RU2627273C1 - Method for arachidonic acid production - Google Patents

Abstract

FIELD: pharmacology.

SUBSTANCE: method for preparation of arachidonic acid from an animal raw material includes alkaline hydrolysis of lipids isolated from the raw material, followed by hydrolyzate acidification, iodine-lactonization reaction, iodine-lactone extraction from the reaction mixture with an organic solvent, iodine-lactone dilution with a silicone agent, neutralisation of the released iodine with sodium thiosulfate, fatty acids extraction with an organic solvent, where, prior to the iodine-lactonization reaction, lipid hydrolyzate is treated with lithium alkali in acetone and saturated acids are isolated as a precipitate, Iodine-lactonization is carried out by transferring lithium salts of unsaturated fatty acids to sodium salts, which are treated with iodine solution to form iodine-lactones of fatty acids at a ratio of the acids sum (arachidonic, eicosapentaenoic and docosahexaenoic):iodine equal to 1.0:1.0-1.2 mol/mol, at mixture temperature of 18-22°C, with duration of iodine solution addition to the reaction mixture of 30-60 minutes, followed by solutions maintaining for 60 minutes; after iodine-lactones extraction from the reaction mixture with an organic solvent, delta- and gamma-iodo-lactone separation is further carried out by chromatography on silica gel using benzene as the eluent; and the disclosure of delta-iodine-lactones of arachidonic and eicosapentaenoic acids is carried out with silicide at a ratio of arachidonic acid iodine-lactone + eicosapentaenoic acid/silicide = 1.0:1.0-1.2 mol/mol, mixture temperature of 18-22°C, reaction time of 120-180 minutes; after fatty acids extraction with an organic solvent, they are esterified with ethyl alcohol and arachidonic acid is isolated as ethyl ether by reverse phase high performance liquid chromatography (C-18) using an ethanol-water eluting system of 8.0:1.0-2.5 vol./vol.

EFFECT: easy and reproducible way to separate pure arachidonic acid from animal tissues, available for sale in laboratories and industrial plants using arachidonic acid in biochemical, medical and dietetic studies.

4 cl, 2 dwg, 1 tbl, 5 ex

Description

The invention relates to the food and pharmaceutical industries and can be used to obtain compositions with a high content of arachidonic acid (AA), which has a pronounced biological effect.

At the moment, we do not know from the scientific and patent literature about methods for producing pharmaceutical compositions with a high AK content, the preparation of which would take place at the same time as high yield and high purity of the target product, while maintaining the efficiency of the process - radically simplifying the process steps, reducing the process time , increasing its environmental friendliness. Thus, an intensification of the process is achieved, which gives pharmaceutical companies the opportunity to obtain new drugs (compositions) with a high content of AA, which have a therapeutic and prophylactic effect in case of lipid metabolism disorders in the body.

Arachidonic acid is a biochemically important essential acid, a precursor of various eicosanoids and other active mediators. In animals, AK is formed in several stages from linoleic acid by elongation and desaturation (Sprecher N. "Metabolism of highly unsaturated n-3 and n-6 fatty acids" // BBA. - 2000. - Vol. 1486. - P. 219- 231), or comes with food. In nature, AK accumulates in phospholipids of cell membranes, from which it is released by phospholipase A ₂ under the action of inflammatory agents (interleukin-1, -6; tumor necrosis factor; phorbol esters, etc.) or other stimulants. Arachidonic acid is the starting material for the biosynthesis of a number of biologically active oxidized derivatives (especially hydroxy, hydroperoxy and epoxy compounds), known as eicosanoids. They are divided into various groups in the arachidonic acid cascade, which is shown in FIG. one.

Among the known "signaling" compounds of arachidonic acid, mention should be made of isoprostanes (non-enzymatic oxidation pathway), 2 - arachidonoyl-glycerol and arachidonic acid ethanolamide (anandamide) - endogenous cannabinoid (Sergeeva MG, Varfolomeeva A. T. "Cascade of arachidonic acid". M .: Public education, 2006. - 256 s).

Studies of arachidonic acid, as a potential substrate for the synthesis and biosynthesis of biologically active substances, continue from year to year at a steady pace. However, for most laboratories, the issue of acquiring AK for research is an acute issue, since the cost of pure AK preparations is extremely high. For example, the company Sigma-Aldrich offers AK (98.5%) at 1130 euros / g. Almost any AK-related research starts with significant costs. This situation has arisen, mainly due to the fact that most experimenters, even having the necessary devices, do not have experience in obtaining AK.

There are many laboratory and industrial methods for producing AK, but their application in a particular process is not obvious and should be justified by the use of appropriate raw materials, the capabilities of the method itself and the presence of certain equipment.

Earlier in world practice, such methods as low-temperature crystallization of fatty acids (FAs) from solvents, molecular distillation of fatty acid methyl esters (FAMEs) in vacuum, complexation of FAs with urea, and chromatographic separation of FAs on sorbents, including silica gel impregnated with salts, were used to obtain AAs. silver. A good illustration of these methods are the work below.

A known method for the isolation of AK from lipids of the adrenal glands of cattle (Herb S.F., Riemenschneider R.W., Donaldson J. "Isolation of natural arachidonic acid as its methyl esters" // JAOCS. 1951. Vol. 28, N 2. P 55-58). The method was carried out as follows: the isolated lipids from the adrenal glands were hydrolyzed to free fatty acids (8-9% AK), crystallized from acetone sequentially at 0 ° C, -18 ° C, -40 ° C, the FA was converted to methyl esters, and then the AK was fractionated on silica gel (silicic acid), according to the authors, an AK concentrate of 99.15% purity was obtained (too optimistic result for that time and the possibility of a real analysis).

The disadvantages of the method:

1) the method of crystallization of FA from acetone has the only possibility - separation of saturated acids from a mixture of FA; other modifications with lower temperatures do not carry an effective concentration of polyunsaturated FAs;

2) fractionation of FAs on silica gel, as practice shows, is not a productive method; in principle, the method is feasible, but the load on the column is small, and the consumption of organic solvents is high;

3) the authors didn’t make a reservation, but judging by the tables given, it is very insignificant, and the cleanliness of the AK raises serious doubts.

A known method of producing AA from mammalian adrenal lipids (A.S. USSR No. 247461, IPC A61K 31/22, A61P 3/00, C11C 1/02, published 01.01.1969), in which the lipids are hydrolyzed with alkali, acidified to excrete free fatty acids, extracted with toluene, the solvent is evaporated, and the lipids are redissolved in acetone and the saturated fraction is precipitated from it at minus 15 ° С, filtered, the fatty acids are esterified with ethyl alcohol (AK content 11.6%) and subjected to distillation at a temperature of 193-205 ° C and a residual pressure of 3 mm RT. Art.

AK content in the final product - 25%.

General exit - 20-25%.

