Oral microemulsions with high epa content and methods of making and using the same

Abstract

The present application relates to a kind of oral microemulsion of high EPA content and its preparation method.The oral microemulsion includes 1%~50% EPA glyceride raw material, 0.1%~10% high iodine value phospholipid (including PC) by weight percentage, other emulsifiers 0.01~10%, the first auxiliary material for assisting EPA liver enrichment 0~10%, other food neutralizing and / or pharmaceutically acceptable raw materials 0~40% and appropriate amount of water, wherein, EPA glyceride raw material provides high content EPA, the first auxiliary material provides liver targeting component and / or PEG modified component.The oral microemulsion provided by the present application can realize the synergistic absorption of EPA and phospholipid, further with the help of the first auxiliary material, EPA entering blood circulation can be reduced by lipoprotein lipase hydrolysis, also can improve the bioavailability of EPA while promoting its enrichment in liver, therefore, can be used for the prevention and / or treatment of fatty liver and other diseases.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of oral preparations, in particular to an oral microemulsion with high EPA content and a preparation method and application thereof. BACKGROUND

[0002] Fatty liver refers to a pathological change of excessive accumulation of fat in liver cells caused by various reasons. Fat deposition in liver cells, starting from simple fatty liver, can develop into fatty hepatitis with different degrees of liver fibrosis, eventually leading to cirrhosis, and even evolving into primary hepatocellular carcinoma. In recent years, with the change of people's lifestyle, fatty liver has become a global problem. At present, the incidence rate of fatty liver in China is more than twice that of western countries, and its management and treatment are very important.

[0003] The pathogenesis of fatty liver is very complex, and common causes include long-term heavy drinking or overnutrition, obesity, etc. The medical community believes that the initial development of fatty liver is caused by triglyceride accumulation, and the excess triglyceride in liver cells has multiple sources, including excessive intake of fatty acids in diet, peripheral fat increase caused by insulin resistance (IR) of adipose tissue, and liver neogenesis of fat increase caused by hyperinsulinemia. Fat accumulation makes liver cells vulnerable to various hepatotoxic damage factors, including lipid peroxidation, oxidative stress of the body, mitochondrial dysfunction, etc., eventually leading to inflammatory response, cell damage and tissue fibrosis at the liver site.

[0004] At present, there is no specific drug or preparation approved for long-term treatment of fatty liver. Statins (lipid-lowering drugs), glitazones (insulin sensitizers), antioxidants and metformin, etc. can be used as potential effective drugs for treating fatty liver. However, statins themselves can cause various common types of drug-induced liver damage, which may cause double damage during use. Glitazones can improve steatosis while causing significant weight gain in patients, and their long-term safety is still unknown. Randomized clinical trials of antioxidants (vitamin E and N-acetylcysteine, etc.) cannot determine the improvement of drug on liver histological state, and their effects may vary with age, dose and lifestyle changes. The results of metformin drug treatment of non-alcoholic fatty liver in clinical practice are still not consistent and clear.

[0005] In addition to drug therapy, lifestyle changes are often recommended as the primary clinical recommendation and the first step in the management of fatty liver disease. Regular physical exercise and reducing total fat intake can effectively reduce steatosis. In addition, the composition of the diet also affects metabolism and endocrine function and overall energy balance. It is generally recommended to reduce the intake of saturated fatty acids, trans fatty acids and fructose. On the contrary, increasing the intake of polyunsaturated fatty acids (PUFA) has been shown to reduce the risk of fatty liver, and this class of substances is more acceptable to people and has fewer adverse reactions than drugs, and can be taken for a long time.

[0006] Studies have shown that Omega-3 polyunsaturated fatty acids (Omega-3 PUFA), mainly including eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), alpha-linolenic acid (ALA) and the like, can participate in lipoprotein decomposition, lipid oxidation, lipid metabolism and other processes, and show certain beneficial effects in the treatment of liver disease. Omega-3 PUFA can be synthesized from ALA in the human body, but the endogenous conversion efficiency is low, so it must be regularly ingested from the diet to ensure sufficient supply in the body. Common Omega-3 PUFA-rich oils include algal oil, fish oil, krill oil, seal oil, etc. High Omega-3 PUFA content can be obtained by enzymatic transesterification in industry.

