute AGE. The following concentrates of unsaturated AGE were obtained (by selachyl alcohol) - 47.8% for Katran, 59.4% for the Greenland shark and 53.6% for the chimera. Other specifying data on the release of AGE are absent.

The disadvantages of the method: 1) the implementation of this method rather illustrates the very presence of AGE in the liver of aquatic organisms than actually offers a rational way to isolate them;

2) chromatographic separation of AGE, without preliminary concentration, for obtaining large quantities of drugs is not acceptable - low efficiency of the method, problems with sorbent regeneration, and, most importantly, high cost of the resulting product;

3) the use of methyl alcohol reduces interest in the method, as it uses an extremely toxic solvent, not acceptable in food production.

A known method for the isolation of alkyl glycerol esters from the fat of Commander squid Berryteuthis magister (RF Patent No. 2415125 IPC C07C 43/13, C07C 41/16, C07C 41/40, publ. 03/27/2013). The method includes hydrolysis of sea fat to isolate free fatty acids and release an unsaponifiable fraction containing AGE, neutralizing the resulting fat mixture with acid, washing with water and sequential double crystallization from an organic solvent, filtering and drying the precipitate formed after each crystallization. For the first and second crystallization, the fat mixture in an organic solvent is kept first for 10-12 hours at room temperature, and then for 12-15 hours at 0-4 ° C.

Acetone or hexane is used in the process, and only one solvent can be used in the whole process, or a combination thereof.

When using hexane as an organic solvent, the ratio of fat mixture to hexane in the first crystallization is 1:10, kg / l, and in the second crystallization of the intermediate product 1:50, kg / l.

When using acetone as an organic solvent, the ratio of fat mixture to acetone is 1:10, kg / l, both in the first and second crystallization of the intermediate product.

The method allows to obtain saturated AGE with a yield of 50-59.0% and a purity of more than 99%.

Disadvantages:

1) the method uses significant volumes of solvents, which requires the use of large containers, appropriate filtering systems and continuous regeneration of solvents;

2) the two crystallizations of the method are separated by drying the precipitate so that when changing the solvent there is no contamination of one solvent with another; drying really interrupts the process and postpones the subsequent second crystallization for several hours;

3) the duration of the entire process - from the initial fat to the target product (AGE) - is at least 59 hours, an average of 65 hours, despite the fact that the process between the stages is inextricable. Such a long process time has an extremely negative effect on the performance of the AGE production line and does not allow for quick and timely processing of incoming raw materials.

4) AGE released by this method is 98-99% composed of saturated compounds, all unsaturated AGEs with the highest biological activity remain in an unused residue and are lost due to the undeveloped technology.

A known method of isolation and concentration of alkyl glycerol esters from marine fats (Patent GB 1,076,706, IPC C07C 43/02, publ. 07/19/1967). The method consists in the following: sea fat (shark liver oil) by transesterification with methyl alcohol (ethyl and isopropyl allowed) and an alkaline catalyst (KOH or Na-OCH ₃ ) were transferred to fatty acid methyl esters at a temperature of 70 ° C for 16 hours and stirring, while the alkyl glycerol esters were released into the mixture, the methanol remaining in the mixture was evaporated in vacuo, and the AGE was repeatedly extracted from 90% water-methanol or water-ethanol solutions in a mixture-extractant ratio of 0.6: 1, kg / l, p At 50 ° С and stirring for 15 minutes and settling for 2 hours, the phase with the solvent was separated and the mixture was extracted again under the same conditions (6 times), the combined water-alcohol solutions were evaporated, obtaining the desired AGE.

The yield of total AGE (saturated and unsaturated) is 71%, the concentration in the product (purity) is 40%.

The disadvantages of the method:

1) the use of methanol is unacceptable in food production and undesirable in pharmaceutical;

2) the method involves the use of a well-established technological line, which complicates its implementation in small and other fishery and food enterprises of the domestic industry;

3) the formula indicates the use of low molecular weight alcohols for extraction of 1-3 carbon atoms, however, no examples other than methanol are given, and methanol is preferred in the patent description;

4) the method also allows the use of acetic acid and acetonitrile in the extraction mixture, such possibilities only complicate the regeneration of solvents and their removal from the final product;

5) using this method, it is possible to obtain total AGE with a purity of only 47.7% (the authors indicate that the same experiment, Example 4, was somewhat better in the laboratory), which is insufficient for commercial use and requires further additional purification stages;

6) the authors in the patent description allow the release of AGE with 7-8-fold extraction up to 90%, but no convincing evidence is given, although in example 4, in which seven-fold extraction was used, a yield of 71% is achieved;

7) there can be no talk of any concentrate of unsaturated AGE, since the extraction method is not selective and the concentration of saturated and unsaturated AGE in the product is equally effective, and the authors do not perform preliminary removal of saturated AGE;

8) the method is quite laborious and, judging by the description, requires increased economic costs for its functioning.

