RU2537252C1 - Method of obtaining docosahexaenoic acid - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: method of obtaining docosahexaenoic acid includes alkaline hydrolysis of marine fats, processing salts of fatty acids with iodine solution, extraction of iodine-lactones of fatty acids with non-polar organic solvent, removal of free fatty acids which did not react from mixture, opening iodine-lactones of fatty acids with silylating agent, neutralisation of released free iodine with sodium thiosulphate and extraction of free fatty acids with non-polar organic solvent at certain conditions.

EFFECT: method makes it possible to obtain end high-purity product with increased output.

5 cl, 4 dwg, 1 tbl, 6 ex

Description

The invention relates to the food and pharmaceutical industries and can be used to obtain compositions with a high content of docosahexaenoic acid (DHA), which has a pronounced biological effect. The formula of docosahexaenoic acid is shown in figure 1.

Omega-3 polyunsaturated fatty acids from marine sources, primarily eicosapentaenoic and docosahexaenoic acids, are known as prophylaxis and treatment of cardiovascular diseases (Simopoulos A.R. Omega-3 Fatty Acids in Inflammation and Autoimmune Diseases // J. Of the American College of Nutrition, 2002. Vol.21, No. 6, P. 495-505). However, despite the increase in human life expectancy, the prevalence of diseases associated with aging has also increased, in particular neurodegenerative diseases such as Alzheimer's disease, multiple sclerosis and Parkinson's disease (Habeck C., Stern Y. Alzheimer's Disease Neuroimaging Initiative. Multivariate data analysis for neuroimaging data : overview and application to Alzheimer's disease // Cell Biochem. Biophys., 2010. Vol. 58, P. 53-67). These diseases, progressing with age, are also associated with socio-economic status, work, physical activity, genetics, and in the last two decades, nutrition has also been identified as an important factor (Stampfer MJ Cardiovascular disease and Alzheimer's: common links // J Inter. Med., 2006. Vol. 260, R. 211-223). This conclusion has the general opinion that a healthy diet can prevent the development of many diseases, such as obesity, hypertension, diabetes mellitus, stroke, some cancers, and now neurodegenerative diseases (Massaro M. et al. Nutraceuticals and prevention of atherosclerosis: focus on omega-3 polyunsaturated fatty acids and Mediterranean diet polyphenols // Cardiovasc. Ther., 2010. Vol. 28, P. 13-19). Epidemiological studies show that populations that consume significantly fish and other foods rich in omega-3 polyunsaturated fatty acids (ω3 PUFAs) have low levels of neurodegenerative diseases (Tully A.M. et al. Low serum cholesterol ester docosahexaenoic acid levels in Alzheimer's disease : a case - control study // Br. J. Nutr., 2003. Vol. 89, P. 483-489). ω3 PUFAs, especially docosahexaenoic acid (DHA, Δ4,7,10,13,16,19-C22: 6, 22: 6ω3), play a fundamental role in the development and maintenance of the nervous system, and in recent years there has been conclusive evidence of their participation in prevention and / or treatment of neurodegenerative diseases (Ikemoto A. et al. Reversibility of n-3 fatty acid deficiency - induced alterations of learning behavior in the rat: level of n-6 fatty acids as another critical factors // J Lipid Res, 2001. Vol. 42, R. 1655-1663). DHA is found in high concentrations in the nervous tissue, especially in phospholipids of neurons and glial membranes. However, aging and the progression of neurodegenerative diseases significantly reduce the content of DHA in the brain (Tully et al., 2003), especially in the cerebral cortex, cerebellum and hypothalamus, which leads to a significant decrease in fluidity of neuronal membranes and a change in neural homeostasis (Sodeberg M. et al Fatty acid composition of brain phospholipids in aging and in Alzheim's disease // Lipids, 1991. Vol. 26, P. 421-425; Kalminj S. et al. Dietary intake of fatty acid and fish in relation to cognitive performance at middle age // Neurology, 2004. Vol.62, R. 275-280). The greatest amount of evidence regarding the neuroprotective effect of DHA is observed in Alzheimer's disease. DHA can suppress the cytotoxic effects of the accumulation of β-amyloid peptide, which is the main mechanism associated with the neuroprotective action of fatty acids (Bazan NG Neuroprotectin D1-mediated anti-inflammatory and survival signaling in stroke, retinal degenerations, and Alzheimer's disease // J Lipid Res, 2009. Vol. 50 (Suppl.), P. 400-405).

Based on the foregoing, it is advisable to consider the positive effect of increasing the consumption of DHA both in the form of foods rich in this fatty acid (fish, etc.) and containing DHA preparations with its high concentration. Unfortunately, most pathologies, when they begin to treat them, are in a state of neglect. Therefore, some preventive measures that would have been effective earlier, as a balanced diet on DHA, no longer have the desired result. According to many researchers and doctors, ^° in this case, the use of highly enriched DHA preparations, in which there would be no other "ballast" fatty acids, is effective. Such compositions with a high content of DHA are considered by scientists as the most promising for widespread use in neurogenital diseases.

