Composition containing eicosapentaenoic acid ethyl ester, preparation method therefor and use thereof

Abstract

The present application relates to the field of biochemical engineering, and discloses a composition containing eicosapentaenoic acid ethyl ester, a preparation method therefor, and a use thereof. The preparation method for the composition containing eicosapentaenoic acid ethyl ester disclosed in the present application comprises the following steps: transesterification, multi-stage molecular distillation, urea inclusion, rectification and preparative chromatographic purification. In the present application, by means of performing molecular distillation and urea inclusion before rectification, a liquid mixture can be separated, removing high-boiling-point saturated fatty acid ethyl esters and most fatty acid ethyl esters with one double bond, thereby effectively lowering the rectification temperature and preventing the formation of excessive isomers. Furthermore, the composition obtained by means of the described steps can achieve an eicosapentaenoic acid ethyl ester content of as high as 96.5 area% to 99.5 area%, with low isomer and impurity content, making it suitable for use in the preparation of food and pharmaceuticals.

Description

Composition containing eicosapentaenoic acid ethyl ester and preparation method and application thereof

[0001] This application claims priority to Chinese patent application No. 2024100276265, filed on January 8, 2024. This application incorporates the entirety of the aforementioned Chinese patent application. Technical Field

[0002] The present application relates to the field of biochemical engineering, and specifically to a composition containing eicosapentaenoic acid ethyl ester, and a preparation method and application thereof. Background Art

[0003] As people become increasingly concerned about their health, they are consuming more nutritional and health supplements, with polyunsaturated fatty acids (PUFAs) playing a significant role. PUFAs are straight-chain fatty acids containing two or more double bonds and carbon chains of 18-22 carbon atoms. They are an important component of metabolism, particularly in infant brain development. They are a constituent of cell membranes, primarily maintaining cell membrane fluidity, promoting cholesterol esterification, lowering cholesterol and triglycerides, reducing blood viscosity, and improving circulation. They also improve thinking and memory. The human body cannot synthesize PUFAs on its own and must obtain them through diet and other means.

[0004] There are many types of polyunsaturated fatty acids (PUFA), mainly including ω-3 polyunsaturated fatty acids (ω-3 PUFA), ω-6 polyunsaturated fatty acids (ω-6 PUFA), ω-9 polyunsaturated fatty acids (ω-9 PUFA) and other forms, such as α-linolenic acid (ALA), eicosapentaenoic acid (EPA), henicosapentaenoic acid (HPA), docosahexaenoic acid (DHA), docosapentaenoic acid (DPA), linoleic acid (LA), conjugated linoleic acid (CLA), γ-linolenic acid (GLA), arachidonic acid (AA), etc. Among them, ω-3 polyunsaturated fatty acids (ω-3 PUFA) represented by eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are the most well-known and accepted by the public. They can be used together with a low-fat diet to reduce hypertriglyceridemia.

[0005] Typically, crude fish oil (referred to as crude fish oil or raw fish oil) with a content of about 30% can be obtained from various marine raw materials by squeezing, extraction, and other means. EPA and DHA are usually present in the form of triglycerides, and in a small amount in the form of wax esters and phospholipids. Because other fatty acid molecules are also connected to the long chains of triglycerides, it is difficult to obtain a specific component with a higher content when purifying it in the form of triglycerides. Therefore, before separation and purification, it is often subjected to ethyl esterification to convert it into the form of eicosapentaenoic acid ethyl ester (EPA-EE) and docosahexaenoic acid ethyl ester (DHA-EE), so that the active ingredient is separated (referred to as ethyl esterification). Then, through a series of means, a crude product with a total content of EPA and DHA of more than 70% is obtained (referred to as initial extraction). EPA and DHA have different mechanisms of action on the human body and influence each other, so it is often necessary to further separate the two to obtain a product with a higher content of a specific fatty acid. For this reason, it is necessary to further increase the content of EPA or DHA in the refining process to more than 90% (referred to as high-purity extraction).

[0006] The drug developed by Amarin Corporation in the United States for the treatment of hypertriglyceridemia and dyslipidemia uses eicosapentaenoic acid ethyl ester (EPA-EE) with a purity greater than 96.5%. It is an omega-3 acid ethyl ester prescription drug for the treatment of hypertriglyceridemia and dyslipidemia in the United States and has good therapeutic effects.

[0007] High-purity eicosapentaenoic acid ethyl ester API is mainly obtained by purifying ethyl ester fish oil. Currently, the methods for separating and purifying EPA-EE API from low-content ethyl ester fish oil mainly include a combination of the following: urea inclusion method, low-temperature freezing method, metal salt precipitation method, vacuum distillation method, supercritical extraction method, high-performance liquid chromatography and column chromatography.

[0008] At the same time, considering that as a bulk drug, not only must the main ingredient, eicosapentaenoic acid ethyl ester (EPA-EE), be present in a content greater than 96.5%, but other impurities must also meet these requirements. Since fatty acids containing a high number of double bonds, such as EPA-EE, are extremely sensitive to heat, they are prone to isomerization, cracking, and polymerization at high temperatures. For example, Europeana J. of Lipid Sci. and Tech., 108 (2006) 589-597, and JAOCS, 66 (1989) 1822-1830 describe that EPA-EE, when heated, is prone to isomerization of one or two of its five double bonds into trans fatty acids, and these isomers have been identified.

[0009] Therefore, during the production of API EPA-EE, in addition to minimizing the removal of fatty acid ethyl esters other than EPA from the raw fish oil, the separation and purification of EPA-EE requires minimal process conditions. This minimizes the production of EPA-EE isomers and other impurities while minimizing the removal of other fatty acid ethyl esters. Generally speaking, the content of other known fatty acid ethyl esters in the API should be less than 0.50%, and the content of each unknown impurity should be below 0.1%.