Closest to the claimed method according to the achieved result is a method of separating unsaturated selachyl alcohol from a mixture of alkyl-glycerol ethers (US Patent 7,368,603, IPC C07C 51/42, publ. 06.05.2008). The method is as follows.

Prepare an ion exchange resin (Amberlite 15 sorbent), saturating it with silver nitrate ions, AgNO ₃ , to a concentration of 4.6 mEq / g (501.4 mg / g sorbent) (column 4, paragraph 1; column 7, paragraphs 2nd and 3rd). Next, the resin is mixed with a mixture of separable compounds (saturated and unsaturated alkyl glycerol esters - chymyl, butyl and selachyl alcohols) in an organic solvent (Claim 6), which can be alcohols, ethers, ketones and other solvents (Claims , paragraphs 12 and 13). Then, the solvent from the system is filtered off, and from the complex formed by the resin and separable substances, the substances are successively eluted with organic solvents of different polarities according to their properties, including unsaturated alkyl glycerol ether - selachyl alcohol. To obtain the desired products, the resulting eluates are evaporated.

The disadvantages of the method.

1. The main objective of this invention is to obtain pure saturated compounds. Only two examples are given in the description and both are devoted to the separation of derivatives of saturated myristic (C14: 0), palmitic (C16: 0) and stearic (C18: 0) acids.

2. Mention of the separation of the mixture of alkyl-glycerol ethers is not given in the examples, but is given only in paragraph 6 of the Formula. In this case, no process conditions are given, for example, the ratio of the resin to the mixture to be separated, the concentration of unsaturated selachyl alcohol in the mixture, the solvent for primary application to the resin, a solvent or a group of solvents or mixtures thereof for washing substances sorbed on the resin.

There is no yield and concentration (in terms of the basic substance) of the released substances.

3. The authors do not mention anything about the regeneration of the resin (and deposited silver, in particular). Whether the resulting resin is reusable or intended for single experiments. The economic side of the process is not completely affected, it should be borne in mind that any technological research related to impregnated silver without taking into account its regeneration is not profitable, and the authors declare the use of this method in the manufacture of pharmaceutical preparations (column 1, paragraphs 3 and 4 below).

4. From the description of the invention it is impossible to determine the purity of the unsaturated alkyl-glycerol ether (selachyl alcohol), it is also impossible to determine the yields and productivity of the process. Therefore, the proposed method should be qualified as potential for these purposes, but the real possibilities of which are not confirmed.

The technical problem posed by the invention is the development of an effective and economical method for the production of unsaturated alkyl-glycerol ethers from sea fats with the aim of extracting them from the prepared raw materials with the high quality of the target product.

The technical problem posed is solved by the fact that in the known method for the separation of unsaturated alkyl glycerol esters (AGE), which includes treating a mixture of saturated and unsaturated AGE with a sorbent in an organic solvent solution, removing the organic solvent from the system by filtration, eluting the AGE with the sorbent with organic solvents of different polarity, evaporation of the obtained eluate, characterized in that as a mixture of saturated and unsaturated AGE use a hydrolyzate of sea fat obtained as a result of Christmas tree hydrolysis, which is acidified, washed with water, then saturated AGE is crystallized from acetone, the precipitate is filtered off, and the filtrate is evaporated and then passed through a sorbent. As a sorbent, a silica gel column and an eluting system of organic solvents are used to increase their polarity, the eluate is evaporated, then unsaturated AGE is extracted, for which one stripped off the eluate is dissolved in a non-polar organic solvent to an AGE concentration of 0.5-2%, mixed with nitric acid solutions silver in 20-60% aqueous ethanol with a ratio of liquid phases non-polar: polar = 1: 1-2.8, vol./about., and a ratio of unsaturated AGE: AgNO ₃ = 1: 3-7, mol / mol, mix, centrifuged, separate the lower water-ethanol layer, acidified with hydrochloric acid to pH = 4, filter the solution, evaporate the organic solvent, releasing unsaturated AGE.