At present, we do not know from the scientific and patent literature about methods for producing pharmaceutical compositions with a high content of DHA, 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 — drastically 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 DHA, which have a therapeutic and prophylactic effect in neurodegenerative diseases.

A known method for producing polyunsaturated fatty acid esters from fish oils and preparations based on them (EP Application No. 0 292 846 A2 "A process for the extraction of polyunsaturated fatty acid esters from fish oils and pharmaceutical and / or dietetic compositions containing said esters", 18.05 .88, Int. Cl. ⁴ C11C 3/04, A61K 31/23, A23D 5/00, A23L 1/30). The method is based on different volatility of fatty acid esters in vacuum at high temperature and is carried out as follows. To convert fatty acids from triglycerides to ethyl esters, fish oil is treated with a 5% solution of sulfuric acid in ethanol, cooled, water is added, ethyl esters of fatty acids are extracted with hexane, the hexane solution is chromatographed to remove oxidized derivatives, the hexane is evaporated, and the product itself used for molecular distillation at a temperature of 80-125 ^° C and a residual pressure of 10 ^-3 mm RT. Art. The original fish oil (as well as the fatty acid ethyl esters derived from it) contains 10.7% of DHA. After the procedures described, the final product contains 39.9% DHA.

The method has several disadvantages:

1. A rather laborious preparation for the implementation of the method is acid ethanolysis and chromatographic purification of oxidized impurities.

2. High temperatures, up to 125 ^° C, cause phenomena such as the oxidation of polyunsaturated fatty acids, the formation of diene conjugates, the migration of double bonds in the chain of fatty acids, the polymerization of the product, as well as the degradation of acid to short-chain volatile products.

3. The use of the final product obtained by this method is permissible only with a very thorough chromatographic purification, which the authors, for some reason, do not indicate, since introducing a product modified in the chemical composition into the pharmaceutical composition will be dangerous for consumers.

4. The method does not provide a high concentration of DHA in the final product (39.9%), the observed increase is only 3.7 times. The resulting composition, after purification, can indeed be used for food purposes, but not as a highly effective drug.

A known method of producing a concentrate of ethyl esters of eicosapentaenoic and docosahexaenoic acids (Copyright certificate SU No. 1581737 A1 "Method for producing a concentrate of ethyl esters of eicosapentaenoic and docosahexaenoic acids", 07/30/90, MKI ⁵ C11C 1/00, C11 B 7/00). This method of concentrating polyunsaturated fatty acids is based on the physical principle of the preferred complexation of urea crystals with saturated and monounsaturated fatty acids in the form of a precipitate, while polyunsaturated fatty acids remain in the initial solution. The method is as follows. Fish oil is subjected to alkaline hydrolysis to obtain free fatty acids, then free fatty acids are dissolved in a solution of urea in ethanol in a ratio of 1: 3: 12 (fatty acids-urea-ethanol, g / g / ml), the mixture is kept for 1-3 hours at room temperature, then 8-12 hours at 0 ^° C, the precipitate is filtered off, and a concentrate of unsaturated fatty acids is isolated from the alcohol solution. So, from 4 g of free fatty acids (Example 2) obtained by hydrolysis of fish oil with a DHA content of 5.3%, 330 mg of a polyunsaturated fatty acid concentrate with a DHA content of 37.0% were obtained by the method. The yield of DHA is 57.8%.

The method has the following disadvantages:

1. To maximize the concentration of DHA by the method, it is assumed that large volumes of solvent and urea are used, which requires appropriate hardware design — large apparatuses with jackets for sequential heating and cooling of the reaction mixture, additional volumetric containers, etc. For the regeneration of the solvent, it is necessary to create specialized distillation due to the large the amount of precipitate in the evaporated solution.

2. The precipitation of the urea complex with saturated and monounsaturated acids is a waste in this process and its disposal is problematic. There are methods of decomposing this precipitate into free fatty acids and urea with the possibility of its regeneration, but this process is carried out in a boiling hydrocarbon, for example, isooctane. The implementation of this concomitant process, given fire safety criteria, is likely to be more complex than the underlying technology. The environmental component in this process is obvious, and its solution has not yet been found.

3. The concentration of DHA does not exceed 37% in the final product, which is very low for using it as a source of this acid. In addition, more than 40% of DHA is lost during the implementation of the method, which is extremely not economical.

A known method of obtaining a composition containing docosahexaenoic acid (UK Patent Application GB 2 098 065 "Antithrombotic compositions containing docosahexaenoic acid", 11/17/82, Int. Cl. ³ A61K 31/20, A61K 31/16). The method is based on a combination of various methods - low-temperature crystallization, molecular distillation and chromatography. The method of low-temperature crystallization uses the physical properties of individual fatty acids - different points of their melting, both in free form and in solutions of organic solvents at various temperatures, which provides the primary concentration of polyunsaturated fatty acids. The resulting concentrate is subjected to enrichment according to the 2nd method - molecular distillation in vacuum and finally use the 3rd method - chromatography. The method is as follows (example 1): fat (15 kg) is extracted from fresh mackerel meat (100 kg), it is hydrolyzed to obtain free fatty acids (9 kg), the mixture is cooled to -60 ^° C, obtaining 4 kg of unsaturated fatty acids, then the concentrate is subjected to molecular distillation at a residual pressure of 10 ^-2 mm RT. Art. and further concentrated by column chromatography to obtain 900 g of a DHA concentrate with a purity of 82%.