The disadvantages of the method:

1) in the extraction of hydrolyzed lipids, the authors use toluene, poorly subjected to further distillation (boiling point 110 ° C), moreover, it is very toxic;

2) crystallization of free FAs from acetone is an inefficient process, and the temperature (-15 ° С) is not so low that it is possible to significantly increase the concentration of AA;

3) the molecular distillation method causes the destruction and oxidation of AA, especially at such high temperatures and insufficiently complete vacuum;

4) AK obtained with 25% purity hydrochloric ^_ - low rate for any biochemical studies, and 20-25% yield reflects the inefficiency of the methods set.

A known method of extracting AK from lipids of the testes of agricultural animals (A.S. USSR No. 641963, IPC A61K 31/557, A61K 31/20, A61K 31/202, A61P 15/00, C07C 57/03, publ. 15.01. 1979) by the consistent application of the methods of low-temperature crystallization (-20 ° C), molecular distillation (3 mmHg, 220 ° -235 ° C) and column chromatography on silica gel with AgNO ₃ . The yield of 96-99% AK did not exceed 9.2%.

The disadvantages of the method should be considered:

1) the use of molecular distillation at concentration of AA at high temperatures, contributing to the rapid oxidation and destruction of the structure of arachidonic acid;

2) the use of chromatography with AgNO ₃ - the load on the column is small, 1:10, for such a significant content of silver salt on the sorbent -50%, moreover, full-fledged regeneration for reuse of such an “expensive” column is problematic, and its efficiency is already critically reduced after the first use;

3) a small yield of the final product (9.2%), almost 9/10 AK is lost as a result of using the method.

A known method for the isolation of AK from adrenal lipids (AS USSR No. 643496, IPC C07C 57/02, C11B 13/00, publ. 01/25/1979) by fractionation of the LC mixture twice in petroleum ether (-28 ° C 78 ° C) and twice in acetone (-78 ° C), and then using column chromatography on AgNO ₃ . The yield of a 96-99% AK is just over 20%.

The disadvantages of the method are:

1) excessive, in our opinion, application of the non-selective method - low-temperature crystallization - four times, and as a result, an increase from 16-18% to 65-67%, and the total duration of these operations is 40-45 hours, the yield is only 24% ;

2) "silver" chromatography greatly increases the cost of the method, because it makes the use of the column disposable;

3) the total output of the AK is low - about 20%.

A known method for the isolation of AK from pancreatic lipids (A.S. USSR No. 585152, IPC C07C 57/02, C11B 13/00, publ. 12/25/1977), including a complex set of methods: first fractional low-temperature crystallization of FA (-65 ° C ), then the complexation of FA with urea, 2-fold molecular distillation, and final AA refining on an AgNO ₃ column. The yield of AK with a purity of 97-98% was 27-28%.

The disadvantages of the method:

1) low-temperature crystallization of the liquid crystal is a low-productivity and rather laborious method, since it requires manipulation at very low temperatures;

2) complexation of FAs with urea is a "compact" and convenient method, but it has efficiency limits - no more than a 2-3-fold increase in polyunsaturated fatty acids (PUFAs) in concentrate and large losses of PUFAs during the implementation of the method (some valuable acids remains in complex with urea);

3) the molecular distillation of polyunsaturated fatty acids is extremely detrimental to them, since a high temperature (150 ° C) provokes the migration of double bonds in the carbon chain and the degradation of polyunsaturated fatty acids;

4) chromatography on sorbents with AgNO ₃ is a low-throughput procedure that does not justify itself at cost;

5) as a result of such an extensive set of methods, a low yield is not more than 28%.

There is a method of producing AK from insulin production wastes (AS USSR No. 897766, IPC С07С 57/02, С07С 51/00, publ. 15.01.1982) using the following set of methods: low-temperature crystallization of FA from diethyl ether (-25 ° - 50 ° C) three times, then molecular distillation of the FA at 0.05-1 mm Hg. Art. and a temperature of 126 ° - 131 ° C, and then - the method of "silver" chromatography. The yield of 99.9% AA, calculated on its initial content in the mixture, is 42.2%. The disadvantages of the method:

1) the use of diethyl ether as a solvent in the fractionation of FAs is not the best solution, since the separation efficiency of FAs in it leaves much to be desired, therefore, no one currently uses diethyl ether for these purposes, moreover, it is extremely fire hazard and its use in technology is not desirable;

2) molecular distillation at temperatures up to 130 ° C does not contribute to the preservation of highly labile AA and, it should be noted that this method requires appropriate hardware design, not available for many laboratories;

3) the method of "silver" chromatography is the most inefficient link in the method, significantly limiting the volume of AK production;

4) as a result of the foregoing, a low yield of 42.2%. A known method of concentrating AK from pancreatic fatty acids (AS USSR, No. 908356, IPC A61K 31/20, publ. 02.28.1982) using only one stage - the complexation of FA with urea at temperatures up to -20 ° C. The yield of 40-60% AA was 60-78%.

The disadvantages of the method:

1) as mentioned earlier, the complexation of FAs with urea is not a method of obtaining individual PUFAs, its effectiveness is limited by a 2-3-fold increase in their content in the concentrate, but no more, and the loss of the target substance is very significant;

2) the concentration of AK to 60% does not meet the needs of biochemical and medical research, since this requires high-purity drugs; in fact, the method provides only an initial enrichment of the AK mixture for further purification.

Among other new methods that appeared in 1980-90, the method of iodine-lactonization (IL) Δ 4 and Δ 5 PUFA is remarkable for its manufacturability and certain simplicity. The essence of the method lies in the possibility of the formation by such acids as AK, EPA and DHA (docosahexaenoic acid, 22: 6n-3) of iodine-lactones with 5 and 6 membered cycles (delta-iodlactones for AK and EPA, gamma-iodine-lactone for DHA) unlike other acids. In FIG. 2 shows a chemical diagram of the formation and disclosure of delta-iodine-lactone AK.

Iodine-lactones of FAs have other physical and chemical properties than FAs; therefore, their isolation from the reaction mixture and their further opening to the initial FAs are not so laborious.

A known method of producing AK (Corey EJ, Wright SW "A simple process for the purification of arachidonic acid" // Tetrahedron letters. - 1984. - Vol. 25, No. 26. - P. 2729-2730) from a mixture of FA, where AK was the only polyunsaturated FA (50% AK). The method was carried out as follows: the reaction of the formation of AK iodine-lactones (IL-AK) was carried out in tetrahydrofuran, after which IL-AK was separated by extraction, opened in acetonitrile, adding trimethyl iodosilane, TMSi-I, and 97% AA was extracted from the mixture.

The disadvantages of the method:

1) it is difficult to find a mixture of natural lipids, where AK was the only polyunsaturated acid, since EPA and DHA, also forming lactones, are always present in the mixtures, which makes significant adjustments to the implementation of the method;

2) the authors do not indicate a general yield in the implementation of the method, but it is known that the first stage - the formation of IL-AK - is the most problematic and requires the greatest knowledge in the implementation, even minor flaws at this stage lead to significant losses in AK.