[0010] In the prior art, a large number of studies have been conducted to purify high-purity eicosapentaenoic acid ethyl ester from the perspective of process or equipment innovation, but the removal of impurities in the high-purity eicosapentaenoic acid ethyl ester has not been considered.

[0011] CN102391112B discloses a method for producing high-purity EPA-EE by molecular distillation, salt precipitation, and preparative chromatography. The obtained EPA-EE content is above 96%, but no attention is paid to other fatty acid impurities therein.

[0012] CN106795452A discloses a method for obtaining EPA-EE by controlling the vacuum degree and temperature of a distillation tower in combination with preparative chromatography. The main purpose is to control the production of isomers in EPA-EE and to remove arachidonic acid ethyl ester (AA) as much as possible. However, other unknown impurity peaks with retention times of 0.613, 0.836, and 0.966 relative to EPA-EE are not controlled. However, in pharmaceutical-grade raw materials, the content of these unknown impurities is subject to strict requirements.

[0013] In general, previously prepared high-content eicosapentaenoic acid ethyl ester combinations and their preparation technologies have several deficiencies: either they only focus on increasing the eicosapentaenoic acid ethyl ester (EPA-EE) content to above 96%, without paying attention to the impurities therein; or they only focus on the isomer impurities of EPA-EE, without controlling other fatty acid impurities or unknown impurities. Such products are not suitable for use as APIs.

[0014] In addition, the API eicosapent ethyl is of natural origin, and its impurity types and levels vary greatly depending on the fish sourced from different fishing areas. For example, when using raw fish oil from Peru as raw material and previous processes to produce pharmaceutical-grade eicosapent ethyl, some impurity peaks are difficult to remove to the ideal level, especially the unknown impurities with relative retention times of 0.613, 0.728, 0.836, 0.858, 0.868, and 0.966, which are difficult to remove using conventional processes.

[0015] In view of the shortcomings of previous technologies and the differences in composition caused by the origin of natural raw materials, it is necessary to find a process that is efficient, suitable for industrial production, and suitable for using fish oils from different origins to prepare eicosapentaenoic acid ethyl ester. This process can not only minimize the generation of impurities during the process, but also effectively remove impurities other than EPA-EE, such as ethyl stearate (SDA-EE), ethyl arachidonic acid (ethyl AA-EE), ethyl eicosatetraenoic acid (ETA-EE), ethyl docosahexaenoic acid (DHA-EE), ethyl docosapentaenoic acid (DPA-EE), and ethyl eicosapentaenoic acid (HPA-EE), as well as other unidentified unknown impurities, especially those with relative retention times of 0.613, 0.728, 0.836, 0.858, 0.868, and 0.966, so that the produced EPA-EE meets the requirements of food-grade raw materials. Summary of the Invention

[0016] The main purpose of the present application is to provide a composition containing eicosapentaenoic acid ethyl ester, and its preparation method and application, in order to solve the problem of high impurity content in the composition containing eicosapentaenoic acid ethyl ester prepared from fish oil in the prior art, especially high content of unknown impurities with retention times of 0.613, 0.728, 0.836, 0.858, 0.868, and 0.966 relative to eicosapentaenoic acid ethyl ester.

[0017] In order to achieve the above object, according to the first aspect of the present application, a method for preparing a composition containing eicosapentaenoic acid ethyl ester is provided, and the preparation method comprises the following steps:

[0018] Transesterification: fish oil is subjected to transesterification reaction to obtain fatty acid ethyl ester products;

[0019] Multi-stage molecular distillation: subjecting the fatty acid ethyl esterification product to multi-stage molecular distillation to obtain a first fatty acid ethyl ester composition, wherein the multi-stage molecular distillation is at least three stages of molecular distillation;

[0020] Urea inclusion: performing urea inclusion on the first fatty acid ethyl ester composition to obtain a second fatty acid ethyl ester composition;

[0021] Distillation: distilling the second fatty acid ethyl ester composition to obtain a third fatty acid ethyl ester composition;

[0022] Preparative chromatography: The third fatty acid ethyl ester composition is subjected to preparative chromatography purification to obtain a composition containing eicosapentaenoic acid ethyl ester.

[0023] Furthermore, the above-mentioned urea inclusion complex is as follows: mixing a first fatty acid ethyl ester composition with a fatty alcohol and urea to obtain a mixture; heating the mixture to 70-80°C, keeping it warm for 0.5-1h, then cooling it to -2-4°C for crystallization, filtering it to obtain a filtrate; washing the filtrate with water, degassing it or extracting it with an extractant, and then concentrating it to obtain a second fatty acid ethyl ester composition.

[0024] Furthermore, the mass ratio of the first fatty acid ethyl ester composition, the fatty alcohol, and the urea is 1:(4-6):(0.8-2); and / or the mass ratio of the filtrate to the extractant is 1:(1-2.5); and / or the fatty alcohol is ethanol or tert-butanol; and / or the extractant is n-hexane or isooctane.

[0025] Furthermore, the fatty alcohol is tert-butanol; and / or the extractant is isooctane.

[0026] Furthermore, the transesterification step comprises: dissolving the fish oil in anhydrous ethanol, adding a catalyst, reacting at 75-85° C. for 0.5-3 hours, washing with water, standing to separate layers, and degassing the upper layer liquid to obtain a fatty acid ethyl ester product; and / or

[0027] The multi-stage molecular distillation is a six-stage molecular distillation, and the temperature of the six-stage molecular distillation is 100-160° C.; and / or

[0028] The distillation conditions are: top vacuum degree <30Pa, kettle temperature 160-180°C; and / or

[0029] The preparative chromatography conditions were as follows:

[0030] Chromatographic column: C18 reverse phase silica gel column;

[0031] Mobile phase: a mixture of methanol and water, wherein the volume ratio of methanol to water is (85-95): (5-15);

[0032] The sample loading amount is 2%-15% of the filler mass.