The disadvantages of the method include:

1. The process at a very low temperature (-60 ^° C) is very costly, because low temperatures require specialized facilities designed for this, and, possibly, the functioning of large rooms at this temperature, if we are talking about getting industrial batches of the drug.

2. The second stage - molecular distillation, using high temperatures (up to 110 ^° C), contributes to a significant oxidation of DHA and general losses in its production.

3. The method initially involves a significant formation of oxidized and polymerized compounds, why adsorption chromatography is used at the final stage. This is a rather expensive procedure for obtaining the product, given that the sorbent (usually silica gel) does not regenerate further.

4. The concentration of DHA is only 82% when the DHA yield is approximately 24.6% (the authors do not indicate the content of DHA in the fat of Japanese mackerel, but based on known data, it can be assumed that this is within 30% (Bae JH Heavy Metal Contents and Chemical Compositions of Atlantic (Scomber scombrus), Blue (Scomber australasicus), and Chub (Scomber japonicus) Mackerel Muscles // Food Sci. Biotechnol., 2011. Vol.20, P. 709-714).

A known method of producing methyl esters of polyunsaturated fatty acids from fish oil (Adolf RO et al., "The Isolation of Omega-3 Polyunsaturated Fatty Acids and Methyl Esters of Fish Oils by Silver Resin" // JAOCS, 1985. Vol.62, P. 1592-1595). The implementation of this method is based on the ability of polyunsaturated compounds to form coordination bonds with silver atoms - the more double bonds, the stronger the stability of such compounds. By applying silver salts to chromatographic sorbents, it is possible to regulate the equilibrium in the system of saturated - unsaturated fatty acids. The method is as follows: fish oil (American herring) is converted to methyl esters of fatty acids, then using a chromatographic column with a resin containing silver ions, they are chromatographed in a system of polar solvents to obtain a concentrate containing 20.4% of DHA during elution (initial DHA content - 11.1%). If for “silver” chromatography a ready-made concentrate of polyunsaturated fatty acids with a content of 20.5% DHA was used, then the best indicator was the composition in one of the fractions - 95.4% of DHA.

The method has the following disadvantages:

1. Despite the possibility of obtaining highly enriched concentrates of polyunsaturated fatty acids, the method is intended, rather, for laboratory use, since it does not give high yields of the final product.

2. The use of silver salts in the method extremely increases the cost of the final product. Elution systems wash impregnated silver salts from the sorbent, and further regeneration of solutions with silver salts, as well as sorbent with their residues in the complex of the process, is impossible.

3. The productivity of the process is very small. Significant in volume columns (340-750 ml) are used to separate 0.5-10.0 g of substance. Significant amounts of solvents are required, their constant regeneration.

4. The purity of the best sample does not exceed 95.4%, while it must be borne in mind that it is obtained from previously prepared concentrate. If we consider examples with methyl esters of the original fish oil, the final product contains 20.4% of DHA, and the yield is only 49.5%.

A known method for producing docosahexaenoic acid from cod liver oil (Medina A.R. et al. Concentration and Purification of Stearidonic, Eicosapentaenoic, and Docosahexaenoic Acids from Cod Liver Oil and the Marine microalga Isochrysis galbana // JAOCS, 1995. Vol. 72, P 575-583). The method is based on different mobility of fatty acids under high pressure chromatography on specialized sorbents. The method is as follows: cod liver oil is hydrolyzed with the release of free fatty acids (the content of DHA is 11%), then it is separated by the composition of the fatty acids on a reverse phase chromatography column (C-18), eluting the column with various solvent systems. The method allows to obtain DHA concentrates with a yield of 70.5-100%, but with the content of this acid not more than 72-85%.

The method has many disadvantages:

1. Column chromatography provides the separation of the desired products, but it is known that the ratio of the shared mixture-sorbent is usually 1: 10-100, g / g This is a very large use of the sorbent for the separation of small amounts of substances. In addition, after the separation of substances, the sorbent on the column must be regenerated, and this requires solvents in no less volume than for the isolation of the target product.

2. The method uses various elution systems at different ratios of solvent to water. This involves the regeneration of the solvent, and, based on the volumes given, it is very significant.

3. The use of methanol as a solvent, namely, in the examples with its participation in elution systems, the concentration of DHA is the highest, causing concerns about the further use of such a product in the food industry.

4. The implementation of the method does not provide preparations with a high content of DHA, and taking into account the costs of their production and low productivity, it cannot pretend for mass use.