A known method of producing AK, described in RF Patent No. 1631067, IPC С11С 1/02, publ. 02/28/1991. The method was carried out as follows: rat liver lipids (AK - 17%, DHA - 7%) were saponified with an alkali solution, then acidified with acid to isolate free fatty acids, then they were converted into potassium salts and treated with iodine solution (with the addition of KI) in the calculation on DHA with a ratio of DHA / I ₂ = 1: 1.1 mol / mol, stirred at 25 ° C for 5 hours, the formed iodine-lactone DHA was extracted with hexane, and potassium iodide and iodine were added to the aqueous layer (the amount of iodine was calculated on AK, contained in the feedstock, more stoichiometric in 5 RA h, the ratio of potassium iodide to iodine is KI / I2 = 0.9 mol / mol) and stirred at 10 ° C for 2 hours, unreacted iodine was neutralized with an aqueous solution of sodium thiosulfate (Na ₂ S ₂ O ₃ ), extracted with IL-AK with hexane -ethyl acetate, the extract was evaporated, the mixture was redissolved again in hexane, the free FAs were washed from AK iodine-lactone with aqueous-alcoholic solutions of bicarbonate or potassium carbonate, hexane was evaporated, and AK iodine-lactone was used in anhydrous acetonitrile with a sizing agent (trimethyl iodosilane, in TMSi-I) ratio of iodine-lactone AK / silylating agent = 1: 0.86, mol / mol, for 3 minutes, the iodine released was neutralized with sodium thiosulfate solution and the AA was extracted from the mixture with chloroform. The purity of the isolated AK is 98.5%, the total yield is 50.0%.

1) Use of potassium iodide (KI) in the mixture for iodine-lactonization — it is known that its addition to the iodine solution slows down the iodine-lactonization reaction and allows for a more “smooth” process. Therefore, iodine is even added in large excess to the content of AA to 5 mol / mol, and the reaction time is limited to 2 hours, while the process temperature should be lowered (10 ° C) due to the possibility of side reactions that reduce the purity of AA. This stage of the process under the mentioned conditions has major drawbacks - unreasonably high use of reagents (iodine, potassium iodide), as well as the need for special control over the temperature regime;

2) The authors use only trimethyl iodosilane (TMSi-I) as the enhancing agent necessary for opening iodine-lactone AK, while the most affordable and inexpensive trimethylchlorosilane (several times cheaper on the market than TMSi-I) for opening iodine -lactone AK is not used, which increases the cost of the product and narrows the scope of the process.

A known method for producing AK by high performance liquid chromatography, HPLC (Yuan C., Wang X., Yu Z. "Separation and preparative purification of arachidonic acid from microbial lipids by urea inclusion reaction and HPLC" // Preparative Biochemistry and Biotechnology. 2007. Vol . 37, N 2.

P. 149-159). The method was carried out as follows: microbial lipids with an AA content of 38.3% were hydrolyzed with a solution of sodium alkali, acidified with hydrochloric acid to isolate free FAs, which were complexed with urea in the ratio FA: urea: methanol - 1: 2: 8, by weight at - 10 ° C (AK - 77.3%, yield at the stage of 68.0%), then the resulting AK concentrate was transferred to FA methyl esters and pure AA (99.5%) was obtained using HPLC on a C-18 preparative column (300 mm × 30 mm id, particle diameter of the sorbent 15 μm), elution system 95: 5, methanol-water, vol./about., de Tector - refractometer.

The total yield in the process of obtaining AK is not specified.

The total implementation time of the method is 15 hours.

The disadvantages of the method:

1) the use of the complexation method of FA with urea dramatically reduces the final yield of the target product - AK;

2) the use of methanol in the complexation of FAs with urea, an extremely toxic compound, does not allow the use of this technique in the medical and food industries;

3) it is known that the performance of HPLC strongly depends on the particle size of the sorbent, therefore, the use of 15 μm particles for the isolation of AK is probably acceptable for reducing the pressure in the system, but at the same time imposes low productivity on the target substance (the speed of the mobile phase is only 5 ml / min ), we implement this method, as this example shows, but it will provide very small outputs for AK;

4) the authors use methanol in the eluting system, although the task of most researchers who offer their own methods for producing active substances is to minimize the use of toxic solvents; the method is interesting because it allows you to select AK, but can not be duplicated due to the requirements for clean production.

Closest to the claimed method according to the number of essential features is the method for producing arachidonic acid described by Gaiday et al. "Separation of Natural Polyunsaturated Fatty Acids by Means of Iodolactonization," JAOCS, v. 68, no 4, 1991, p. 231). The method was carried out as follows: rat liver lipids were hydrolyzed, the hydrolyzate was acidified, and free fatty acids were isolated by low-temperature crystallization, then the resulting polyunsaturated fatty acid concentrate was converted into potassium salts and treated with a mixture of iodine and potassium iodide in the iodine-lactonization reaction, at the end of the iodine-lactone reaction AK was extracted with an organic solvent and then TMS-I was opened to the original AK, the iodine released was neutralized with sodium thiosulfate and organic fatty acids were extracted. solvent.

The disadvantages of the method:

1. In the article, the authors do not give the kinetics of the formation of AK iodine-lactone, while for other acids (EPA and DHA) it is given even in tabular form, only because the process of formation of AK iodine-lactone proposed by the authors is not perfect and must be subjected to comprehensive critical examination;

2. The process of obtaining AK is very long - more than 2 days (55+ hours);

3. Throughout the whole process, different temperature conditions are used (-25 ° С, + 10 ° С, + 15 ° С), which creates certain difficulties in the hardware design when scaling;

4. during the process of AK isolation, the following results were obtained:

- yield 40.5% (based on the fact that 222 mg AA was taken (1 g with a content of 22.2% AA), and 92 mg 97.8% AA or 100% AK 90 mg were obtained;

- AK purity - 97.8%;

the data obtained fully reflect the incompleteness of the study, since most of the AK is lost, and the purity of the product does not meet the requirements currently imposed for use in biochemistry.

The problem solved by the invention is the development of a simple and economical method for producing arachidonic acid, with high quality and product yield while reducing production costs and time of the technological cycle.