[0007] Regarding the application of Omega-3 polyunsaturated fatty acid preparations in the treatment of liver disease, patent document CN101757497A describes a combination capsule of EPA and traditional Chinese medicine, the oily substance of which is not conducive to human digestion and absorption, patent document CN104856985A describes an EPA ethyl ester soft capsule, which has not yet obtained positive clinical results in the treatment of fatty liver, and patent document CN110312509A describes an EPA / DHA composition for treating or preventing liver disease, but provides a type of injectable fat emulsion. The existing Omega-3 PUFA preparations reported so far generally have the following problems: (1) The existing products are mostly in the form of capsules, which are not conducive to human absorption and cannot reach the required liver active fatty acid concentration for treatment. The digestion and absorption of Omega-3 PUFA in the capsule are affected by food intake, and the bioavailability in the fasting state is less than 50% of that in the fed state, so the method of taking the medicine needs to be strictly controlled. (2) Although the injectable fat emulsion reported in some documents improves the bioavailability by improving the dosage form, it cannot achieve high concentration of active fatty acids in the liver, and the improvement effect on fatty liver is not good.

[0008] Therefore, it is necessary to develop a new preparation that can efficiently enrich active fatty acids in the liver. SUMMARY

[0009] Based on this, one object of the present application is to provide an oral microemulsion capable of efficiently enriching eicosapentaenoic acid (EPA) in the liver, which has a high content of EPA and phospholipids (including phosphatidylcholine, PC) with a high iodine value, controls the appropriate ratio of EPA and PC, and can be synergistically absorbed with EPA and PC, thus helping to absorb EPA orally and enrich it in the liver, and can fully exert the preventive and / or therapeutic effect of EPA on diseases such as fatty liver. Another object of the present application is to provide the use of the above-mentioned oral microemulsion, including but not limited to the use in medical foods, health foods, drugs, and the like.

[0010] The above-mentioned objects of the present application can be achieved by the following technical solutions.

[0011] According to a first aspect of the present application, an oral microemulsion is provided, which has a total weight percentage of 100% and comprises the following components in terms of weight percentage:

[0012]

[0013] In the above-mentioned EPA glyceride raw material, the mass content of EPA is ≥45%, and the mass percentage of triglyceride in the glyceride component is ≥58%;

[0014] The first emulsifier is phospholipid with an iodine value > 70;

[0015] In the phospholipid component of the first emulsifier, the mass percentage of phosphatidylcholine is ≥50%;

[0016] The mass ratio of EPA to phosphatidylcholine is ≤8:1;

[0017] The components of the second emulsifier are all different from those of the first emulsifier;

[0018] The first auxiliary material contains at least one of a targeting component and a PEG-modified component;

[0019] The second auxiliary material is a food and / or pharmaceutically acceptable raw and auxiliary material, and is different from the first emulsifier, the second emulsifier, and the first auxiliary material.

[0020] In some preferred embodiments of the present application, the oral microemulsion comprises the following components in terms of weight percentage:

[0021]

[0022] In some embodiments of the present application, in the oral microemulsion, the minimum weight percentage of water is 65%; and / or,

[0023] The oral microemulsion comprises oil phase components and water phase components, wherein the weight percentage of the oil phase components is ≤30%; and / or,

[0024] The mass content of EPA in the EPA glyceride raw material is ≥ 57%; and / or,

[0025] The mass content of monoglyceride in the glyceride component of the EPA glyceride raw material is less than 30%, and the mass content of diglyceride in the glyceride component is less than 30%; and / or,

[0026] The first emulsifier is a phospholipid with an iodine value ≥ 90; and / or,

[0027] The mass content of phospholipid with an iodine value > 70 in all phospholipid components of the oral emulsion is greater than 90%; and / or,

[0028] The mass content of phosphatidylcholine in the phospholipid component of the first emulsifier is ≥ 70%; and / or,

[0029] The mass ratio of EPA to phosphatidylcholine is ≤ 5:1; and / or,

[0030] The liver targeting component is selected from one or more of a liver targeting molecule and a modified liver targeting molecule containing one or more of a PEG modified lipid unit and a non-PEG modified lipid modification unit; and / or,

[0031] The oral microemulsion is a PEG modified microemulsion, and the mass percentage of the PEG modified raw material in the oral microemulsion is 0.01% to 10%; and / or,

[0032] The PEG modified component is selected from one or more of a polyethylene glycol modified lipid component and a polyethylene glycol monomethyl ether modified lipid component; and / or,

[0033] The oral microemulsion is an oil-in-water structure; and / or,

[0034] The average droplet size in the oral microemulsion is less than 500 nm.