A known method for producing docosahexaenoic acid from cod liver oil (Wright SW, Kuo EY, Corey EJ An Effective Process for the Isolation of Docosahexaenoic Acid in Quantity from Cod Liver Oil // J. Org. Chem., 1987. Vol. 52, P 4399 -4401). The method was carried out as follows: cod liver oil (200 g) was subjected to alkaline hydrolysis, unsaponifiables were separated, acidified to isolate free fatty acids (180 g). Next, polyunsaturated fatty acids were concentrated by the separation of lithium salts of fatty acids in acetone (a concentrate was obtained with a content of 42% DHA, 50 g). Then, the reaction of iodine-lactonization of fatty acids in tetrahydrofuran with iodine (ratio of 1.2 mol of iodine per 1 mol of DHA) and potassium iodide was carried out for 48 hours, after which iodine-lactones were extracted with ethyl acetate, evaporated, redissolved in hexane, washed unreacted fatty acid with a water-methanol solution of potassium carbonate, the hexane solution was washed successively with water and brine, evaporated to obtain 24.11 g of iodine-lactone DHA (81% yield based on iodine-lactone of DHA). Next, the obtained iodine-lactone DHA was opened with the release of the starting DHA in a solution of sodium iodide and trimethylchlorosilane (ratio of iodine-lactone DHA / trimethylchlorosilane, TMS-Cl - 1: 2, mol / mol) in acetonitrile. The liberated iodine was bound with a solution of sodium sulfite and sodium citrate. Fatty acids were extracted with a mixture of hexane and dichloromethane, the combined extracts were washed with water, brine, dried and evaporated to obtain 13.55 g of DHA with a purity of 100% (yield at the stage of 78%).

General output:

1) when calculating from 180 g of fatty acids with an average DHA content in cod liver oil of 12.5% - 58.9% (the authors do not indicate the initial concentration of DHA, but according to published data, the liver oil of Atlantic cod Gadus morhua contains 10.5- 14.3% DHA, Gruger EH “Fatty acid composition. In: Fish oils / Ed. Stansby ME Westport, Connecticut: The AVI Publ. Comp., Inc., 1967. P. 15);

2) when calculating from 50 g of polyunsaturated fatty acid concentrate - 63.2%.

The method has the following disadvantages:

1. The solvents used in the implementation of the method - tetrahydrofuran and methanol, are extremely toxic and should not be used to obtain drugs prescribed to humans. The preparations obtained in this way should be attributed to substances for biochemical studies.

2) The method is carried out in the 4th stage, which significantly complicates the process and increases the time of its implementation to almost 3 days (66 hours or more).

3) Preconcentration using the method of precipitation of lithium salts of saturated acids from acetone is a purely laboratory method, since the volume of solvent is 20 times the volume of the mass to be separated. Scaling such a method for production is unreasonable and not effective.

4) The iodine-lactonization reaction was carried out with a low yield (81%), that is, part of the DHA from the concentrate obtained in the previous stage is simply lost. This increases the cost of releasing this drug.

5) The output at the stage of disclosure of iodine-lactone of DHA is also low - 78%. It can be concluded that the reaction conditions are not optimal. This depends on the ratio of components in the medium, and on the time of the reaction, and on other important details.

6) The authors indicate that the product isolated in this way is pure (100%). However, the 4-stage production of DHA is ineffective due to the low yields at each stage, the overall yield is very low - 58.9%, although the advantage of any process is the maximum use of the feedstock, minimizing the loss of product, labor and time. This method for possible use in industrial production should be critically accepted and substantially modified.

Closest to our claimed method is a method for producing docosahexaenoic acid described in the Copyright Certificate SU 1631067 of 02.28.1991 "A method for producing docosahexaenoic, eicosapentaenoic and arachidonic acids or a mixture thereof" (authors N.A. Latyshev, A.B. Imbs, N .V. Gaidai, D.V. Kuklev, S.P. Kasyanov). The method is as follows: fish oil is saponified with an alkali solution, then acidified with acid to isolate free fatty acids, then they are converted into potassium salts and treated with iodine solution (with the addition of KI) based on DHA at a ratio of DHA / I ₂ = 1: 0 , 5-1.2, mol / mol, for 3-15 minutes, unreacted iodine is neutralized with an aqueous solution of sodium thiosulfate, the resulting iodine-lactone DHA is extracted with organic solvents (benzene, hexane, diethyl ether, chloroform, ethyl acetate), the solvent is evaporated, P dissolve the mixture in hexane, wash free fatty acids from iodine-lactone of DHA with aqueous-alcoholic solutions of bicarbonate or potassium carbonate, evaporate hexane and open iodine-lactone of DHA in anhydrous acetonitrile with a bonding agent (trimethyl iodosilane, TMS-I or trimethylchlorosilane) the ratio of iodine-lactone DHA / silylating agent = 1: 1.2-1.5, mol / mol, for 20-30 minutes, neutralize the released iodine with sodium thiosulfate solution and extract DHA from the mixture with an organic solvent.