[0033] Furthermore, the mass ratio of fish oil, anhydrous ethanol, and catalyst is 1:(2-5):(0.003-0.035); and / or

[0034] The catalyst is one of a liquid caustic soda aqueous solution with a mass fraction of 25% to 35% of liquid caustic soda, a potassium hydroxide aqueous solution with a mass fraction of 15% to 25% of potassium hydroxide, and sodium ethoxide; and / or

[0035] Before washing with water, the reaction product is concentrated.

[0036] Furthermore, the temperatures of the six-stage molecular distillation are 100-110°C, 110-120°C, 120-130°C, 130-140°C, 140-150°C, and 150-160°C, respectively.

[0037] According to a second aspect of the present application, there is provided a composition containing eicosapentaenoic acid ethyl ester prepared by the above method, wherein when the following gas chromatography method is used for detection, the retention time of eicosapentaenoic acid ethyl ester is 1, and the sum of the areas of the peaks of the components is 100%. In the composition, the area percentage is:

[0038] Containing 96.5%-99.5% of eicosapentaenoic acid ethyl ester, a first impurity with a relative retention time of 0.613 is less than 0.07%, a second impurity with a relative retention time of 0.836 is less than 0.53%, a third impurity with a relative retention time of 0.966 is less than 0.51%, a fourth impurity with a relative retention time of 0.728 is less than 0.5%, a fifth impurity with a relative retention time of 0.858 is less than 0.5%, and a sixth impurity with a relative retention time of 0.868 is less than 0.5%;

[0039] Gas chromatography detection conditions:

[0040] Instrument: Agilent 6890;

[0041] Chromatographic column: HP-INNOWAX, 30m×0.25mm×0.25μm;

[0042] Carrier gas: helium;

[0043] Inlet temperature: 250°C;

[0044] Column temperature: start at 170°C, hold for 2 min, increase at 3°C / min to 240°C, hold for 7 min;

[0045] Detector: FID, 270°C;

[0046] Injection volume: 1 μL;

[0047] Split ratio: 100:1;

[0048] Flow rate: 1.8 mL / min;

[0049] Furthermore, in the above composition, the sum of the area percentages of the isomers of eicosapentaenoic acid ethyl ester is less than 2%.

[0050] Furthermore, in the above composition, substances with relative retention times of 0.989, 1.013, 1.017, 1.024, and 1.03 are all isomers of eicosapentaenoic acid ethyl ester.

[0051] Furthermore, in the above composition, calculated by area percentage, the content of the first impurity is less than 0.07%, the content of the second impurity is less than 0.53%, the content of the third impurity is less than 0.51%, the content of the fourth impurity is less than 0.35%, the content of the fifth impurity is less than 0.3%, and the content of the sixth impurity is less than 0.3%.

[0052] Furthermore, in the above composition, calculated by area percentage, the content of the first impurity is less than 0.03%, the content of the second impurity is less than 0.15%, the content of the third impurity is less than 0.1%, the content of the fourth impurity is less than 0.1%, the content of the fifth impurity is less than 0.1%, and the content of the sixth impurity is less than 0.15%.

[0053] Furthermore, in the above composition, calculated by area percentage, the content of ethyl stearate is less than 1%, the content of ethyl docosapentaenoate is less than 0.1%, the content of ethyl docosahexaenoate is less than 0.1%, and the content of ethyl henicosapentaenoate is less than 0.3%.

[0054] According to a third aspect of the present application, there is provided a use of the above-mentioned composition containing eicosapentaenoic acid ethyl ester in the field of food preparation.

[0055] According to a fourth aspect of the present application, there is provided a use of the above-mentioned composition containing eicosapentaenoic acid ethyl ester in the field of preparing medicines.

[0056] By applying the technical solution of the present application, through transesterification, multi-stage molecular distillation, urea inclusion complex, distillation and preparative chromatography purification, the contents of eicosapentaenoic acid ethyl ester isomers, other fatty acid ethyl esters and several unknown impurities with relative retention times of 0.613, 0.728, 0.836, 0.858, 0.868 and 0.966 in the composition containing eicosapentaenoic acid ethyl ester are effectively reduced, so that the above-mentioned composition containing eicosapentaenoic acid ethyl ester can be used as a raw material for preparing food or medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] FIG1 is a chromatographic data diagram of the composition containing eicosapentaenoic acid ethyl ester in Example 1. DETAILED DESCRIPTION

[0058] To make the purpose, technical solutions and advantages of the examples of the present application clearer, the technical solutions in the examples of the present application will be described clearly and completely below. Where specific conditions are not specified in the examples, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0059] As described in the background of this application, although the prior art provides a method for preparing high-purity eicosapentaenoic acid ethyl ester, the content of impurities therein is not limited. Some impurities may have adverse effects on the human body. Therefore, there is an urgent need to provide a composition containing eicosapentaenoic acid ethyl ester with a low impurity content.

[0060] In order to solve the above technical problems, in a typical embodiment of the present application, a method for preparing a composition containing eicosapentaenoic acid ethyl ester is provided, and the preparation method comprises the following steps:

[0061] Transesterification: fish oil is subjected to transesterification reaction to obtain fatty acid ethyl ester products;

[0062] Multi-stage molecular distillation: subjecting the fatty acid ethyl esterification product to multi-stage molecular distillation to obtain a first fatty acid ethyl ester composition, wherein the multi-stage molecular distillation is at least three stages of molecular distillation;

[0063] Urea inclusion: performing urea inclusion on the first fatty acid ethyl ester composition to obtain a second fatty acid ethyl ester composition;

[0064] Distillation: distilling the second fatty acid ethyl ester composition to obtain a third fatty acid ethyl ester composition;

[0065] Preparative chromatography: The third fatty acid ethyl ester composition is subjected to preparative chromatography purification to obtain a composition containing eicosapentaenoic acid ethyl ester.