The claimed technical result is achieved by the fact that in the known method for producing arachidonic acid from raw materials of animal origin, including alkaline hydrolysis of lipids isolated from raw materials, followed by acidification of the hydrolyzate, the iodine-lactonization reaction, the extraction of iodine-lactones from the reaction mixture with an organic solvent, the disclosure of iodine -lactones with a silylating agent, neutralization of iodine released by sodium thiosulfate and extraction of fatty acids with an organic solvent according to the invention, before the reaction iodine-lactonization lipid hydrolyzate is treated with lithium alkali in acetone and saturated acids are separated in the form of a precipitate; iodine-lactonization is carried out by converting lithium salts of unsaturated fatty acids into sodium salts, which are treated with iodine solution to form iodine-lactones of fatty acids with a ratio of the sum (arachidonic, eicosapentaenoic and docosahexaenoic) acids: iodine equal to 1.0: (1,0- 1.2) mol / mol, at a temperature of the mixture 18-22 ° C, with the duration of adding iodine solution to the reaction mixture for 30-60 minutes, followed by exposure of the solutions for 60 minutes; after extraction of iodine-lactones from the reaction mixture with an organic solvent, the delta and gamma-iodine-lactones are further separated by silica gel chromatography using benzene as an eluent; and the disclosure of delta-iodine-lactones of arachidonic and eicosapentaenoic acids is carried out with a silylating agent at a ratio of iodine-lactones of arachidonic + eicosapentaenoic acid / silylating agent = 1.0: (1.0-1.2) mol / mol, the temperature of the mixture is 18-22 ° C, the duration of the reaction is 120-180 minutes; after extraction of the fatty acids with an organic solvent, they are esterified with ethyl alcohol and arachidonic acid is isolated in the form of ethyl ether by reverse phase HPLC (C-18) using an ethanol-water 8.0 elution system: (1.0-2 5) v / v

The claimed method to isolate the pure target product by HPLC, in contrast to the prototype, includes the 3rd stage: 1)

precipitation in the form of Li salts of the saturated fraction; 2) the process of iodine-lactonization in new technological conditions; 3) the use of chromatographic separation of gamma and delta iodine lactones on silica gel.

Precipitation in the form of lithium salts allows you to remove saturated fatty acids from the hydrolyzate, which, in the final result, helps to obtain the target product with a high degree of purity. The translation of fatty acids into Li-salts is carried out by a known method, in particular from acetone according to Hildich (Hildich TP, “The Chemical constitution of natural fats.” London: Chapman & Hall Ltd., 1949. - P. 471).

Carrying out iodine-lactonization under the proposed conditions provides not only the isolation of a mixture of arachidonic, eicosapentaenoic and docosahexaenoic acids from the hydrolyzate of unsaturated fatty acids, since only they form iodine-lactones, but also the separation of docosahexaenoic acid from this mixture, which forms gamma-iodine-lactone. Docosahexaenoic acid is separated using chromatographic separation of gamma and delta-iodine-lactones on silica gel.

The ratio of the components of the reaction mixture during the formation of iodine-lactones (the sum of arachidonic, eicosapentaenoic and docosahexaenoic acids) / I ₂ = 1 / 1.2 mol / mol) provides a high yield of FA iodine-lactones. However, with a decrease in the amount of iodine taken in the reaction, the yield of iodine-lactones decreases in proportion to its deficiency, but with an excess of more than 1.2 mol 1 g per 1 mol of LCs forming iodine-lactones, this leads, on the one hand, to unnecessary spending on reagents , and on the other, to an irreparable loss of AK due to adverse reactions. The application of this approach allowed us to achieve a new technical result that provides a high yield of AA while reducing the reaction time and saving chemicals.

The temperature regime of the reaction of iodine-lactonization, 18 ° C-22 ° C, guarantees a high yield of iodine-lactones LC. When the reaction temperature decreases below 18 ° C, the formation process slows down and the implementation of the method increases significantly. When the temperature rises above 22 ° C, chemical processes of deterioration of the target product proceed.

The duration of the iodine-lactonization process is very important, since it determines the necessary measure of the effect of iodine on the iodine-lactones forming LC. Modeling the process, it was determined that the best conditions for this method are in the range of 90-120 minutes. Less than 90 minutes, as it turned out, iodine-lactonization does not pass completely, and over 120 minutes no increase in yield occurs anymore.

The ratio of the components of the reaction medium upon the discovery of iodine-lactones to free FAs - (sum of iodine-lactones AK, EPA and DHA): silylating agent = 1: 1.2 mol / mol), in these ratios guarantees a high yield of FA. With a decrease in the molar ratio of the silylating agent of less than 1 mol, the opening of iodine-lactones of the FA decreases sharply, and with an increase of more than 1.2 mol per 1 mol of iodine-lactones of the FA, adverse reactions occur, resulting in a decrease in the purity of the target product.

As a sizing agent, trimethyl iodosilane (TMS-I) or trimethylchlorosilane (TMS-Cl) can be used. The stage of disclosure of iodine-lactones of fatty acids with a silylating agent, TMS-Cl, in acetonitrile containing sodium iodide helps to reduce production costs for obtaining the target product (AK), due to the low cost of these reagents.

The temperature regime of the disclosure of iodine-lactones FA is optimal at 18-22 ° C. Carrying out the process less than the lower limit (18 ° C) does not interfere with the reaction process itself, but makes this process longer, and exceeding the temperature of more than 22 ° C not only does not accelerate the process, but also activates the degradation of FAs and, first of all, AA. In the final result, this leads not only to an increase in the duration of the whole process of obtaining arachidonic acid, but also negatively affects its output.

As an organic solvent in the method for producing AA during the extraction of fatty acids and iodine-lactones, in particular, petroleum ether, hexane, heptane can be used.

To simplify the method and reduce production costs for producing AK, it is advisable to extract iodine-lactone EPA, DHA, and AK formed during iodine-lactonization from the reaction mixture with pentane or hexane. These solvents are less soluble in the reaction mixture, do not form emulsions and have a high extraction capacity for iodine-lactones. An advantage of using these solvents is also that after extraction, their combined solutions do not need to be evaporated and re-redissolved iodine-lactone FA in hexane in order to remove unreacted free fatty acids from the solution. The combined extraction solutions are immediately treated to remove free fatty acids.

Compliance with the claimed operational parameters at all stages of the process provides a high yield and quality of the target product - AK.

The use of silica gel chromatography makes it possible to separate the gamma-iodine-lactones of DHA from the delta-iodine-lactones of EPA and AK. Elution with pure benzene subsequently makes it possible to reuse the regenerated solvent, which makes the whole process more cost-effective.

The final purification of AA in the form of ethyl ether by HPLC is carried out on the reverse phase (C-18) using a water-ethanol eluent, ethanol-water, 8.0: (1.0-2.5) vol./about.

The use of water-ethanol eluent allows you to abandon the use of harmful substances in the process, unlike the prototype, polluting as the target product, but also makes the process of obtaining AK harmless and environmentally friendly. The proposed new eluting system allows you to allocate pure AK in the form of ethyl ether, while the system is non-toxic (ethanol-water 8.0: (1.0-2.5) vol./about.), Is well regenerated and provides a high-quality target drug .