The disadvantages of this method include:

1) Use of a ready-made mixture of free fatty acids. However, when introducing the method into semi-industrial or industrial production, this starting point is completely not economical, since the iodine-lactonization of fatty acids, in particular DHA, with iodine occurs when the fatty acids are in solution in the form of salts (soaps). And with this approach, the following is obtained: first, fish oil is subjected to alkaline hydrolysis and, instead of immediately using this mixture for iodine-lactonization, it is acidified with acid, free fatty acids are isolated and the same acids are then dissolved in alkaline agents and put into reaction. In fact, an additional unnecessary stage is formed in the process, which significantly takes away the reagents, time and leads to the formation of acid-salt by-products, which require neutralization efforts and attention to ensure environmental safety.

2) Use in a mixture for iodine-lactonization of potassium iodide (KI). It is known that its addition to iodine solution slows down the iodine-lactonization reaction and allows for a more “smooth” process. Therefore, iodine is even added in excess of the content of DHA to 1.2 mol / mol, the reaction time is limited to 3-15 minutes. But these ratios do not realize themselves when scaling the process from a laboratory flask to a reactor. Such insignificant temporal indicators lead to a change in the composition of the formed iodine-lactones of fatty acids - not only the iodine-lactone of DHA, but also the iodine-lactones of eicosapentaenoic and arachidonic acids are formed, moreover, the ratio of iodine to DHA contributes to this, much more than required by stoichiometry - 1,2 mol I _2/1 mol DHA. This stage of the process under the mentioned conditions has one very big drawback - even a small discrepancy in the process leads to the formation of iodine-lactone-DHA with a high content of impurities, which not only precludes its further use to obtain a highly enriched DHA concentrate, but also significantly reduces the yield of DHA .

3) The mentioned organic solvents and their mixtures, which are used for the extraction of iodine-lactone-DHA, especially benzene, chloroform, diethyl ether and ethyl acetate, are not preferred for such extraction, since they take with them many other impurities, primarily free fatty acids. In the future, it is required to remove these solvents, dissolve the mixture in hexane and carry out further procedures, which reduces the efficiency of the process, requires additional volumes of solvents, complexity of the process and unjustified use for this production equipment.

4) The authors, citing TMS-I and TMS-C1 as the enhancing agents necessary for the disclosure of DHA iodine-lactone, actually use only trimethyl iodosilane (TMS-I). Moreover, the most affordable and inexpensive trimethylchlorosilane (several times cheaper on the market than TMS-I) is not used for the disclosure of iodine-lactone DHA, which increases the cost of the product and narrows the scope of the process.

The problem solved by the invention is the development of a simple and economical method for producing docosahexaenoic 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 docosahexaenoic acid, including alkaline hydrolysis of sea fat, treatment of fatty acid salts with iodine solution to form iodine-lactones of fatty acids, extraction of iodine-lactone of DHA fatty acids with a non-polar organic solvent, removal of unreacted free solvent from the mixture fatty acids, the disclosure of iodine-lactone of fatty acids with a silylating agent, the neutralization of the liberated iodine by sodium thiosulfate, acidification of the reaction mixture si and extraction of free fatty acids, according to the invention, the treatment of salts of fatty acids with an iodine solution is carried out immediately after hydrolysis at a ratio of docosahexaenoic acid: iodine equal to 1: 0.80-0.95, mol / mol, at a temperature of the mixture 18-22 ° C, the duration of the addition of iodine solution to the reaction mixture is 30-60 minutes, with a total duration of the iodine-lactonization process of 90-120 minutes; and the disclosure of iodo-lactones of docosahexaenoic acid (DHA) is carried out at a ratio of iodine-lactone DHA / silylating agent = 1: 1.55-2.20, mol / mol, the temperature of the mixture is 18-22 ° C and the reaction time is 3-5 hours.

Treatment with iodine of salts of fatty acids, i.e. carrying out the iodine-lactonization stage directly in the reaction mixture after alkaline hydrolysis of sea fat ensures the continuity of the DHA production process, which proceeds according to the scheme: fish oil → hydrolysis with the formation of salts of fatty acids → iodine-lactonization reaction → opening of iodine-lactones with the release of DHA. This greatly simplifies and shortens the process of obtaining DHA compared with the prototype. The process in the prototype provides several more unnecessary additional manipulations - acidification, the allocation of free fatty acids and their repeated conversion to salt form.

Alkaline hydrolysis of sea fat is a solution of alkali metal hydroxides. It was experimentally found that the iodine-lactonization reaction is not affected by the salt in which the fatty acids are present in the mixture. In further technological experiments on iodine-lactonization, the form in which the fatty acid was found depended only on the alkaline agent with which the fish oil was hydrolyzed. As an alkaline agent, in particular, NaOH, KOH, LiOH can be used, as a result of which the range of substances used for hydrolysis is expanded. As shown by the examples of the application, the most preferred alkaline agents are potassium and sodium hydroxides, since hydrolysis proceeds very quickly in comparison with lithium alkali; in addition, this reagent is several times more expensive than NaOH and KOH (2-3 times), which affects the efficiency of the process.