[0035] In some embodiments of the present application, the EPA glyceride raw material is selected from one or more of the following EPA glycerides having the following compositions: EPA 57%~60%+DHA 0~10%, EPA 57%~60%+DHA 10~20%, EPA 57%~60%+DHA 20~30%, EPA 57%~60%+DHA 30%~40%, EPA 60%~70%+DHA 0~10%, EPA 60%~70%+DHA 10~20%, EPA 60%~70%+DHA 20~30%, EPA 60%~70%+DHA 30% above, EPA 70%~80%+DHA 0~10%, EPA 70%~80%+DHA 10~20%, EPA 70%~80%+DHA 20% above, EPA 80%~90%, and EPA >90%; and / or,

[0036] The phospholipid component in the first emulsifier is selected from one or more of the following: soybean phospholipid, sunflower phospholipid, egg yolk phospholipid, and synthetic phospholipid; and / or,

[0037] The second emulsifier is selected from one or more of the following: phospholipid different from the first emulsifier, sucrose ester, citric acid fatty acid glyceride, fatty acid glyceride, polysorbate, fatty acid sorbitan, polyoxyethylene fatty acid ester, span, poloxamer, alginate, and caseinate; and / or,

[0038] The liver targeting component is selected from one or more of the following: glycyrrhetic acid and its derivatives, galactose and its derivatives, mannose and its derivatives, hyaluronic acid and its derivatives, bile acid and its derivatives; and / or,

[0039] The PEG modified component is selected from one or more of the following: distearoyl phosphatidyl ethanolamine-polyethylene glycol, polyethylene glycol-stearate, vitamin E succinate polyethylene glycol ester, soybean phosphatidyl ethanolamine-polyethylene glycol monomethyl ether, polyethylene glycol oleate, polyethylene glycol laurate; and / or,

[0040] The second adjuvant is selected from one or more of the following: nutritional supplements, antioxidants, co-emulsifiers, oils, flavoring agents, and pH adjusting agents; and / or,

[0041] The average droplet size of the oral microemulsion is ≤300 nm; and / or,

[0042] In the oral microemulsion, the weight percentage of water is 65%~95%.

[0043] In some embodiments of the present application, the phospholipid component in the first emulsifier is a polyene phospholipid of soybean origin; and / or,

[0044] the second emulsifier does not contain a phosphatidylcholine with a high iodine value; and / or,

[0045] the liver targeting component contains glycyrrhetinic acid and derivatives thereof selected from the group consisting of liposoluble derivatives of glycyrrhetinic acid; and / or,

[0046] the liver targeting component contains one or more of DSPE-PEG-galactose, DSPE-PEG-mannose, DSPE-glycyrrhetinic acid, DSPE-PEG-glycyrrhetinic acid, and DSPE-hyaluronic acid;

[0047] the PEG-modified component is selected from one or more of PEG-modified phospholipids, PEG-modified vitamin E esters; and / or,

[0048] the nutritional supplement is selected from one or more of vitamin A, vitamin E, vitamin B complex, vitamin D, silymarin, glucomannan, and branched chain amino acids; and / or,

[0049] the oil is selected from one or more of soybean oil, medium-chain triglycerides, olive oil, flaxseed oil, walnut oil, sea buckthorn oil, Job's tears oil, grape seed oil, ginger oil, coconut oil, camellia oil, rose oil, peppermint oil, and lemon oil; and / or,

[0050] the antioxidant is selected from one or more of sodium sulfite, sodium bisulfite, sodium metabisulfite, vitamin C and esters thereof, tocopherol and esters thereof.

[0051] in some embodiments of the present application, the second emulsifier does not contain a phospholipid component; and / or,

[0052] the liposoluble derivative of glycyrrhetinic acid is selected from one or more of DSPE-glycyrrhetinic acid, DSPE-PEG-glycyrrhetinic acid, glycyrrhetinic acid fatty acid ester, and glycyrrhetinic acid succinate; and / or,

[0053] the PEG-modified component is selected from one or more of distearoylphosphatidylethanolamine-polyethylene glycol, vitamin E succinate polyethylene glycol ester; and / or,

[0054] the vitamin E is selected from one or more of a-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol, a-tocotrienol, β-tocotrienol, γ-tocotrienol, and δ-tocotrienol; and / or,

[0055] the vitamin B complex is selected from one or more of vitamin B1, vitamin B2, niacin, pantothenic acid, vitamin B6, vitamin B12, folic acid, and vitamin B7.