The claimed system parameters (ethanol-water 8.0: (1.0-2.5) vol./about.) Allow the wide use of a polar component (H ₂ O) to increase the overall polarity of the system, and in isocratic used in HPLC AK isolation mode does not require changes (gradient) in the content of the eluting system - from the beginning to the end of the process, the parameters remain unchanged.

This eluting system has great advantages over the use of methanol-based systems, as it allows the use of this method in pharmaceutical and, most importantly, in food production without any restrictions, which opens up great prospects for producing a product (AA) without toxic methyl alcohol, using an environmentally friendly method .

The availability of such an eluting system is not in doubt, and its cost is less than traditional with methyl alcohol.

The most preferred elution system is the ethanol-water system at a ratio of 8.0: (1.8-2.2) v / v, because it is with this ratio of components that, along with the preparation of arachidonic acid with a high degree of purity, the yield is further reduced the time of the selection of AK by HPLC.

Compliance with the claimed operational parameters at all stages of the technological process ensures the achievement of the claimed technical result - obtaining AK with high quality and easy and economical way out while reducing production costs and time of the technological cycle.

The claimed method is illustrated by the following figures: in FIG. 1 shows a cascade of arachidonic acid; in FIG. 2 is a reaction diagram of the formation and opening of iodine-lactone of arachidonic acid.

The method is as follows.

Extraction

Raw materials of animal origin were crushed on a homogenizer and lipids were extracted with a mixture of ethanol-chloroform 1: 1 vol./about., In the ratio of liver - extraction mixture 1: 3 kg / l. Next, the extract was filtered through a dense tissue, the precipitate was squeezed. The separated precipitate was re-extracted with the same volume of chloroform. Both filtrates were combined, evaporated, an equal volume of water was added, and the chloroform layer was taken. The water-alcohol portion was re-extracted with chloroform. The extracts were combined and evaporated.

Lipid hydrolysis

The lipids were hydrolyzed with a 7% KOH solution in 50% ethanol in the ratio of lipids - solution, 1: 4, kg / l, at a temperature of 50 ° C for 60 minutes. The mixture was cooled and the alkali was neutralized with concentrated hydrochloric acid to pH 3. Then it was diluted 2 times with water and the fatty acids and other lipids were extracted with petroleum ether and evaporated.

The deposition of FA in the form of Li-salts

Saturated fatty acids were removed by the precipitation procedure in the form of lithium salts from acetone according to Hildich (Hildich, T. P. “The Chemical constitution of natural fats.” London: Chapman & Hall Ltd., 1949. P. 471).

Hydrolyzed lipids were dissolved in a 4-fold volume of pure acetone. 2 ml of a 2% solution of phenolphthalein in acetone was added and, upon heating (40 ° C), free fatty acids were titrated with saturated aqueous LiOH (≈ 4N) until a characteristic raspberry color appeared. The volume of lithium alkali used was recorded. Further, acetone was added to the mixture until the water content in it was 5% by volume. The mixture was cooled to room temperature, held for 1 hour, the precipitate was filtered on a paper filter, the acetone filtrate was acidified with hydrochloric acid to pH 3, and evaporated.

Iodine Lactonization

The reaction of iodine-lactonization of FA was carried out based on all fatty acids that form lactones.

A concentrate of fatty acids and other lipids was dissolved in a 5-fold volume of 95% ethanol, and an aqueous solution of sodium bicarbonate was added according to stoichiometry to convert free fatty acids to Na-salt.

Then, according to stoichiometry, the required amount of iodine was calculated, dissolved in 95% ethanol to a solution concentration of 5% and added from the separatory funnel in portions to the stirred mixture of salts of FA in the ratio (AK + EPA + DHA) / I ₂ = 1.0 / 1- 1.2 mol / mol, at room temperature for 30 minutes, after adding, they stood for another 90 minutes.

Unreacted iodine was neutralized to colorless with a saturated aqueous solution of sodium thiosulfate, the mixture was diluted with 2 volumes of water, and the iodine-lactones of the LC were extracted three times with a non-polar organic solvent. The combined extracts were evaporated twice and was extracted not forming iodine-lactones FA 2% NaCO ₃ in 40% ethanol. The organic solvent layer was washed with water, acidified with concentrated hydrochloric acid to pH 3, filtered through sodium sulfate for dehydration, and evaporated.

Chromatographic separation of iodine-lactones

Silica gel, 100-160 μm, was suspended in benzene, applied to a chromatographic column, the total FA iodine-lactones (ratio of iodine-lactones FA: silica gel = 1:10 g / g) were loaded and eluted with benzene. Control was carried out by TLC.

Fractions with pure δ-iodine-lactone AK were combined and evaporated.

Disclosure of iodine-lactone AK

The iodine-lactone AK was disclosed in a 3% solution of anhydrous NaI in acetonitrile. To the mixture, TMSi-Cl was added with stirring in the ratio iodine-lactones LC - TMSi-Cl = 1.0: 1-1.2 mol / mol, dropwise, with a total reaction time of 120-180 minutes, after which the released iodine was neutralized with saturated aqueous sodium thiosulfate solution, the mixture was diluted with 4 volumes of water and the lipids were extracted with a non-polar solvent.

Free FAs were extracted from a non-polar solvent with 2% NaCO ₃ in 40% ethanol, the alkaline extract was washed with a non-polar solvent, an equal volume of water was added, acidified with concentrated hydrochloric acid to pH 3 and the free FAs were extracted with a non-polar solvent, the extract was dried, filtered through sulfate sodium, evaporated.

Obtaining ethyl esters of LCD

Fatty acids were added to a 1% solution of sulfuric acid in absolute ethanol at a ratio of FA: ethanol of 1:10 v / v. The mixture was heated (50 ° C) with stirring for 1.5 hours, then diluted with 3 volumes of water and the ethyl ethers were extracted with a non-polar solvent.

One stripped off extract was purified on a column of silica gel; the eluent was a nonpolar solvent.

The selection of AK in the form of ethyl ether by HPLC

The separation of the mixture of FA ethyl esters was performed on a Supelco Discovery HS C-18 column, 250 mm × 50 mm i.d. (particle size 10 μm) on a Shimadzu-LC8A liquid chromatograph with an SPD-20A UV detector. Elution was carried out with a pre-degassed and filtered solvent system ethanol-water 8: (1.0-2.5) vol./about., At a speed of 50 ml / min.

The method is illustrated by the following figures: FIG. 1, which shows the cascade of arachidonic acid, FIG. 2. - presents a reaction diagram of the formation and disclosure of iodine-lactone arachidonic acid, confirmed by examples of the specific implementation of the method.

Example 1

The cattle liver was used in the experiment. Prior to use, all material was stored at a temperature of minus 20 ° С.

All reagents and solvents used were brand name “hch”; silica gel manufactured by Chemapol (Czech Republic), anhydrous NaI (Sigma-Aldrich, USA), trimethylchlorosilane, TMSi-Cl (Acros Organics, Belgium), lithium hydroxide, ethanol and petroleum ether, 40 ° -60 ° С ( "Reagent", Moscow).