Carrying out iodine-lactonization in the claimed operating parameters allows, in the final result, to increase the yield and purity of the obtained target product. The reaction with iodine solution is based on docosahexaenoic acid in the ratio DHA / I ₂ = 1: 0.80-0.95, mol / mol, at a temperature of the mixture 18-22 ° C and the duration of the addition of iodine solution to the reaction mixture 30-60 minutes, with a total duration of the iodine-lactonization process of 90-120 minutes.

The use of less iodine in the iodine-lactonization reaction than the content of DCG in the feedstock is very important for the formation of pure iodine-lactone of DHA, since it is known that gamma-iodine-lactones (including iodine-lactone-DHA) are formed in the first first, and delta-iodine-lactones (including iodine-lactones of arachidonic and eicosapentaenoic acids) are much slower. Therefore, the obvious lack of iodine is justified not only for all acids that form iodine-lactones, but even for DHA, which forms iodine-lactone faster than others. The framework of the taken ratio of DHA / I ₂ = 1: 0.80-0.95, mol / mol, ensures that in the iodine-lactone reaction mixture, DHA will be practically the only iodine-lactone formed. The ratio of DHA / I ₂ = 1: 0.80, mol / mol, is the lower limit to the decrease in the amount of iodine in the reaction, since a further decrease leaves the purity of the product exactly the same, and the yield of formation of iodine-lactone of DHA decreases in proportion to the decrease in the added iodine. The ratio of DHA / I ₂ = 1: 0.95, mol / mol, limits the upper limit of iodine use, because, despite the general increase in the output of iodine-lactone DHA, excessively added iodine begins to react with arachidonic and eicosapentaenoic acids, forming their iodine-lactones . Thus, a mixture of iodine-lactones is obtained, which further does not lead to the isolation of the pure target product - DHA.

The duration of adding iodine solution to the reaction mixture - 30-60 minutes - was determined as the most optimal. When iodine solution is added to the medium where the iodine-lactonization reaction occurs, iodine is always added dropwise, since an extremely undesirable local increase in its concentration can occur. At the same time, there is no longer a lack of iodine in this particular place, and this, in turn, provokes the formation of three iodine-lactones - iodine-lactone-DHA, and iodine-lactone of arachidonic acid, and eicosapentaenoic acid iodine-lactone. This situation can only be avoided by slowly adding iodine solution to the mixture, several times larger than in the prototype — 30-60 minutes. At the same time, an increase in the rush time of the iodine solution for more than 60 minutes no longer affects the quality of the product, and a decrease of less than 30 minutes is fraught with the occurrence of the side reactions described above.

The overall conduct of the iodine-lactonization reaction was determined in 90-120 minutes due to the fact that a very insignificant part of iodine, even in conditions of its lack in the reaction medium, still remains unreacted. Therefore, additional time is required for the complete passage of the iodine-lactonization reaction. Usually this is 30-60 minutes after adding the last drops of iodine solution to the mixture or, considering the entire iodine-lactonization reaction, 90-120 minutes. The end point of the reaction is determined quite simply - the lack of smell of iodine from the reaction mixture. The complete completion of the reaction makes it possible to exclude from the process the stage of neutralizing unreacted iodine with an aqueous solution of sodium thiosulfate (Na ₂ S ₂ O ₃ * 5H ₂ O), which is an obligatory step in the prototype.

The disclosure of iodine-lactone of DHA is carried out at a ratio of iodine-lactone of DHA / silylating agent = 1: 1.55-2.20, mol / mol, at a temperature of the mixture 18-22 ° C and the reaction time is 3-5 hours.

As the sizing agent, in particular, trimethyl iodosilane (TMS-I), trimethylchlorosilane (TMS-C1), triethylchlorosilane (TES-C1), triethyl iodosilane (TES-I) can be used. It is preferable to use trimethylchlorosilane as a sizing agent, since this agent is inexpensive and affordable, which, in the end, not only simplifies, but also reduces the cost of the process of obtaining the target product.

A decrease in the concentration of a silylating agent of less than 1.55 mol will not allow the entire DHA iodine-lactone to open up, which will lead to a decrease in yield and additional purification procedures.

The upper boundary of the DHA / silylating agent = 1.55: 2.20, mol / mol, determines the ratio of TMS-C1 to iodine-lactone of DHA. Since TMS-C1 in the reaction medium transforms rather slowly into the active form of trimethyl iodosilane (TMS-I), a certain excess of TMS-C1 is required to shift the equilibrium towards the formation of trimethyl iodosilane. An increase in this excess of more than 2.20 mol of TMS-C1 per 1 mol of iodine-lactone-DHA had no effect on the course of the reaction and was excessive from an economic point of view.

The stage of the disclosure of iodine-lactones of fatty acids with silylating agents is carried out in acetonitrile, which helps to reduce the production costs of obtaining the target product (DHA) due to the low cost of these reagents. When silylating chlorosilane agents are used for the disclosure of iodine-lactones of fatty acids, sodium iodide is additionally introduced into the medium.