[0056] In some embodiments of the present application, the oral microemulsion is a PEG modified microemulsion, and the PEG modified raw material accounts for 0.01% to 10% of the total mass.

[0057] According to a second aspect of the present application, a method for preparing the oral microemulsion of the first aspect of the present application is provided, comprising the following steps:

[0058] Preparation of an oil phase under heating conditions, including oil phase components of the EPA glyceride raw material;

[0059] Preparation of an aqueous phase under heating conditions, including mixing the aqueous phase components including phospholipids with an aqueous solvent;

[0060] Mixing the oil phase and the aqueous phase, and shearing to prepare an oil-in-water microemulsion;

[0061] High pressure homogenization treatment of the oil-in-water microemulsion to obtain a nanoemulsion with a droplet particle size of less than 500 nm;

[0062] Optionally, the nanoemulsion is filtered, packaged, and sterilized.

[0063] According to a third aspect of the present application, the oral microemulsion of the first aspect of the present application, or the oral microemulsion obtained by the preparation method of the second aspect of the present application, is provided.

[0064] In some embodiments of the present application, the oral microemulsion is used in the preparation of a medicine for preventing and / or treating a fat accumulation related disease, or in a medical food or a health food.

[0065] In some preferred embodiments of the present application, the fat accumulation related disease is selected from one or more of fatty liver, liver damage, and hepatitis, and preferably, the fatty liver is non-alcoholic fatty liver.

[0066] The inventors have found through experiments that the EPA active ingredient plays an important role in PUFA, and the therapeutic effect on fatty liver is significantly related to the purity of EPA in fish oil raw materials. Moreover, compared with EPA EE microemulsion, the glyceride type EPA microemulsion combined with other components in the present application can better enrich EPA in the liver. Therefore, the present application provides an oral microemulsion prepared from a glyceride type raw material with high EPA content.

[0067] The oral microemulsion provided by the present application can make up for the blank that there is no specific drug or preparation approved for long-term treatment of fatty liver at present. The oral microemulsion provided by the present application uses EPA glyceride raw material with high EPA purity, thereby providing high content of active EPA, and high iodine value phospholipid (including phosphatidylcholine, PC) provided by phospholipid raw material, wherein the content of EPA in the EPA glyceride raw material is greater than or equal to 45%, the mass content of EPA glyceride in the glyceride component is greater than or equal to 58%, the iodine value of phospholipid in the first emulsifier is greater than 70, and the mass ratio of EPA to PC is less than or equal to 8:1, so that the synergistic absorption of EPA and phospholipid can be achieved, the EPA is promoted to enter the liver, and the phospholipid content in the oral emulsion of the administration dose is much lower than the administration amount of polyene phosphatidylcholine capsules on the market. The polyene phosphatidylcholine in the commercial product is about 1.8-2.3 times of the oral microemulsion of the present application. The first auxiliary material (liver targeting component and / or PEG modified component) is further added in the oral microemulsion provided by the present application, which can promote the enrichment of EPA in the liver through the targeting effect and / or the inhibition of enzymatic hydrolysis. Compared with the first auxiliary material with strong water solubility, the first auxiliary material with strong fat solubility is more easily combined with the EPA microemulsion and the chylomicron formed after entering the body, and can better improve the bioavailability of EPA and promote the enrichment of EPA in the liver. The oral microemulsion provided by the present application can have good effects on reducing body weight, plasma triglyceride level, body insulin resistance, oxidative stress and inflammatory response for 4 to 6 weeks or even longer administration period for diseases related to fat accumulation, especially for non-alcoholic fatty liver disease (NAFLD).

[0068] The oral microemulsion provided by the present application can improve the bioavailability of EPA by about 3 times compared with the EPA soft capsule.

[0069] The present application can provide PEG modified raw materials through the first emulsifier, the second emulsifier and / or the first auxiliary material, so as to obtain PEG modified oral microemulsion, which can reduce the hydrolysis of EPA by lipoprotein lipase when entering the blood circulation, thereby promoting the aggregation of EPA in the liver. Further combined with the liver targeting component, the enrichment of EPA in the liver can be greatly improved, and the bioavailability can also be effectively improved.