[0066] Natural fish oil contains nearly 200 components, including over 100 fatty acids with varying carbon chain lengths, ranging from C8 to C24. EPA accounts for only about 15% of this total. Creating a composition with an EPA content as high as 96.5% requires multiple purification methods. First, the fatty acids in fish oil are typically bound to glycerol in the form of triglycerides. Triglycerides have a high boiling point, making separation of the different fatty acids difficult. Therefore, prior to EPA purification, the triglycerides must be transesterified to form fatty acid ethyl esters, facilitating subsequent separation and purification.

[0067] Secondly, molecular distillation is a method used to separate the components of a liquid mixture. Operating under a high vacuum, it allows the mean free path of vapor molecules to be greater than the distance between the evaporation surface and the condensation surface. This allows the liquid mixture to be separated by exploiting the differences in evaporation rates of the various components in the liquid. While less efficient than rectification, molecular distillation can lower the heating temperature of the material, removing C8-C16 fatty acid ethyl esters, which have a significant boiling point difference from EPA-EE, as well as a small amount of C18 fatty acid ethyl ester, thereby increasing the EPA-EE content from approximately 20% to approximately 40%.

[0068] The presence and number of double bonds in fatty acids lead to different spatial configurations in their molecular structures. Saturated fatty acid carbon chains are curved, and the more double bonds, the less flexible they are. The urea inclusion method utilizes the urea molecule to form relatively stable crystalline inclusion complexes with saturated or monounsaturated fatty acid ethyl esters during crystallization. Polyunsaturated fatty acid ethyl esters, due to their numerous double bonds, are not easily incorporated by urea. This inclusion complex can be separated through filtration, resulting in a filtrate containing higher-purity polyunsaturated fatty acid ethyl esters, thereby increasing the EPA-EE content.

[0069] Distillation utilizes the difference in boiling points of fatty acids with different carbon chain lengths and different numbers of double bonds. Through the reflux of liquid and gas phases, the gas and liquid phases are brought into reverse multi-stage contact. Under the constraints of thermal energy drive and phase equilibrium, the volatile components are continuously transferred from the liquid phase to the gas phase, while the non-volatile components are transferred from the gas phase to the liquid phase, so that the mixture is continuously separated, which can further increase the EPA-EE content.

[0070] Preparative chromatography refers to the use of chromatographic technology to prepare pure substances. By adopting the above-mentioned purification methods, the present application can obtain an eicosapentaenoic acid ethyl ester composition with a high eicosapentaenoic acid ethyl ester content from natural fish oil, wherein the eicosapentaenoic acid ethyl ester content can be as high as 96.5%-99.5%, and it is beneficial to reduce impurities in the composition, avoiding side effects on the human body after the above-mentioned composition is used in food and medicine.

[0071] Typically, but not limiting, the urea inclusion method is as follows: mixing a first fatty acid ethyl ester composition with a fatty alcohol and urea to obtain a mixture; heating the mixture to 70-80°C, keeping warm for 0.5-1h, then cooling to -2-4°C for crystallization, filtering to obtain a filtrate; washing the filtrate with water, degassing it, or extracting the filtrate with an extractant, and then concentrating it to obtain a second fatty acid ethyl ester composition.

[0072] As a preferred embodiment of the present application, the mass ratio of the first fatty acid ethyl ester composition, the fatty alcohol, and the urea is 1:(4-6):(0.8-2); and / or the mass ratio of the filtrate to the extractant is 1:(1-2.5); and / or the fatty alcohol is ethanol or tert-butanol; and / or the extractant is n-hexane or isooctane.

[0073] The above-mentioned restrictions on the ratio of the components allow for good inclusion of saturated fatty acid ethyl esters and monounsaturated fatty acid ethyl esters, resulting in the filtrate being primarily composed of polyunsaturated fatty acid ethyl esters, thereby increasing the purity of eicosapentaenoic acid ethyl ester. Furthermore, extracting the filtrate can further increase the purity of eicosapentaenoic acid ethyl ester compared to methods without extraction.

[0074] As a preferred embodiment of the present application, the fatty alcohol is tert-butanol. Compared with ethanol used in conventional urea complexation, the use of tert-butanol is beneficial for increasing the content of eicosapentaenoic acid ethyl ester in the composition and can reduce the total content of unknown impurities to below 2%.

[0075] As a preferred embodiment of the present application, the filtrate is subjected to extraction, and the content of icosapentaenoic acid ethyl ester in the extracted product is higher. In addition, the extraction solvent is preferably isooctane. Compared with n-hexane, the use of isooctane for extraction results in a higher content of icosapentaenoic acid ethyl ester in the final obtained composition product, and the content of unknown impurities in the product is effectively reduced.

[0076] As a preferred embodiment of the present application, the transesterification is to dissolve the fish oil in anhydrous ethanol, add a catalyst, react at 75-85°C for 0.5-3h, wash with water, let stand and separate, and degas the upper liquid to obtain a fatty acid ethyl ester product; and / or

[0077] The multi-stage molecular distillation is a six-stage molecular distillation, and the temperature of the six-stage molecular distillation is 100-160° C.; and / or

[0078] The distillation conditions are: top vacuum degree <30Pa, kettle temperature 160-180°C; and / or

[0079] The preparative chromatography conditions were as follows:

[0080] Chromatographic column: C18 reverse phase silica gel column;

[0081] Mobile phase: a mixture of methanol and water, wherein the volume ratio of methanol to water is (85-95): (5-15);

[0082] The sample loading amount is 2%-15% of the filler mass.