The process of crystallization of saturated AGE from acetone allows you to separate the bulk of saturated AGE from the mixture, as a result of which, it helps to obtain a pure target product - unsaturated AGE.

The use of AgNO ₃ in the process allows selective binding of unsaturated compounds (substances having one or more double bonds in a molecule), while silver ions are inert with respect to saturated compounds. In contrast to the prototype, in which an ion exchange resin impregnated with a silver salt is used, the inventive method provides both significant convenience in the separation of substances and high yields of the target product. Silver nitrate, which is readily soluble in water-ethanol mixtures, but not soluble in nonpolar solvents (hexane, petroleum ether, etc.) in the solution, dissociates with the formation of active silver ions (Ag +), which, when the mixture is treated with this solution, bind coordination bonds with double bonds bonds in unsaturated AGE and, in fact, “drag” these compounds into a water-alcohol solution. This approach is more effective than the use of a resin impregnated with a silver salt, since each time the load (loaded mixture) on the resin must be selected individually due to differences in the initial composition, which requires additional trial studies and, for the same reason, adjustments must be made in a set of solvents, their ratio when leaching substances from the resin.

It is advisable to use sequentially chloroform → chloroform + ethanol (ethanol + acetic acid) as organic solvents with increasing polarity in the elution system for chromatography, and pentane, hexane, and petroleum ether are used as a non-polar organic solvent for the separated mixture of alkyl-glycerol ethers.

These non-polar organic solvents are most suitable since they do not form emulsions with a polar fraction, thereby creating a homogeneous medium and increasing the purity of the emitted unsaturated AGE.

To increase the yield of unsaturated AGE, the extraction of AGE from a nonpolar organic solvent is carried out from one to three times.

Description of the General scheme of the claimed method.

Hydrolysis of Fat from the Digestive Gland of Commander Squid

The initial stage of AGE isolation is the alkaline hydrolysis of squid digestive gland lipids, which results in cleavage of the FA from the alkyl-diacyl-glycerol (ADAG) molecule; 2.

075 L of 96% ethanol and 0.5 L of a 40% KOH solution are added to 1 kg of fat. The reaction is carried out at 60 ° C for 1 hour. The end of the hydrolysis reaction is controlled by thin layer chromatography.

At the end of the reaction, the mixture was transferred to a separatory funnel, 2 L of water was added and neutralized with an aqueous solution of H ₂ SO ₄ to pH = 4 on indicator paper. Hydrolyzed lipids are washed twice with 1.5 L of a 1% NaCl solution.

Precipitation (crystallization) of saturated alkyl glycerol esters

0.5 kg of hydrolyzed lipids were taken, dissolved in 1.4 l of acetone, stirred vigorously and placed in a freezer (-15 ° C) for 18 hours to precipitate (crystallize) saturated AGE. At the end of the process, the precipitate of saturated AGE is filtered off, and the acetone solution is evaporated, obtaining the initial product of unsaturated AGE for further concentration.

Isolation of total saturated and unsaturated AGE by chromatography 50 g of the product from the previous step are loaded onto a chromatographic column with 300 g of silica gel (particle size 100 μm). Elution is carried out sequentially with solvent mixtures - 1) chloroform (1000 ml), 2) chloroform-ethanol, 9: 1, v / v. (800 ml), 3) ethanol-acetic acid, 1: 0.02, vol./about. (800 ml). The control of fractions containing total AGE is carried out by thin layer chromatography. Fractions with AGE are combined and evaporated.

Extraction of unsaturated AGE solutions with AgNO ₃

A mixture of total saturated and unsaturated AGE from the previous stage, 600 mg, is dissolved in a non-polar organic solvent to a concentration of 0.5-2% alkyl glycerol esters and mixed with solutions of silver nitrate in 20-60% aqueous ethanol with a non-polar liquid phase ratio : polar = 1: 1-2.8, vol./about. and the ratio of unsaturated alkyl glycerols: AgNO ₃ = 1: 3-7, mol / mol, stirred, centrifuged the mixture at 3000 rpm./min, separated the lower water-ethanol layer, repeated extraction of unsaturated AGE from non-polar solvent an additional 1-2 times under the same conditions, the combined water-ethanol extracts are acidified with hydrochloric acid to pH = 4 to precipitate a silver salt, the solution is filtered and the solvent is evaporated, releasing unsaturated alkyl glycerol esters.

When developing the method, solutions were found that made it possible to implement it in the most economical, and most importantly, simple way.