[0070] The inventors have found through experiments that the content of phosphatidylcholine and the selection of unsaturation degree of phospholipid have an effect on drug efficacy. In the present application, when the iodine value of phospholipid is greater than or equal to 70 and the mass ratio of EPA to phosphatidylcholine is less than or equal to 8:1, the blood lipid-lowering effect is better, and the treatment effect on fatty liver is better.

[0071] The fish oil oral microemulsion can reduce the ALT and AST levels of NASH induced by high-fat diet, relieve inflammatory reaction, prevent lipid peroxidation, and reduce the serum TG and TC levels, and therefore, the fish oil oral microemulsion can be used as a potential drug for preventing and / or treating fatty liver, especially non-alcoholic fatty liver disease (NAFLD). BRIEF DESCRIPTION OF DRAWINGS

[0072] In order to more clearly illustrate the technical solutions in the embodiments of the present application, more completely understand the present application and its beneficial effects, the drawings needed to be used in the embodiment description will be briefly introduced.

[0073] Figure 1 NAS score results of rats with fatty liver in an embodiment of the present application for different preparations;

[0074] Figure 2 Blood glucose content-time curve of rats after four weeks of administration of different preparations in an embodiment of the present application;

[0075] Figure 3 AUC calculation results of blood glucose content of rats after four weeks of administration of different preparations in an embodiment of the present application;

[0076] Figure 4 NAS score results of rats with fatty liver in an embodiment of the present application for different preparations;

[0077] Figure 5 HE staining results of liver tissue morphology of rats in an embodiment of the present application for different preparations. DETAILED DESCRIPTION

[0078] The present application will be further described in detail below in conjunction with the drawings, embodiments and examples. It should be understood that these embodiments and examples are only used to illustrate the present application and not used to limit the scope of the present application, and the purpose of providing these embodiments and examples is to make the disclosure of the present application more thorough and comprehensive. It should also be understood that the present application can be realized in many different forms and is not limited to the embodiments and examples described herein, and those skilled in the art can make various modifications or changes without departing from the connotation of the present application, and the equivalent forms obtained are also within the protection scope of the present application. In addition, in the following description, a large number of specific details are given in order to provide a more complete understanding of the present application, and it should be understood that the present application can be implemented without one or more of these details.

[0079] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description herein is for the purpose of describing only the embodiments and examples and is not intended to be limiting of the application.

[0080] Terms

[0081] Unless otherwise indicated, or unless the context of use indicates otherwise, terms or phrases used herein have the meanings provided below:

[0082] The term "and / or", "or / and", "and / or" used herein is a selective range including any one of two or more relevant listed items, and also including any and all combinations of the relevant listed items, including any two relevant listed items, any more relevant listed items, or a combination of all relevant listed items. It should be noted that when at least two conjunctions selected from "and / or", "or / and", "and / or" are combined to connect at least three items, it should be understood that in this application, the technical solution undoubtedly includes the technical solution connected by "logical and", and also undoubtedly includes the technical solution connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For another example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C and D (i.e. the technical solution connected by "logical or"), and also includes any and all combinations of A, B, C and D, i.e. includes the combination of any two or any three of A, B, C and D, and also includes the four-item combination of A, B, C and D (i.e. the technical solution connected by "logical and").

[0083] In the present application, "multiple", "plurality" and the like, if not specifically limited, refer to greater than or equal to two in number. For example, "one or more" means one or greater than or equal to two.

[0084] As used herein, "combinations thereof", "any combination thereof", "any combination manner thereof" and the like include all suitable combination manners of any two or more of the listed items.

[0085] As used herein, "suitable", "suitable manner", "any suitable manner" and the like in "suitable combination manner", "suitable manner", "any suitable manner" and the like refer to the ability to implement the technical solutions of the present application, solve the technical problems of the present application, and achieve the intended technical effects of the present application.

[0086] As used herein, "preferably", "more preferably", "more preferably", "preferably" only describe the embodiments or examples with better effects, and it should be understood that it does not constitute a limitation on the scope of protection of the present application.

[0087] In the present application, "further", "still further", "in particular" and the like are used for the purpose of description, indicating differences in content, but should not be understood as limiting the scope of protection of the present application.

[0088] In the present application, "optionally", "optional" and "optional" mean optional, that is, selected from "yes" or "no" two parallel schemes. If there are multiple "options" in a technical solution, unless otherwise specified, and there is no contradiction or mutual restriction, each "option" is independent of each other. In the present application, "optionally contains", "optionally contains" and the like indicate "contains or does not contain". "Optional component X" means that component X exists or does not exist.