[0083] The above-mentioned transesterification, multi-stage molecular distillation, and preparative chromatography conditions are conventionally selected in the art, but the distillation conditions are lower than the temperature commonly used in the art. The reason is that high distillation temperatures can cause isomerization or polymerization of ethyl eicosapentaenoic acid, resulting in a lower content of ethyl eicosapentaenoic acid in the composition. The reason why the distillation temperature can be lowered in the present application is that multi-stage molecular distillation and urea inclusion complexation are pre-performed before distillation, removing other saturated fatty acid ethyl esters or monounsaturated fatty acid ethyl esters with a boiling point close to that of ethyl eicosapentaenoic acid, thereby reducing the content of ethyl eicosapentaenoic acid isomers in the composition.

[0084] Typically, but not limiting, during the transesterification process, the mass ratio of fish oil, anhydrous ethanol, and catalyst is 1:(2-5):(0.003-0.035); and / or, the catalyst is one of a 25%-35% by mass aqueous caustic soda solution, a 15%-25% by mass potassium hydroxide aqueous solution, and sodium ethoxide; and / or, the reaction product is concentrated before washing, which can improve the recovery efficiency of anhydrous ethanol and reduce the recovery cost.

[0085] Typically, but not limiting, the temperatures of the six-stage molecular distillation are 100-110° C., 110-120° C., 120-130° C., 130-140° C., 140-150° C., and 150-160° C. The use of multi-stage molecular distillation can gradually increase the concentration of the intermediate product, shorten the distillation time, and improve the distillation efficiency.

[0086] In another typical embodiment of the present application, a composition containing eicosapentaenoic acid ethyl ester is provided. When the following gas chromatography method is used for detection, the retention time of eicosapentaenoic acid ethyl ester is 1, the sum of the areas of the peaks of each component is 100%, and the composition is calculated as the area percentage.

[0087] Containing 96.5%-99.5% of eicosapentaenoic acid ethyl ester, a first impurity with a relative retention time of 0.613 is less than 0.07%, a second impurity with a relative retention time of 0.836 is less than 0.53%, a third impurity with a relative retention time of 0.966 is less than 0.51%, a fourth impurity with a relative retention time of 0.728 is less than 0.5%, a fifth impurity with a relative retention time of 0.858 is less than 0.5%, and a sixth impurity with a relative retention time of 0.868 is less than 0.5%;

[0088] Gas chromatography detection conditions:

[0089] Instrument: Agilent 6890;

[0090] Chromatographic column: HP-INNOWAX, 30m×0.25mm×0.25μm;

[0091] Carrier gas: helium;

[0092] Inlet temperature: 250°C;

[0093] Column temperature: start at 170°C, hold for 2 min, increase at 3°C / min to 240°C, hold for 7 min;

[0094] Detector: FID, 270°C;

[0095] Injection volume: 1 μL;

[0096] Split ratio: 100:1;

[0097] Flow rate: 1.8 mL / min;

[0098] The composition containing eicosapentaenoic acid ethyl ester is prepared by the above-mentioned preparation method and can be used in preparing food.

[0099] As a preferred embodiment of the present application, in the composition, the sum of the area percentages of the isomers of eicosapentaenoic acid ethyl ester is less than 2%.

[0100] In addition, the present application tested and found that in the composition, the substances with relative retention times of 0.989, 1.013, 1.017, 1.024, and 1.03 were all isomers of eicosapentaenoic acid ethyl ester.

[0101] As a preferred embodiment of the present application, in the composition, calculated by area percentage, the content of the first impurity is less than 0.07%, the content of the second impurity is less than 0.53%, the content of the third impurity is less than 0.51%, the content of the fourth impurity is less than 0.35%, the content of the fifth impurity is less than 0.3%, and the content of the sixth impurity is less than 0.3%.

[0102] In order to further improve the application scope of the composition, as a preferred embodiment of the present application, in the composition, calculated by area percentage, the content of the first impurity is less than 0.03%, the content of the second impurity is less than 0.15%, the content of the third impurity is less than 0.1%, the content of the fourth impurity is less than 0.1%, the content of the fifth impurity is less than 0.1%, and the content of the sixth impurity is less than 0.15%.

[0103] As a preferred embodiment of the present application, in the composition, calculated by area percentage, the content of ethyl stearate is less than 1%, the content of ethyl docosapentaenoate is less than 0.1%, the content of ethyl docosahexaenoate is less than 0.1%, and the content of ethyl henicosapentaenoate is less than 0.3%.

[0104] By limiting the amount of each component in the composition as above, the composition containing eicosapentaenoic acid ethyl ester can be used in both food and pharmaceutical fields.

[0105] In a typical embodiment of the present application, a use of the above-mentioned composition containing eicosapentaenoic acid ethyl ester in the field of food preparation is disclosed, such as the preparation of health foods for enhancing memory, preventing myopia, etc.

[0106] In a typical embodiment of the present application, the application of the above-mentioned composition containing eicosapentaenoic acid ethyl ester in the field of preparing medicines is disclosed, such as preparing drugs for lowering blood lipids, lowering blood pressure, preventing atherosclerosis, etc.

[0107] In order to make those skilled in the art better understand the technical solution of the present application, the present application is further described in detail below in conjunction with the examples. Unless otherwise specified, the raw materials in the present application can be obtained through commercial channels.

[0108] The fish oil in the examples is raw fish oil from Peru.