The optimal duration of the reaction of the disclosure of iodine-lactone-DHA is 3-5 hours. This range is determined based on the possibility of transforming TMS-C1 into TMS-I, which then quickly reacts with iodine-lactone-DHA. Reducing the reaction time of less than 3 hours does not provide the formation of the required amount of TMS-I required for the disclosure of iodine-lactone DHA. As a result, the overall DHA yield is reduced and additional purification procedures are required. An increase in the reaction time of more than 5 hours does not affect the yield of the product (DHA), since all the iodine-lactone-DHA during this time is fully disclosed.

The temperature of the reaction of iodine-lactonization and the reaction of the opening of iodine-lactones - 18-22 ° C ensures the achievement of a technical result - obtaining a pure target product with a good yield. In addition, this temperature range is close to the temperatures of real industrial premises, since it is precisely this temperature that is the most comfortable for the work of production personnel. At the same time, no additional efforts are necessary to thermally regulate the ongoing processes - since both reactions are not exothermic, their temperature corresponds to an ambient temperature of 18-22 ° C.

As a non-polar organic solvent in the inventive process can be used, in particular, pentane, hexane, petroleum ether, heptane. To simplify the method and reduce production costs for obtaining DHA, it is advisable to extract iodine-lactone-DHA formed during the iodine-lactonization process 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 ability for iodine-lactone-DHA. An advantage of using these solvents is also that after extraction, their combined solutions do not need to be evaporated and redissolved iodine-lactone-DHA 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 - DHA.

The method is as follows.

Example 1

To obtain compositions with DHA, marine (fish) fat was used with the following composition of fatty acids forming iodine-lactones (see Table 1).

Each experiment was carried out using 100 g of fish oil of the above composition as a feedstock. The content of DHA in 100 g of fat is 6.4 g.

The process of obtaining compositions with DHA was carried out in stages as follows.

1) Fat hydrolysis

A schematic diagram of fat hydrolysis * is shown in FIG. 2

100 g of fish oil was loaded into a 2 L glass container with heating and stirrer and a solution of 14.5 g of NaOH in 500 ml of 70% ethanol was added. The mixture was stirred for 1 hour at a temperature of 65 ° C. The end of the fat hydrolysis reaction was monitored by thin layer chromatography of the reaction products on plates with a layer of silica gel (Sorbfil, Russia) in a 80: 20: 1 system, hexane-diethyl ether-acetic acid (vol%). Next, the reaction mixture was cooled to room temperature.

2) Iodine-lactonization reaction

In FIG. Figure 3 shows a schematic diagram of the production of DHA by iodine-lactonization.

A solution of 4 g of iodine in 100 ml of 95% ethanol was added to the reaction mixture from the previous step with stirring for 60 minutes. The temperature of the process is 18 ° C. The ratio of DHA / I ₂ = 1: 0.95, mol / mol. Next, the reaction mixture was given an additional exposure of 30 minutes. The total process time is 90 minutes.

Iodine-lactone-DHA was extracted with pentane 3 times × 100 ml. The extracts were combined. Next, free fatty acids were removed from the combined extract by washing it with a 2% solution of Na ₂ CO ₃ , 3 times × 30 ml. The pentane extract was evaporated.

Received 8.40 g of iodine-lactone-DHA, yield 94.2%, purity 99.8%.

3) Disclosure of DHA iodine-lactone

Iodine-lactone-DHA was added to a solution of 10 g of NaI in 100 ml of acetonitrile, and trimethylchlorosilane, TMS-C1, was added with stirring at a temperature of 18 ° C for 5 hours, with a ratio of iodine-lactone DHA / enhancing agent = 1: 2, 20, mol / mol. The completion of the reaction for the detection of iodine-lactone-DHA was monitored by thin-layer chromatography of the reaction products on plates with a layer of silica gel (Sorbfil, Russia) using pure benzene as the eluting system (double run of the plate).

At the end of the reaction, the released free iodine was neutralized with a 30% solution of sodium thiosulfate, Na ₂ S ₂ O ₃ , until the reaction mixture was discolored. Docosahexaenoic acid, DHA, was extracted with pentane, 3 times × 75 ml. The combined pentane extracts were evaporated.

Received 5.93 g of DHA, yield at the stage of 98.1%, purity 99.7%.

The total yield of 92.4%.

Example 2

All operations in this example were carried out in the same way as in example 1, except that:

- hydrolysis of fish oil was carried out with potassium alkali, KOH;

- the iodine-lactonization reaction was carried out at a ratio of DHA / I ₂ = 1: 0.80, mol / mol, for 30 minutes at 22 ° C and a total process time of 120 minutes. DHA iodine-lactone was extracted with hexane. Received 6.97 g of iodine-lactone DHA, yield 78.1%, purity 99.9%;

- in the iodine-lactone opening reaction, the trimethylchlorosilane silylating agent, TMS-C1, was used in the ratio of iodine-lactone DHA / silylating agent 1: 1.55, mol / mol, reaction temperature 22 ° C, the duration of the process was 3 hours. DHA was extracted with hexane. Received 5.06 g of DHA, yield at the stage of 97.9%, purity 99.9%.

The total yield of 76.5%.