[0089] In the present application, in the terms "first", "second", "third", "fourth" and the like in the "first aspect", "second aspect", "third aspect", "fourth aspect" and the like, the terms "first", "second", "third", "fourth" and the like are only used for the purpose of description, and cannot be understood as indicating or implying relative importance or quantity, nor can it be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth" and the like only serve the purpose of non-exhaustive enumeration description, and should be understood as not constituting a closed limitation on the quantity.

[0090] In the present application, the technical features described in an open manner include both the closed technical solution consisting of the listed features and the open technical solution containing the listed features.

[0091] In the present application, with respect to numerical intervals (i.e. numerical ranges), if not otherwise specified, the optional numerical distribution within the above numerical interval is considered to be continuous, and includes the two numerical endpoints (i.e. the minimum value and the maximum value) of the numerical range, as well as every numerical value between the two numerical endpoints. If not otherwise specified, when the numerical interval only points to the integers within the numerical interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In addition, when multiple ranges are provided to describe a feature or characteristic, these ranges can be combined. In other words, unless otherwise indicated, the ranges disclosed herein should be understood to include any and all sub-ranges subsumed therein.

[0092] In the present application, with respect to the approximate number, if not otherwise specified, the fluctuation range is generally ±10%, and can further indicate ±8%, ±5%, ±3% and the like. In the present application, the approximate number provides the listed numerical value, and also provides the numerical interval represented by the approximate number. For example, approximately 200nm provides a technical solution of "200nm", and also provides a technical solution of the numerical interval constituted by "200nm ± fluctuation range".

[0093] The temperature parameters in the present application, if not particularly limited, allow for constant temperature treatment as well as for variations within a certain temperature interval. It is to be understood that the constant temperature treatment allows for fluctuations within the accuracy of the instrument control. Fluctuations within a range of, for example, ±5°C, ±4°C, ±3°C, ±2°C, ±1°C are allowed.

[0094] In the present application, % (w / w) and wt% both mean weight percentage.

[0095] All the documents mentioned in the present application are incorporated by reference in the present application as if each document was individually incorporated by reference. Unless and to the extent that the incorporated documents conflict with the subject matter and / or technical solutions of the present application, the incorporated documents are incorporated by reference in their entirety and for all purposes. When the present application refers to the incorporated documents, the definitions of the relevant technical features, terms, names, phrases, etc. in the incorporated documents are also incorporated by reference. When the present application refers to the incorporated documents, the examples and preferred modes of the relevant technical features that are incorporated by reference can also be incorporated by reference in the present application, but are limited by the implementability of the present application. It should be understood that when the incorporated content conflicts with the description in the present application, the present application is the priority or is amended according to the description in the present application.

[0096] The abbreviations in the present application, if not particularly limited, have the following meanings: PUFA means polyunsaturated fatty acid, Omega-3 PUFA means Omega-3 polyunsaturated fatty acid, EPA means eicosapentaenoic acid, DHA means docosahexaenoic acid, ALA means alpha-linolenic acid, DSPE means distearoylphosphatidylethanolamine, PEG means polyethylene glycol, TG means triglyceride, TPGS means vitamin E succinate polyethylene glycol ester, DSPE means distearoylphosphatidylethanolamine, PC means phosphatidylcholine, ALT means glutamic-pyruvic transaminase, AST means glutamic-oxaloacetic transaminase, SOD means superoxide dismutase, MDA means malondialdehyde, and NAFLD means non-alcoholic fatty liver disease.

[0097] In the present application, "EPA" is used alone, if not particularly limited, to mean an EPA molecule or an EPA unit in a certain molecule. For example, the mass content of EPA (EPA unit) in an EPA glyceride raw material, the mass ratio of EPA (PEA unit) to phosphatidylcholine in the first emulsifier.

[0098] In the present application, the unsaturation degree of phospholipids is mainly characterized by iodine value, if not particularly limited, which means average iodine value.

[0099] In the present application, "high-iodine-value phospholipids", if not particularly limited, are phospholipids with an iodine value > 70, and can be phospholipids with a higher iodine value, such as phospholipids with an iodine value ≥ 90.

[0100] The "enzymatic hydrolysis" and "hydrolysis" referred to in the present application, if not otherwise specified, refer to the hydrolysis of oil by pancreatic lipase after EPA enters the gastrointestinal tract and recombination, and the hydrolysis by lipoprotein lipase after entering the blood.