Thin layer chromatography (TLC) of lipids for monitoring the processes was carried out on plates with a fixed layer of silica gel (Sorbfil, Russia) with eluting systems: 1) 80: 20: 1, hexane-diethyl ether-acetic acid, v / v / vol., to determine the composition of neutral lipids, 2) 2-fold elution with benzene to identify iodine-lactones FA.

Methyl fatty acids of lipids for GLC analysis were obtained according to the methodology (Carreau JP, Dubacq JP "Adaptation of a macro-scale method to the micro-scale for fatty acid methyl transesterification of biological lipid extracts" // J. Chromatogr. - 1978. - Vol. 151, No. 3. - 384-390). Fatty acid methyl esters were analyzed by gas chromatography on a Shimadzu GC-17A chromatograph (Shimadzu, Kyoto, Japan) with a flame ionization detector and a 30 m × 0.25 mm i.d capillary column. Supelcowax 10 (Bellefonte, USA), analysis conditions: column temperature 190 ° С, injector and detector 240 ° С, helium carrier gas. Peaks of methyl (ethyl) fatty acid esters were identified by retention times of individual fatty acid esters and using standard PUFA mixtures (Supelco, Bellefonte, USA) and equivalent chain lengths (Christie WW Lipid Analysis: Isolation, separation, identification and structural analysis of lipids. ”- The oily Press, Bridgwater (UK), 2003. - 416 p.).

For lipid extraction, frozen beef liver (500 g) was ground. on a homogenizer and lipids were extracted with a mixture of ethanol-chloroform 1: 1 vol./about. 1.5 liters Next, the extract was filtered through a dense tissue, the precipitate was squeezed. The separated precipitate was re-extracted with 1.5 L of chloroform. Both filtrates were combined, evaporated to 2 L, an equal volume of water was added, and the chloroform layer was taken. The water-alcohol portion was re-extracted with chloroform, 500 ml. The extracts were combined and evaporated. Received 52.5 g of lipids.

For lipid hydrolysis from the previous stage, 210 ml of a 7% solution of KOH in 50% ethanol at a temperature of 50 ° C for 60 minutes were used. The mixture was cooled and the alkali was neutralized with concentrated hydrochloric acid to pH 3. Then it was diluted with water, 500 ml, and the fatty acids and other lipids were extracted with petroleum ether, 2 × 200 ml, and evaporated. Received 45.6 g of LCD.

Saturated fatty acids were removed from the FA mixture by the deposition procedure in the form of lithium salts from acetone as follows: hydrolyzed lipids were dissolved in 180 ml of acetone, 2 ml of a 2% solution of phenolphthalein in acetone was added, and when heated (40 ° C), free fatty acids were titrated with saturated aqueous solution of LiOH (≈ 4N), 36 ml, until a characteristic raspberry color appears. Next, another 500 ml of acetone was added to the mixture. The mixture was cooled to room temperature, kept for 1 hour, the precipitate was filtered on a paper filter, the acetone filtrate was acidified with hydrochloric acid to pH 3, and evaporated. Received 25.4 g of the LCD.

The reaction of iodine-lactonization of FA was carried out based on all fatty acids that form lactones.

A concentrate of fatty acids and other lipids, 25.4 g, was dissolved in 125 ml of 95% ethanol and an aqueous solution of sodium bicarbonate (7 g in 150 ml) was added by stoichiometry to convert free fatty acids to Na-salt.

Next, the required amount of iodine was calculated by stoichiometry ((AK + EPA + DHA) / I ₂ = 1.0 / 1.0 mol / mol), 4.1 g of iodine were dissolved in 8 0 ml of 95% ethanol and added from separatory funnel in portions to a stirred mixture of salts of FA at a temperature of 18 ° C for 30 minutes, after addition was held for another 60 minutes.

Unreacted iodine was neutralized until colorless with a saturated aqueous solution of sodium thiosulfate (20 g in 100 ml of water), a mixture of 400 ml of water was diluted, and 3-fold LC iodine-lactones were extracted with 150 ml of petroleum ether. The combined extracts were evaporated by a factor of 2 and the non-iodine-lactones forming FAs were extracted with 2% NaCO ₃ in 40% ethanol, three times in 100 ml each. The petroleum layer was washed with 200 ml of water, acidified with concentrated hydrochloric acid to pH 3, filtered through sodium sulfate for dehydration, and evaporated. Received 6.96 g of iodine-lactones FA.

Chromatographic separation of LC iodine-lactones was carried out on silica gel, 100-160 μm suspended in benzene: silica gel was applied to a chromatographic glass column (50 cm × 4 cm), total LCD iodine-lactones, 6.9 g, were loaded (ratio of iodine-lactones LC - silica gel, 1: 10, g / g) and was eluted with benzene. Control was carried out by TLC. Fractions with pure δ-iodine-lactones. AK and EPA were combined and evaporated. Received 5.54 g of δ-iodine-lactones FA.

Disclosure of δ-iodine-lactones FA, 5.54 g, was carried out in a solution of anhydrous NaI - 6 g of salt in 200 ml of acetonitrile. TMSi-Cl was added to the mixture with stirring, taken according to stoichiometry in the ratio iodine-lactones LC - TMSi-Cl = 1.0: 1.0, mol / mol, dropwise over 60 minutes, then kept for another 60 minutes, after which the separated iodine was neutralized with a saturated aqueous solution of sodium thiosulfate (10 g in 100 ml of water), the mixture was diluted with 1.2 L of water and the lipids were extracted with petroleum ether, 3 × 150 ml.

Free FAs were extracted from petroleum ether with 2% NaCO ₃ in 40% ethanol, 3 × 100 ml, the alkaline extract was washed with 100 ml of petroleum ether, 300 ml of water was added, acidified with concentrated hydrochloric acid to pH 3 and the free FAs were extracted with petroleum ether , 3 × 100 ml, the extract was dried, filtered through sodium sulfate, evaporated. Received 3.93 g of the LCD.

Fatty acids, 3.93 g, were converted into FA ethyl esters by adding them to a 1% solution of sulfuric acid in absolute ethanol, 40 ml. The mixture was heated (50 ° C) with stirring for 1.5 hours, then was diluted with 220 ml of water and the ethyl esters were extracted with LC petroleum ether, 3 × 100 ml.

One stripped off extract was purified on a column of silica gel (10 g of silica gel suspended in benzene), the eluent was petroleum ether, and evaporated. Received 3.85 g of ethyl esters of FA.