Example 3

All operations in this example were carried out in the same way as in example 1, except that:

- the iodine-lactonization reaction was carried out at a ratio of DHA / I ₂ = 1: 1.0, mol / mol. Received 8.79 g of iodine-lactone DHA, yield 98.5%, purity 99.6%.

Upon disclosure of the iodine-lactone of DHA, 6.22 g of DHA were obtained, yield at the stage of 98.2%, purity 99.5%.

The total yield of 96.7%.

Example 4

All operations in this example were carried out in the same way as in example 2, except that:

- the reaction of the disclosure of iodine-lactone of DHA was carried out using trimethyl iodosilane, TMS-I, as the enhancing agent, with the ratio of iodine-lactone-DHA / enhancing agent = 1: 1.0, mol / mol. The reaction was carried out in acetonitrile medium without the addition of sodium iodide.

Received 6.96 g of iodine-lactone DHA, yield 78.1%, purity 99.8%.

Upon the disclosure of the iodine-lactone of DHA, 4.90 g of DHA was obtained, yield at the stage of 97.8%, purity 99.7%.

The total yield of 76.4%.

Example 5

All operations in this example were carried out as in example 1, except that:

- hydrolysis of sea fat was carried out with lithium hydroxide for 1.5 hours;

- extraction of iodine-lactone-DHA was performed with heptane;

- extraction of DHA after the disclosure of the iodine-lactone of DHA was carried out with heptane.

Received 8.32 g of iodine-lactone DHA, yield 93.3%, purity 99.8%.

Upon the discovery of iodine-lactones, 5.85 g of DHA was obtained, yield at the stage of 96.7%, purity 99.7%.

The total yield of 90.2%.

In FIG. 4. shows the chromatogram of the obtained compound (DHA) and quantitative analysis results (table), which confirm that the resulting product has a high degree of purity and does not contain critical impurities.

Analysis of mixtures of fatty acids was carried out in the form of their methyl esters. Analysis conditions: Shimadzu-14A gas-liquid chromatograph with flame ionization detector, Supelcowax-10TM capillary column, 0.25 mm × 30 mm, helium carrier gas, thermostat temperature 190 ° C, injector and detector temperature 240 ° C.

Example 6

All operations in this example were carried out in the same way as in example 1, except that:

- extraction of iodine-lactone DHA was carried out with petroleum ether;

- when opening the lactone, a silylating agent, triethylchlorosilane, was used;

- the ratio of the components of the iodine-lactone of DHA and the silylating agent upon opening is 1: 1.55 (mol / mol);

- extraction of DHA after the disclosure of iodine-lactone-DHA was carried out with petroleum ether.

Received 8.38 g of iodine-lactone DHA, yield 94.1%, purity 99.8%.

Upon the discovery of iodine-lactones, 5.78 g of DHA was obtained, yield at the stage of 96.8%, purity 99.2%.

The total yield of 91.1%.

As can be seen from the above examples, the proposed method allows to obtain compositions with a high content of DHA (more than 99% of the content of the main substance) while at the same time high extraction (yield) of this acid from the raw materials used is 76.4-96.7%.

Thus, a new effective and low-cost method for producing a preparation with a high content of docosahexaenoic acid has been developed, taking into account the characteristics of the raw materials used, the nature and conditions of the chemical processes that occur during its processing, and also ensuring a high yield and quality of the resulting product.

Claims (5)

1. A method for producing docosahexaenoic acid by alkaline hydrolysis of sea fats, treatment of salts of fatty acids with an iodine solution, extraction of iodine-lactones of fatty acids with a non-polar organic solvent, removal of unreacted free fatty acids from the mixture, disclosure of iodine-lactones of fatty acids with a silylating agent, neutralization of released free iodine sodium thiosulfate and extraction of free fatty acids with a non-polar organic solvent, characterized in that the treatment of salts of fatty acids with iodine solution lead directly after hydrolysis at a ratio of docosahexaenoic acid (DHA): iodine equal to 1: 0.80-0.95, mol / mol, at a temperature of the mixture 18-22 ° C, while the duration of the addition of iodine solution to the reaction mixture is 30- 60 minutes, with a total duration of the iodine-lactonization process of 90-120 minutes; and the disclosure of iodo-lactones of docosahexaenoic acid (DHA) is carried out in acetonitrile at a ratio of iodine-lactone DHA / silylating agent = 1: 1.55-2.20, mol / mol, at a temperature of the mixture 18-22 ° C and reaction time 3 -5 o'clock. 2. The method according to claim 1, characterized in that the alkaline hydrolysis of sea fats is carried out with solutions of potassium or sodium hydroxides. 3. The method according to claim 1, characterized in that pentane or hexane is used as a non-polar organic solvent for the extraction of iodine-lactones of fatty acids, as well as for the extraction of free fatty acids. 4. The method according to claim 1, characterized in that when used for the disclosure of iodine-lactones of fatty acids, silylating chlorosilane agents, sodium iodide is additionally introduced into the medium. 5. The method according to claim 1, characterized in that trimethylchlorosilane is taken as a sizing agent.

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