It was found that the optimal concentration of AGE in a non-polar solvent should be 0.5-2.0%. This, firstly, ensures good solubility of the substance, and, secondly, contributes to the effective extraction of the unsaturated fraction of AGE from the total mixture. A decrease in the concentration of total AGE in the solvent of less than 0.5% did not increase the yield and quality of the target product, but only led to an increase in the extraction mixture itself and was not justified by the large expenditures of the solvent. An increase in the concentration of AGE in the solution over 2% reduced the quality of the product - the proportion of saturated AGE increased in it.

As a polar solvent for extraction, aqueous solutions of ethyl alcohol were used. As it turned out, the optimal concentration of ethanol is in the range of 20-60%. Moreover, a decrease in alcohol concentration of less than 20% makes the solution so unsuitable for lipid extraction that, despite the content of AgNO ₃ , a significant part of unsaturated AGE remains in a non-polar solvent, and an increase in concentration of more than 60% leads to an excessive transition of saturated AGE to the extractant , reducing the purity of the product.

The following optimal ratios of non-polar organic solvent and aqueous-alcoholic solutions were established experimentally during the extraction of unsaturated AGE - 1: 1-2.8, vol./about, respectively. It turned out that using a ratio of less than 1: 1 leads to contamination of the extractant with saturated AGE, which requires further further purification of the product. An increase in the ratio of more than 1: 2.8, vol./about., Does not affect the recoverable unsaturated AGE, i.e. the increase in extractant will become costly when implementing the method.

A particularly significant result, unexpected in its effectiveness, during experimental studies was the determination of the actual ratio of unsaturated AGE in a nonpolar organic solvent to the amount of AgNO ₃ in the water-alcohol extractant — unsaturated AGE: AgNO ₃ = 1: 3-7, mol / mol. It was established that the minimum limit of the presence of AgNO ₃ in the mixture should be at least 3 mol per 1 mol of unsaturated AGE. Violation of the proportion leads to a decrease in the extraction of unsaturated AGE from a non-polar solvent to the water-ethanol fraction. When the content of AgNO ₃ in the extractant is more than 7 mol, the yield of the product does not increase, and the consumption of reagents increases several times.

During the process, it was found that a single extraction does not provide the maximum extraction of unsaturated AGE. To increase this indicator, an additional 1 or 2 extractions were carried out under the same conditions. The total number of extractions did not exceed 3. By repeated, 4th or more extractions, it was no longer possible to increase the yield of the target product.

The invention is illustrated by the following FIG. and table .; in FIG. 1 is a diagram of alkaline hydrolysis of alkyl diacyl glycerols (ADAG); in FIG. 2. The structural formula of alkyl glycerol esters is given in the table. The composition of lipids in products (and intermediates) at different stages of the isolation of unsaturated AGE is presented.

The method is as follows.

EXAMPLE 1

Isolation of saturated and unsaturated AGE from the digestive gland fat of Commander squid Berryteuthis magister

The main classes of digestive lipids are alkyl-diacyl-glycerols (ADAG) and free fatty acids, 37.5% and 29.7%, respectively. In addition to these components, the composition of lipids also includes triacyl glycerols (18.5%), cholesterol (7.0%). Polar lipids, sterol esters and waxes are present in small quantities.

The qualitative and quantitative composition of lipid mixtures at all stages of the process is evaluated using thin layer chromatography (TLC) (Sorbfil plates, hexane-diethyl ether-acetic acid system 50: 50: 1, v / v / v) using a flatbed scanner and evaluating the density (intensity) of spots shown on TLC chromatograms using the TLC manager version 3.1 2005 @PinSoft program.

The composition of AGE isolated from hydrolyzed lipids, as well as the obtained fractions, was determined by gas chromatography-mass spectrometry (GC-MS).

Take 10 kg of the digestive gland (frozen squid guts) are heated in a screw with a water jacket and separated. 4.85 kg of fat are obtained.

Hydrolysis of squid digestive lipids

0.75 L of 96% ethanol and 0.5 L of a 40% KOH solution are added to 1 kg of fat. The reaction is carried out at 60 ° C for 1 hour. The end of the hydrolysis reaction is controlled by thin layer chromatography.

At the end of the reaction, the mixture was transferred to a separatory funnel, 2 L of water was added and neutralized with an aqueous solution of H ₂ SO ₄ to pH = 4 on indicator paper. The separated lipids are washed twice with 1.5 L of a 1% aqueous NaCl solution.

Received 0.88 kg of hydrolyzed fat.

The yield (to the initial mass of lipids) is 88%.