Separation of the EE-LC mixture, 3.80 g, was carried out by HPLC on a Supelco Discovery HS C-18 column, 250 mm × 50 mm i.d. (particle size 10 μm). Elution was carried out with a pre-degassed and filtered solvent system ethanol-water 8.0: 1.0 vol./about. at a speed of 50 ml / min. The amount of a mixture of FAs in the form of ethyl esters applied to the column was 1 ml (0.95 g); the consumption of the eluting system per separation was on average 2.7 liters. The procedure was repeated 4 times.

After separation, fractions with pure AK in the form of ethyl ether were evaporated.

Received AK in the form of ethyl ether, 3.43 g, with a purity of 99.67%. The total yield, calculated on the content of AK in the feedstock, is 50.9%.

The total implementation time of the method is 12 hours.

Example 2

The raw materials, reagents, solvents and analytical methods used in Example 2 were as in Example 1.

For lipid extraction, frozen beef liver (500 g) was ground on a homogenizer and the lipids were extracted with ethanol-chloroform 1.0: 1.2 vol./vol. 1.5 liters Next, the extract was filtered through a dense tissue, the precipitate was squeezed. The separated precipitate was re-extracted with 1.5 L of chloroform. Both filtrates were combined, evaporated to 2 L, an equal volume of water was added, and the chloroform layer was taken. The water-alcohol portion was re-extracted with chloroform, 500 ml. The extracts were combined and evaporated. Received 52.1 g of lipids.

For lipid hydrolysis from the previous stage, 210 ml of a 7% solution of KOH in 50% ethanol was used at a temperature of 50 ° C for 60 min. The mixture was cooled and the alkali was neutralized with concentrated hydrochloric acid to pH = 3. Then it was diluted with water, 500 ml, and the fatty acids and other lipids were extracted with petroleum ether, 2 × 200 ml, and evaporated. Received 4 5.3 g of LCD.

Saturated fatty acids were removed from the FA mixture by the deposition procedure in the form of lithium salts from acetone as follows: hydrolyzed lipids were dissolved in 180 ml of acetone, 2 ml of a 2% solution of phenolphthalein in acetone was added, and when heated (40 ° C), free fatty acids were titrated with saturated aqueous solution of LiOH (≈ 4N), 36 ml, until a characteristic raspberry color appears. Next, another 500 ml of acetone was added to the mixture. The mixture was cooled to room temperature, held for 1 hour, the precipitate was filtered on a paper filter, the acetone filtrate was acidified with hydrochloric acid to pH 3, and evaporated. Received 25.2 g of LCD.

The reaction of iodine-lactonization of FA was carried out based on all fatty acids that form lactones.

A concentrate of fatty acids and other lipids, 25.2 g, was dissolved in 125 ml of 95% ethanol and an aqueous solution of sodium bicarbonate (7 g in 150 ml) was added to convert free fatty acids to the Na salt.

Next, the required amount of iodine was calculated

(AK + EPA + DHA) / I ₂ = 1.0 / 1.2, mol / mol), 4.9 g of iodine were dissolved in 100 ml of 95% ethanol and added from the separatory funnel in portions to the stirred mixture of salts of FA at 22 ° C for 60 minutes, after addition was held for another 60 minutes.

Unreacted iodine was neutralized until colorless with a saturated aqueous solution of sodium thiosulfate (20 g in 100 ml of water), a mixture of 400 ml of water was diluted, and 3-fold LC iodine-lactones were extracted with 150 ml of petroleum ether. The combined extracts were evaporated by a factor of 2 and the non-iodine-lactones forming FA were extracted with 2% NaCO ₃ in 40% ethanol, 3 × 100 ml. Petroleum layer - washed with water, 200 ml, acidified with concentrated hydrochloric acid to pH = 3, filtered through sodium sulfate for dehydration, evaporated. Received 6.89 g of iodine-lactones FA.

Chromatographic separation of iodine-lactones was carried out on silica gel, 100-160 μm, 69 g suspended in benzene: silica gel was applied to a chromatographic glass column (50 cm × 4 cm), the total LC iodine-lactones, 6.89 g, were loaded and eluted with benzene . Control was carried out by TLC. Fractions with pure δ-iodine lactones AK and EPA were combined and evaporated. Received 5.50 g of δ-iodine-lactones FA.

Disclosure of δ-iodine-lactones FA, 5.50 g, was carried out in a solution of anhydrous NaI - 6 g of salt in 200 ml of acetonitrile. TMSi-Cl, taken in the ratio iodine-lactones LC - TMSi-Cl = 1.0: 1.2 mol / mol, was added dropwise to the mixture with stirring, dropwise for 120 minutes, then it was held for 60 minutes, after which the released iodine was neutralized with saturated aqueous sodium thiosulfate solution (10 g in 100 ml of water), the mixture was diluted with 1.2 l of water and the lipids were extracted with petroleum ether, 3 × 150 ml.

Free FAs were extracted from petroleum ether with 2% NaCO ₃ in 40% ethanol, 3 × 100 ml, the alkaline extract was washed with petroleum ether, 100 ml, 300 ml of water was added, acidified with concentrated hydrochloric acid to pH = 3 and free FAs were extracted petroleum ether, 3 × 100 ml, the extract was dried, filtered through sodium sulfate, evaporated. Received 3.87 g of the LCD.

Fatty acids, 3.87 g, were converted into FA ethyl esters by adding them to a 1% solution of sulfuric acid in absolute ethanol, 40 ml. The mixture was heated (50 ° C) with stirring for 1.5 hours, then was diluted with 220 ml of water and the ethyl esters were extracted with LC petroleum ether, 3 × 100 ml.

One stripped off extract was purified on a column of silica gel (10 g of silica gel suspended in benzene), the eluent was petroleum ether, and evaporated. Received 3.76 g of ethyl esters of FA.

Separation of the EE-LC mixture, 3.76 g, was carried out by HPLC on a Supelco Discovery HS C-18 column, 250 mm × 50 mm i.d. (particle size 10 μm). Elution was carried out with a pre-degassed and filtered solvent system ethanol-water 8.0: 2.2 vol./about. at a speed of 50 ml / min. The amount of a mixture of FAs in the form of ethyl esters applied to the column was 0.94 g; the consumption of the eluting system per separation was on average 2.7 liters. The procedure was repeated 4 times.

After separation, fractions with pure AK in the form of ethyl ether were evaporated.

Received AK in the form of ethyl ether, 3.38 g, with a purity of 99.18%. The total yield, calculated on the content of AK in the feedstock, is 50.1%.

The total implementation time of the method is 12.1 hours.

The table shows the phased concentration of AK from Examples 1 and 2, namely the composition of concentrates AK from raw materials of animal origin (Examples 1 and 2)

The numbers for the production of AK given in the table clearly show that the method provides AK in the form of ethyl ether - with a purity of 99.18-99.67% and a total yield of 50.1-56 of AK in the feedstock; , 2%. The implementation of this method solves the problems that currently exist when receiving AK in laboratory and factory production. The most important indicator is the purity of the product - over 99%, which allows AK to use AK for very thin enzymatic syntheses, where the presence of other acids is unacceptable due to by-products of the reaction, which with other methods of implementation requires additional costs for the purification of the obtained compounds. The total yield of AK in the method is more than 50%, this is more than in other analogues when receiving highly enriched AK.