[0109] Unless otherwise specified, the content of each component in the composition containing eicosapentaenoic acid ethyl ester is the area percentage content measured by the following gas chromatography method (the sum of the peak areas of each component is 100%):

[0110] Instrument: Agilent 6890;

[0111] Chromatographic column: HP-INNOWAX, 30m×0.25mm×0.25μm;

[0112] Carrier gas: helium;

[0113] Inlet temperature: 250°C;

[0114] Column temperature: start at 170°C, hold for 2 min, increase at 3°C / min to 240°C, hold for 7 min;

[0115] Detector: FID, 270°C;

[0116] Injection volume: 1 μL;

[0117] Split ratio: 100:1;

[0118] Flow rate: 1.8 mL / min.

[0119] Example 1

[0120] An embodiment of the composition containing eicosapentaenoic acid ethyl ester of the present application is prepared as follows:

[0121] S1, 6000 kg of fish oil and 12000 kg of anhydrous ethanol were added to a reactor, heated to 80°C for dissolution, 200 kg of a 30% sodium hydroxide aqueous solution was added dropwise, and the mixture was reacted for 0.5 h. 5000 kg of unreacted ethanol was recovered under vacuum, the remaining product was washed with water, allowed to stand for stratification, and the supernatant was degassed to obtain a fatty acid ethyl esterification product;

[0122] S2, subjecting the fatty acid ethyl esterification product to six-stage molecular distillation at temperatures of 100° C., 115° C., 125° C., 135° C., 145° C., and 158° C., to obtain a first fatty acid ethyl ester composition;

[0123] S3, adding 1000 kg of the first fatty acid ethyl ester composition and 2000 kg of urea to 6000 kg of tert-butanol, dissolving at 75 ° C for 1 hour, then cooling to -2 ° C for crystallization, filtering, adding 2500 kg of isooctane to the filtrate for extraction, and taking the upper isooctane layer for concentration to obtain a second fatty acid ethyl ester composition;

[0124] S4, controlling the vacuum degree at the top of the distillation tower to be below 30 Pa and the kettle temperature to be 178° C. to obtain a third fatty acid ethyl ester composition;

[0125] S5, purifying the third fatty acid ethyl ester composition by preparative chromatography to obtain a composition containing eicosapentaenoic acid ethyl ester;

[0126] The specific conditions of the preparative chromatography are as follows, and the chromatographic data of Example 1 is shown in FIG1 :

[0127] Chromatographic column: C18 reverse phase column (Φ50*250), filled with C18 reverse phase silica gel;

[0128] Detector: UV detector, detection wavelength: 210nm;

[0129] Mobile phase: a mixture of methanol and water in a volume ratio of 95:5, with a mobile phase flow rate of 150 mL / min;

[0130] Elution mode: isocratic elution;

[0131] The sample loading amount was 12% of the filler mass.

[0132] Table 1. Content of main components in intermediates and products in each step of Example 1

[0133] Example 2

[0134] An embodiment of the present application of a composition containing eicosapentaenoic acid ethyl ester, the preparation method of which differs from that of Example 1 in that, in S3, ethanol is used instead of tert-butanol, and after filtration, the filtrate is concentrated, washed with water, allowed to stand for stratification, and degassed to obtain a second fatty acid ethyl ester composition.

[0135] Table 2. Content of main components in intermediates and products in each step of Example 2

[0136] Example 3

[0137] An embodiment of the present application of a composition containing eicosapentaenoic acid ethyl ester, the preparation method of which differs from that of Example 1 in that, in S3, after obtaining the filtrate, the filtrate is not extracted, and the filtrate is concentrated, washed with water, allowed to stand for stratification, and degassed to obtain a second fatty acid ethyl ester composition.

[0138] Table 3. Content of main components in intermediates and products in each step of Example 3

[0139] Example 4

[0140] An embodiment of the composition containing eicosapentaenoic acid ethyl ester of the present application has a preparation method different from that of Example 1 in that, in S3, n-hexane is used instead of isooctane for extraction.

[0141] Table 4. Content of main components in intermediates and products of each step in Example 4

[0142] Example 5

[0143] An embodiment of the composition containing eicosapentaenoic acid ethyl ester of the present application has a preparation method different from that of Example 1 in that, in S3, ethanol is used instead of tert-butanol, and n-hexane is used instead of isooctane.

[0144] Table 5. Content of main components in intermediates and products in each step of Example 5

[0145] Example 6

[0146] An embodiment of the composition containing eicosapentaenoic acid ethyl ester in the present application is prepared as follows:

[0147] S1, adding 1000 kg of fish oil and 4000 kg of anhydrous ethanol into a reactor, heating to 80°C for dissolution, adding 3 kg of sodium acetate dropwise, reacting for 3 hours after the addition is complete, recovering the unreacted ethanol, washing the remaining product with water, allowing it to stand for stratification, and degassing the supernatant to obtain a fatty acid ethyl ester product;

[0148] S2, subjecting the fatty acid ethyl esterification product to six-stage molecular distillation at temperatures of 100° C., 115° C., 125° C., 135° C., 145° C., and 158° C., to obtain a first fatty acid ethyl ester composition;

[0149] S3, adding 1000 kg of the first fatty acid ethyl ester composition and 800 kg of urea to 4000 kg of tert-butanol, dissolving at 75 ° C for 0.5 h, then cooling to 4 ° C for crystallization, filtering, adding 1000 kg of isooctane to the filtrate for extraction, and taking the upper isooctane layer for concentration to obtain a second fatty acid ethyl ester composition;

[0150] S4, controlling the vacuum degree at the top of the distillation tower to be below 30 Pa and the kettle temperature to be 180° C. to obtain a third fatty acid ethyl ester composition;

[0151] S5, purifying the third fatty acid ethyl ester composition by preparative chromatography to obtain a composition containing eicosapentaenoic acid ethyl ester;

[0152] The specific conditions of preparative chromatography are as follows:

[0153] Chromatographic column: C18 reverse phase column (Φ50*250), filled with C18 reverse phase silica gel;

[0154] Detector: UV detector, detection wavelength: 210nm;

[0155] Mobile phase: a mixture of methanol and water in a volume ratio of 90:10, with a mobile phase flow rate of 150 mL / min;

[0156] Elution mode: isocratic elution;

[0157] The sample loading amount was 2% of the filler mass.