[0101] The "medium-chain triglyceride" used in the present application, also known as "medium-chain glyceride", is the full name of medium-chain triglyceride in English, and is abbreviated as MCT. In the national food safety standard, medium-chain triglyceride can be used as a food raw material or emulsifier.

[0102] The "branched-chain amino acid" used in the present application is selected from three amino acids with a branched side chain; the branched-chain amino acid used in the present application can be combined with a plurality of amino acids, which can promote the metabolism of the body, for example, selected from leucine, valine and isoleucine.

[0103] In the present application, polyethylene glycol (PEG) and polyoxyethylene (POE) have the same meaning and can be used interchangeably. The molecular weight related to PEG, if not otherwise specified, refers to the average molecular weight, which can be number average molecular weight or weight average molecular weight, if not otherwise specified, refers to the weight average molecular weight.

[0104] In the present application, "above" and "below" each independently contain the number.

[0105] In the present application, "≥" and "greater than or equal to" have the same meaning and can be used interchangeably, both indicating greater than or equal to. "≤" and "less than or equal to" have the same meaning and can be used interchangeably, both indicating less than or equal to.

[0106] First aspect of the invention

[0107] According to the first aspect of the present application, an oral microemulsion capable of efficiently enriching eicosapentaenoic acid (EPA) in the liver is provided, which has a high content of EPA (such as the content of EPA in EPA glyceride raw material ≥45%) and a high iodine value of phospholipid (such as iodine value >70), controls the appropriate ratio of EPA and PC (such as ≤8:1), can synergistically absorb EPA and PC, helps the oral absorption of EPA and the enrichment in the liver, and can fully exert the preventive and / or therapeutic effect of EPA on fatty liver and other diseases.

[0108] In some embodiments of the present application, an oral microemulsion is provided, the total weight percentage of the oral microemulsion is 100%, and the oral microemulsion comprises the following components:

[0109]

[0110] The mass content of EPA in the EPA glyceride raw material is ≥45%, and the mass proportion of triglyceride in the glyceride component is ≥58%;

[0111] The first emulsifier is a phospholipid with an iodine value > 70;

[0112] In the phospholipid component of the first emulsifier, the mass proportion of phosphatidylcholine is ≥50%;

[0113] The mass ratio of EPA to phosphatidylcholine is ≤8:1;

[0114] The components of the second emulsifier are different from those of the first emulsifier;

[0115] The first adjuvant includes at least one of a liver targeting component and a PEG modified component;

[0116] The second adjuvant is a food and / or pharmaceutically acceptable adjuvant, and is different from the first emulsifier, the second emulsifier, and the first adjuvant.

[0117] The first adjuvant and the second adjuvant can be optional components.

[0118] Preferably, the average droplet size in the oral microemulsion is less than 500 nm.

[0119] In some embodiments of the present application, the specific components of the oral microemulsion include: EPA glyceride raw material, phospholipid, other emulsifiers (non-phospholipid), first adjuvant (adjuvant for EPA enrichment in liver), water, and second adjuvant (other food and / or pharmaceutically acceptable adjuvant).

[0120] In some embodiments, the sum of the weight percentages of the EPA glyceride raw material, the first emulsifier, the second emulsifier, the first adjuvant, the second adjuvant, and water is not more than 100%, and preferably one of them is 100%.

[0121] In some embodiments of the present application, the oral microemulsion contains 1% to 50% EPA glyceride raw material, 0.1% to 10% high-iodine-value phospholipid (including PC), 0.01 to 10% other emulsifiers (denoted as second emulsifiers), 0 to 10% first adjuvant for EPA liver enrichment, 0 to 40% other food and / or pharmaceutically acceptable adjuvant, and an appropriate amount of water, in terms of weight percentage, wherein the EPA glyceride raw material provides high content of EPA, and the first adjuvant provides a liver targeting component and / or a PEG modified component.