Example 3

The raw material in this experiment was the liver of a rat (laboratory animals) - 500 g.

The content of arachidonic acid from the total amount of FA of liver lipids is 14.52%, eicosapentaenoic acid - 1.33%, docosahexaenoic acid - 3, 81%.

Solvents, reagents and analytical procedures, as in Example 1.

Lipid extraction, hydrolysis, precipitation of saturated fatty acids in the form of Li-salts, reaction of iodine-lactonization of FA, chromatographic separation of gamma and delta-iodine-lactones of FA, disclosure of delta-iodine-lactones of FA to free fatty acids, from esterification to ethyl LC esters and further separation by HPLC was carried out as in Example 1, except that petroleum ether was used as the non-polar solvent for the extraction of iodine-lactones of fatty acids and FA after the disclosure of iodine-lactones, and elution was carried out with Stem solvent ethanol-water 8.0:. 1.0 v / v.

Received AK in the form of ethyl ether, 4.56 g, with a purity of 99.43%.

The total yield, calculated on the content of AK in the feedstock, is 55.4%.

The total implementation time of the method is 13.0 hours.

Example 4

The raw material in this experiment was the mouse liver (laboratory animals) - 50 g.

The content of arachidonic acid from the total amount of FA of liver lipids is 8.03%, eicosapentaenoic acid - 1.62%, docosahexaenoic acid 14.13%.

Solvents, reagents and analytical procedures, as in Example 2.

Lipid extraction, hydrolysis, precipitation of saturated fatty acids in the form of Li-salts, reaction of iodine-lactonization of FA, chromatographic separation of gamma and delta-iodine-lactones of FA, disclosure of delta-iodine-lactones of FA to free fatty acids, from esterification to ethyl LC esters and further separation by HPLC was carried out as in Example 1, except that pentane was used as a non-polar solvent for the extraction of iodine-lactones of fatty acids and FAs after the opening of iodine-lactones. LC wire or trimethyl iodosilane in the ratio delta-iodine-lactones FA: trimethyl iodosilane = 1: 1 mol / mol, and the elution was carried out with an ethanol-water solvent system of 8.0: 2.0 vol / vol.

Received AK in the form of ethyl ether, 0.44 g, with a purity of 99.55%.

The total yield, calculated on the content of AK in the feedstock, is 51.6%.

The total implementation time of the method is 11.0 hours.

Example 5

The raw material in this experiment was the sale of reindeer liver - 500 g.

The content of arachidonic acid from the total amount of FA of liver lipids is 10.66%, eicosapentaenoic acid - 0.76%, docosahexaenoic acid 1.27%.

Solvents, reagents and analytical procedures, as in Example 1.

Lipid extraction, hydrolysis, precipitation of saturated fatty acids in the form of Li-salts, reaction of iodine-lactonization of FA, chromatographic separation of gamma and delta-iodine-lactones of FA, disclosure of delta-iodine-lactones of FA to free fatty acids, from esterification to ethyl LC esters and further separation by HPLC was carried out as in Example 1, except that hexane was used as the non-polar solvent for the extraction of iodine-lactones of fatty acids and FAs after opening of iodine-lactones, and the elution was carried out using a ra solvent was ethanol-water 8.0:. 2.5 v / v.

Received AK in the form of ethyl ether, 4.41 g, with a purity of 99.60%.

The total yield, calculated on the content of AK in the feedstock, is 56.2%.

The total implementation time of the method is 13.8 hours.

Thus, the applicant has proposed a simple and reproducible method for the isolation of pure AK from animal tissues, available for implementation in laboratories and industrial plants using AK in biochemical, medical and dietetic studies.

* - the first digit indicates the number of carbon atoms in the fatty acid chain, the second - the number of unsaturated bonds, n-3 or others. - the location of the first double bond from the methyl group;

** - the yield is given in the 3rd procedure — iodine-lactonization, chromatographic separation of lactones and their opening;

*** - the output is given for the 2nd procedure - obtaining ethyl esters of LCD and their separation by HPLC.

**** - Σ SFA - the sum of saturated fatty acids, Σ SFA - the sum of monounsaturated fatty acids, Σ PUFA - the sum of polyunsaturated fatty acids.

Claims (4)

1. A method for producing arachidonic acid from animal feed, including alkaline hydrolysis of lipids isolated from the feed, followed by acidification of the hydrolyzate, iodine-lactonization reaction, extraction of iodine-lactones from the reaction mixture with an organic solvent, opening of iodine-lactones with a silylating agent, neutralization of the released iodine with sodium thiosulfate, extraction of fatty acids with an organic solvent, characterized in that before the iodine-lactonization reaction, the lipid hydrolyzate is treated with lithium alkali s in acetone and saturated acids are separated in the form of a precipitate, iodine-lactonization is carried out by converting lithium salts of unsaturated fatty acids into sodium salts, which are treated with a solution of iodine to form iodine-lactones of fatty acids in the ratio of the sum (arachidonic, eicosapentaenoic and docosahexaenoic) acids: iodine equal to 1.0: (1.0-1.2) mol / mol, at a temperature of the mixture 18-22 ° C, with the duration of adding iodine solution to the reaction mixture for 30-60 minutes, followed by exposure of the solutions for 60 minutes; after extraction of iodine-lactones from the reaction mixture with an organic solvent, separation of delta and gamma-iodine-lactones by silica gel chromatography is additionally carried out using benzene as an eluent; and the disclosure of delta-iodine-lactones of arachidonic and eicosapentaenoic acids is carried out with a silylating agent at a ratio of iodine-lactones of arachidonic + eicosapentaenoic acid / silylating agent = 1.0: (1.0-1.2) mol / mol, the temperature of the mixture is 18-22 ° C, the duration of the reaction is 120-180 minutes; after extraction of the fatty acids with an organic solvent, they are esterified with ethyl alcohol and arachidonic acid is isolated in the form of ethyl ether by reverse phase high performance liquid chromatography (C-18) using an ethanol-water 8.0 elution system: (1.0-2.5) about / about 2. The method according to p. 1, characterized in that trichlorosilane or trimethyl iodosilane is used as a enhancing agent for the disclosure of iodo-lactones of arachidonic and eicosapentaenoic acids. 3. The method according to p. 1 or 2, characterized in that the disclosure of iodine-lactones of arachidonic and eicosapentaenoic acids with trichlorosilane is carried out in the presence of sodium iodide in a solution of acetonitrile. 4. The method according to p. 1, characterized in that when performing high-performance liquid chromatography on a reversed phase (C-18), the elution system ethanol-water 8.0: (1.8-2.2) v / v is predominantly used

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