The lipid composition after hydrolysis is shown in summary table 1.

Isolation of saturated alkyl glycerol esters

After alkaline hydrolysis, crystallization of saturated AGE from acetone is carried out according to the previously developed method (RF Patent No. 2476611, IPC C22B 7/04, C22B 15/00, C22B 23/00, C22B 4/06, published on 02/27/2013, “Method for the production of alkyl glycerol esters from sea fats ”/ Kasyanov SP, Latyshev NA, Chu Quang Chuen).

0.5 kg of hydrolyzed lipids was taken, dissolved in acetone, stirred vigorously and placed in a freezer (-15 ° C) for 18 hours to precipitate saturated AGE. The precipitate formed is filtered through filter paper at -15 ° C. The resulting filtrate containing fatty acids and unsaturated alkyl glycerol esters was evaporated in vacuo.

The precipitate formed is recrystallized from acetone under the same conditions, filtered off from the solvent, and dried.

The precipitate obtained is a mixture of saturated AGE with a purity of 99.6%, the main component of which is chymyl alcohol (content 95.7%).

The yield of saturated AGE was 11.0% of the lipids taken for crystallization, or 42.0% of the AGE content in hydrolyzed lipids.

Isolation of total saturated and unsaturated AGE by chromatography

To isolate unsaturated AGE using one stripped off filtrate obtained after the first crystallization of the hydrolyzed mixture from acetone at -15 ° C.

50 g of one stripped off filtrate from the previous step are loaded onto a chromatographic column with 300 g of silica gel (particle size 100 μm). Elution is carried out sequentially with solvent mixtures with an increase in their polarity - 1) chloroform (1000 ml), 2) chloroform-ethanol, 9: 1, vol./about. (800 ml), 3) ethanol-acetic acid, 1: 0.02, vol./about. (800 ml). The control of fractions containing total AGE is carried out by thin layer chromatography. Fractions with AGE are combined and evaporated.

Received 7.06 g of total AGE.

The output (on the initial content of AGE in one stripped off filtrate) is 92%.

The content of total AGE in the product is 99%.

The content of unsaturated AGE, from the total AGE, is 37.6%.

Extraction of unsaturated AGE solutions with AgNO ₃

A mixture of total saturated and unsaturated AGE from the previous step, 600 mg, is dissolved in 120 ml of hexane (concentration of alkyl glycerol esters in a solution of 0.5%) and mixed with a solution of silver nitrate (560 mg) in 120 ml of 20% aqueous ethanol when the ratio of liquid phases is non-polar: polar = 1: 1, vol./about. and the ratio of unsaturated alkyl glycerols: AgNO ₃ = 1: 5, mol / mol, stirred, centrifuged the mixture at 3000 rpm./min, separated the lower water-ethanol layer, repeated extraction of unsaturated AGE from a non-polar solvent an additional 1 time at the same Under conditions, the combined water-ethanol extracts are acidified with hydrochloric acid to pH = 4 to precipitate a silver salt (AgCl), the solution is filtered and the solvent is evaporated, releasing unsaturated alkyl glycerol esters.

Received 204 mg of a concentrate of unsaturated AGE.

The unsaturated AGE concentrate was a slightly brownish oily liquid at 20–25 ° C and a solid margarine-like mass at 0–5 ° C with a faint odor.

The yield of unsaturated AGE at the extraction stage is 90.4%.

The total yield of unsaturated AGE (from the initial content in fat) is 74.8%.

The content of unsaturated AGE in the concentrate is 68.5%.

The main components of the concentrate were isomers of selachyl alcohol (C-18: 1) (53.5%). The results are presented in table.

EXAMPLE 2

To obtain unsaturated AGE was taken 600 mg of the total saturated and unsaturated AGE isolated at the stage of chromatographic purification of AGE in Example 1.

The selection of unsaturated AGE was carried out as in Example 1 with the following changes:

- the concentration of total AGE in a nonpolar organic solvent was 1.15%;

- pentane was used as a non-polar solvent;

- extraction of unsaturated AGE was carried out with 60% aqueous ethanol;

- during extraction, the ratio of the liquid phases is non-polar: polar = 1: 2.8, vol./about .;

- the ratio of unsaturated AGE: AgNO ₃ = 1: 3, mol / mol;

- number of extractions 3.

The yield of unsaturated AGE at the extraction stage is 90.0%.

The total yield of unsaturated AGE (from the initial content in fat) is 74.1%.