[0158] The content of EPA-EE in the obtained composition containing eicosapentaenoic acid ethyl ester is 98.3%, the area percentage contents of impurities with relative retention times of 0.613, 0.728, 0.836, 0.858, 0.868, and 0.966 are 0.021%, 0.086%, 0.112%, 0.089%, 0.092%, and 0.093%, respectively, and the area percentage contents of SDA-EE, HPA-EE, DPA-EE, and DHA-EE are 0.085%, 0.221%, 0.089%, and 0.053%, respectively.

[0159] Example 7

[0160] An embodiment of the composition containing eicosapentaenoic acid ethyl ester in the present application is prepared as follows:

[0161] S1, adding 1000 kg of fish oil and 5000 kg of anhydrous ethanol into a reactor, heating to 80°C to dissolve, adding a 20% potassium hydroxide aqueous solution dropwise, reacting for 3 hours after the addition is complete, recovering the unreacted ethanol, washing the remaining product with water, allowing it to stand for stratification, and degassing the supernatant to obtain a fatty acid ethyl ester product;

[0162] S2, subjecting the fatty acid ethyl esterification product to six-stage molecular distillation at temperatures of 100° C., 115° C., 125° C., 135° C., 145° C., and 158° C., to obtain a first fatty acid ethyl ester composition;

[0163] S3, adding 1000 kg of the first fatty acid ethyl ester composition and 1200 kg of urea to 5000 kg of tert-butanol, dissolving at 75 ° C for 0.5 h, then cooling to 4 ° C for crystallization, filtering, adding 2000 kg of isooctane to the filtrate for extraction, and taking the upper isooctane layer for concentration to obtain a second fatty acid ethyl ester composition;

[0164] S4, controlling the vacuum degree at the top of the distillation tower to be below 30 Pa and the kettle temperature to be 170° C. to obtain a third fatty acid ethyl ester composition;

[0165] S5, purifying the third fatty acid ethyl ester composition by preparative chromatography to obtain a composition containing eicosapentaenoic acid ethyl ester;

[0166] The specific conditions of preparative chromatography are as follows:

[0167] Chromatographic column: C18 reverse phase column (Φ50*250), filled with C18 reverse phase silica gel;

[0168] Detector: UV detector, detection wavelength: 210nm;

[0169] Mobile phase: a mixture of methanol and water in a volume ratio of 85:15, with a mobile phase flow rate of 150 mL / min;

[0170] Elution mode: isocratic elution;

[0171] The sample loading amount was 15% of the filler mass.

[0172] The content of EPA-EE in the obtained composition containing eicosapentaenoic acid ethyl ester is 98.9%, wherein the area percentage contents of impurities with relative retention times of 0.613, 0.728, 0.836, 0.858, 0.868, and 0.966 are 0.027%, 0.095%, 0.052%, 0.063%, 0.102%, and 0.054%, respectively; the area percentage contents of SDA-EE, HPA-EE, DPA-EE, and DHA-EE are 0.035%, 0.289%, 0.043%, and 0.089%, respectively.

[0173] From the above test results, it can be seen that the content of eicosapentaenoic acid ethyl ester in the eicosapentaenoic acid ethyl ester composition prepared in the present application can reach more than 96.5%, and the area percentage of impurities with relative retention times of 0.613, 0.728, 0.836, 0.858, 0.868, and 0.966 are all lower than 0.55%, which is suitable as a raw material for preparing food.

[0174] Furthermore, comparing the test results of Examples 2 and 3, it can be found that, during the urea inclusion purification process, using tert-butanol as the solvent for inclusion is more effective than using ethanol as the solvent, and the total amount of unknown impurities in the product can be significantly reduced. Furthermore, comparing the test results of Examples 2-3 and 4-5, it can be found that extracting the filtrate during the urea inclusion process can further increase the content of eicosapentaenoic acid ethyl ester in the product. Comparing the test results of Examples 4 and 5, it can be found that using tert-butanol as the solvent during the urea inclusion process can significantly improve the purity of eicosapentaenoic acid in the product and reduce the content of each unknown impurity. Comparing the test results of Examples 1, 6, and 7 with those of Example 4, it can be found that the type of extractant also significantly affects the content of each component in the product. When the extractant is isooctane, the content of eicosapentaenoic acid ethyl ester in the product can be effectively increased, while the content of unknown impurities can be sharply reduced. The resulting composition is suitable for use in the preparation of food and medicine.

[0175] In addition, extracting the filtrate can effectively increase the content of eicosapentaenoic acid ethyl ester in the composition, and the effect of using isooctane for extraction is better.

[0176] The above are merely preferred embodiments of the present application and are not intended to limit the present application. Persons skilled in the art will readily appreciate that various modifications and variations are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present application shall be included within the scope of protection of the present application.

Claims

1. A method for preparing a composition containing eicosapentaenoic acid ethyl ester, characterized in that, It includes the following steps: Transesterification: Carry out transesterification on fish oil to obtain a fatty acid ethyl esterification product; Multi-stage molecular distillation: Subject the fatty acid ethyl esterification product to multi-stage molecular distillation to obtain a first fatty acid ethyl ester composition, and the multi-stage molecular distillation is at least three-stage molecular distillation; Urea inclusion: Carry out urea inclusion on the first fatty acid ethyl ester composition to obtain a second fatty acid ethyl ester composition; Rectification: Carry out rectification on the second fatty acid ethyl ester composition to obtain a third fatty acid ethyl ester composition; Preparative chromatography: Carry out preparative chromatography purification on the third fatty acid ethyl ester composition to obtain the composition containing eicosapentaenoic acid ethyl ester.

2. The preparation method according to claim 1, characterized in that, The urea inclusion is as follows: Mix the first fatty acid ethyl ester composition with fatty alcohol and urea to obtain a mixture; Heat the mixture to 70 - 80 °C, keep warm for 0.5 - 1 h, then cool down to -2 - 4 °C for crystallization, filter to obtain a filtrate; Wash and degas the filtrate, or extract the filtrate with an extractant, and then concentrate to obtain the second fatty acid ethyl ester composition.

3. The preparation method according to claim 2, wherein, The mass ratio of the first fatty acid ethyl ester composition, the fatty alcohol, and the urea is 1:(4 - 6):(0.8 - 2); and / or The mass ratio of the filtrate to the extractant is 1:(1 - 2.5); and / or The fatty alcohol is ethanol or tert-butanol; and / or The extractant is n-hexane or isooctane.

4. The preparation method according to claim 3, characterized in that, The fatty alcohol is tert-butanol; and / or, the extractant is isooctane.

5. The preparation method according to any one of claims 1 to 4, wherein, The transesterification step includes: Dissolve the fish oil in absolute ethanol, then add a catalyst, react at 75 - 85 °C for 0.5 - 3 h, wash with water, let it stand for layer separation, and carry out degassing treatment on the upper layer liquid to obtain the fatty acid ethyl esterification product; and / or The multi-stage molecular distillation is six-stage molecular distillation, and the temperature of the six-stage molecular distillation is 100 - 160 °C; and / or The rectification conditions are: The top vacuum degree < 30 Pa, and the kettle temperature is 160 - 180 °C; and / or The preparative chromatography conditions are as follows: Chromatographic column: C18 reversed-phase silica gel chromatographic column; Mobile phase: A mixture of methanol and water, wherein the volume ratio of methanol to water is (85 - 95):(5 - 15); The sample loading amount is 2% - 15% of the mass of the packing.

6. The preparation method according to claim 5, wherein, The mass ratio of the fish oil, the absolute ethanol, and the catalyst is 1:(2 - 5):(0.003 - 0.035); and / or The catalyst is one of an aqueous solution of liquid alkali with a liquid alkali mass fraction of 25% - 35%, an aqueous solution of potassium hydroxide with a potassium hydroxide mass fraction of 15% - 25%, and sodium ethanolate; and / or Before washing with water, first concentrate the reaction product.

7. The preparation method according to claim 5, wherein The temperatures of the six-stage molecular distillation are successively 100 - 110 °C, 110 - 120 °C, 120 - 130 °C, 130 - 140 °C, 140 - 150 °C, 150 - 160 °C.

8. A composition containing eicosapentaenoic acid ethyl ester, characterized in that, When detected by the following gas chromatography method, taking the retention time of ethyl eicosapentaenoate as 1 and the sum of the areas of each component peak as 100%, in the said composition, by area percentage, it contains 96.5%-99.5% of ethyl eicosapentaenoate, the first impurity with a relative retention time of 0.613 < 0.07%, the second impurity with a relative retention time of 0.836 < 0.53%, the third impurity with a relative retention time of 0.966 < 0.51%, the content of the fourth impurity with a relative retention time of 0.728 < 0.5%, the content of the fifth impurity with a relative retention time of 0.858 < 0.5%, the content of the sixth impurity with a relative retention time of 0.868 < 0.5%; Gas chromatography detection conditions: Instrument: Agilent6890; Chromatographic column: HP-INNOWAX, 30m×0.25mm×0.25μm; Carrier gas: helium; Injection port temperature: 250°C; Column temperature: starting at 170°C, holding for 2 min, heating to 240°C at 3°C / min, and holding for 7 min; Detector: FID, 270°C; Injection volume: 1 μL; Split ratio: 100:1; Flow rate: 1.8 mL / min; The composition containing ethyl eicosapentaenoate is prepared by the preparation method described in any one of claims 1 to 7.

9. The composition containing eicosapentaenoic acid ethyl ester according to claim 8, characterized in that, In the said composition, the total area percentage of ethyl eicosapentaenoate isomers < 2%.

10. The composition containing eicosapentaenoic acid ethyl ester according to claim 8 or 9, characterized in that, In the said composition, the substances with relative retention times of 0.989, 1.013, 1.017, 1.024, and 1.03 are all ethyl eicosapentaenoate isomers.

11. The composition containing eicosapentaenoic acid ethyl ester according to claim 8 or 9, characterized in that, In the said composition, by area percentage, the content of the first impurity < 0.07%, the content of the second impurity < 0.53%, the content of the third impurity < 0.51%, the content of the fourth impurity < 0.35%, the content of the fifth impurity < 0.3%, the content of the sixth impurity < 0.3%.

12. The composition containing eicosapentaenoic acid ethyl ester according to claim 11, characterized in that, In the said composition, by area percentage, the content of the first impurity < 0.03%, the content of the second impurity < 0.15%, the content of the third impurity < 0.1%, the content of the fourth impurity < 0.1%, the content of the fifth impurity < 0.1%, the content of the sixth impurity < 0.15%.

13. The composition containing eicosapentaenoic acid ethyl ester according to claim 8 or 9, characterized in that, In the said composition, by area percentage, the content of ethyl stearate < 1%, the content of ethyl docosapentaenoate < 0.1%, the content of ethyl docosahexaenoate < 0.1%, the content of ethyl eicosapentaenoate < 0.3%.

14. Use of the composition containing ethyl eicosapentaenoate according to any one of claims 8 to 13 in the preparation of food.

15. Use of the composition containing ethyl eicosapentaenoate according to any one of claims 8 to 13 in the preparation of medicine.

Images