The content of unsaturated AGE in the concentrate is 67.4%.

The results are presented in table.

EXAMPLE 3

To obtain unsaturated AGE was taken 600 mg of the total saturated and unsaturated AGE isolated at the stage of chromatographic purification of AGE in Example 1.

The selection of unsaturated AGE was carried out as in Example 1 with the following changes:

- the concentration of total AGE in a nonpolar organic solvent was 2.0%;

- as a non-polar solvent used petroleum ether;

- extraction of unsaturated AGE was carried out with 45% aqueous ethanol;

- during extraction, the ratio of the liquid phases is non-polar: polar = 1: 1.6, vol./about .;

- the ratio of unsaturated AGE: AgNO ₃ = 1: 7, mol / mol;

- the number of extractions 2.

The yield of unsaturated AGE at the extraction stage is 91.1%.

The total yield of unsaturated AGE (from the initial content in fat) is 75.4%.

The content of unsaturated AGE in the concentrate is 69.7%.

The results are presented in table.

The proposed methodological approach made it possible to obtain both saturated AGE with a purity of 96.0% (Dietary supplement Lipidomarin) and a concentrate with an unsaturated AGE content of 67.4-69.7%. At the same time, a feature is the use of simple isolation methods that do not require sophisticated equipment and highly qualified personnel, which can be used in any research laboratory to obtain the required amounts of biologically active AGE.

As can be seen from the above examples and the results shown in the table, the claimed method allows to obtain a concentrate of unsaturated AGE of good quality with a high yield, while the method is not only technologically not complicated, but also economically advantageous, since it makes maximum use of valuable marine raw materials.

The silver precipitated during the implementation of the method (in the form of an insoluble salt of AgCl) is filtered off and converted into AgNO _{3 by} known chemical procedures and reused in the method.

The sorbent (silica gel), after isolation of the total alkyl glycerol esters, is washed with a mixture of ethanol-acetic acid, calcined at 650 ° C for 8 hours and re-used to separate lipids.

Solutions of organic solvents are regenerated by reintroducing into the process, and not the utilized bottoms remaining after evaporation and representing free fatty acids, can be used in the technology for the preparation of omega-3 polyunsaturated fatty acid concentrate (RF Patent No. 1581737 dated 05/21/1993 “Method for the preparation of ethyl ester concentrate of eicosapentaenoic and docosahexaenoic acids ”, Kasyanov SP, Latyshev NA, Glushenko TV).

The concentrate of unsaturated AGE obtained by the method expands the possibilities of researchers for its use in biochemical, medical and dietetic experiments on the development of domestic effective anti-cancer drugs, in immunodeficiency states and other socially significant diseases.

Claims (4)

1. The method of separation of unsaturated alkyl glycerol esters, including processing a mixture of saturated and unsaturated alkyl glycerol esters with a sorbent in an organic solvent solution, removing the solvent from the system by filtration, eluting the alkyl glycerol esters from the sorbent with organic solvents with different polarity, evaporating the resulting eluate, which differs in that, as a mixture of saturated and unsaturated alkyl glycerol esters, a marine fat hydrolyzate obtained from an alkaline hydrochloride is used isa, which is acidified, washed with water, then saturated alkyl glycerol ethers are crystallized from acetone, the precipitate is filtered off, the filtrate is evaporated and then passed through a sorbent, which is used as a column with silica gel and an elution system from organic solvents with an increase in their polarity, the eluate is evaporated, then the unsaturated alkyl-glycerol ethers are extracted, for which the one-off eluate is dissolved in a non-polar organic solvent to an alkyl glycerol ester concentration of 0.5-2%, mixed with solutions of silver nitrate in 20-60% aqueous ethanol with a liquid phase ratio of non-polar: polar = 1: 1-2.8, vol./about., and a ratio of unsaturated alkyl glycerins: AgNO ₃ = 1: 3- 7, mol / mol, stirred, centrifuged, the lower aqueous ethanol layer was separated, acidified with hydrochloric acid to pH = 4, the solution was filtered, and the organic solvent was evaporated. 2. The method according to claim 1, characterized in that pentane, hexane, petroleum ether are used as a non-polar organic solvent to dissolve the alkyl glycerol esters. 3. The method according to claim 1, characterized in that chloroform, ethanol, acetic acid and mixtures thereof are used as organic solvents of the eluting system. 4. The method according to claim 1, characterized in that the extraction of unsaturated alkyl glycerol esters from a solution of a non-polar organic solvent is carried out from one to three times.

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