[0122] The oral microemulsion provided by the present application uses EPA glyceride raw material with high EPA purity, thereby providing high content of active EPA, and high iodine value phospholipid (including phosphatidylcholine, PC) is provided by phospholipid raw material, and the mass ratio of EPA to PC is controlled to be a suitable ratio, so that the synergistic absorption of EPA and phospholipid can be realized, and the first auxiliary material (liver targeting component and / or PEG modified component) is further added, so that the enrichment of EPA in the liver can be promoted through the targeting effect and / or the inhibition of enzymatic hydrolysis, thereby improving the bioavailability of EPA and promoting the enrichment of EPA in the liver. The oral microemulsion provided by the present application can achieve good effects on body weight and liver index for a long period of 4 to 6 weeks or even longer for fat accumulation related diseases, especially for non-alcoholic fatty liver disease (NAFLD).

[0123] The inventors found through experiments that among various components of PUFA, EPA is a key active fatty acid for treating diseases such as fatty liver, and the therapeutic effect of treating fatty liver is more related to the purity of EPA in fish oil raw material. Therefore, the present application uses Omega-3 polyunsaturated fatty acid raw material with high EPA content. Further, the inventors also found that compared with EPA EE microemulsion, the glyceride type EPA microemulsion combined with other components in the present application can make the enrichment of EPA in the liver better. Therefore, the inventors selected EPA glyceride type raw material as the active fatty acid component after a large number of experimental investigations. Further, even at high concentrations, the preparation prepared by high-concentration EPA glyceride in the present application is more likely to reach the liver site than the preparation prepared by common high-concentration EPA ethyl ester.

[0124] The preparation provided by the present application is a microemulsion preparation, which can be suitable for oral administration, and is preferably an oil-in-water structure. In the present application, EPA exists in the form of microemulsion, and the solubility in water is greatly improved. The droplets with an average particle size of less than 500 nm help the small intestine to digest and absorb the poorly soluble component EPA, improve the bioavailability, and help EPA to reach the liver site. In addition, the present application found that the microemulsion preparation helps to reduce the difference in bioavailability of EPA under the condition of empty and full stomach. In some embodiments of the present application, the average particle size of the droplets in the oral microemulsion is less than 500 nm, further, the average particle size can be ≤300 nm, further, it can also be ≤250 nm, further, it can also be about 150 nm. In some specific embodiments of the present application, the average particle size of the droplets in the oral microemulsion is about 150 nm to 300 nm.

[0125] EPA glyceride feedstock

[0126] The oral microemulsion provided by the present application contains an EPA glyceride raw material. In some embodiments of the present application, the content of the EPA glyceride raw material in the oral microemulsion is 1% to 30% by weight, and can further be 4% to 20%, and specific examples include 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, and the like.

[0127] The main component of the EPA glyceride raw material used in the present application is polyunsaturated fatty acid ester, which includes a large amount of EPA ester, and can provide a high content of EPA component. In the present application, the mass content of EPA in the EPA glyceride raw material is preferably ≥45%, further preferably ≥50%, and more preferably ≥57%. If the content of EPA in the EPA glyceride raw material is low (such as <20%), it cannot achieve sufficient active substance exposure after taking, and the high content of ineffective fatty acids in the product will increase the burden of fat metabolism in patients. In the present application, the use of high-content EPA glyceride raw material can reduce the intake of ineffective fatty acids and prevent the burden of fat metabolism in the body. While effectively reducing the synthesis of glycerides in the liver, it can reduce the adverse effects on other indicators of the body. In some embodiments of the present application, the mass content of EPA in the EPA glyceride raw material is ≥60%. In some embodiments of the present application, the mass content of EPA in the EPA glyceride raw material is 60% to 70%. Examples of the mass content of EPA in the EPA glyceride raw material include 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, and the like.

[0128] The EPA in the EPA glyceride raw material of the present application mainly exists in the form of EPA glyceride, which can provide a large amount of active EPA. The glyceride component in the EPA glyceride raw material can be any one of monoglyceride, diglyceride, and triglyceride, or any combination thereof. Preferably, the mass content of triglyceride in the glyceride component is ≥58%, and further preferably, the mass content of monoglyceride in the glyceride component is less than 30%, and the mass content of diglyceride in the glyceride component is less than 30%. In some preferred embodiments, the mass content of triglyceride in the glyceride component is ≥70%. In some preferred embodiments, the mass content of triglyceride in the glyceride component is 70%, 71%, 72%, 73%, 74%, 75%, 78%, 79%, 80%, 82%, 85%, 88%, 90%, 92%, 95%, and the like.

[0129] In the present application, the EPA glyceride starting material can be synthesized by hydrolyzing natural EPA triglyceride to release free fatty acids, purifying and then re-attaching EPA to the glycerol backbone. The resulting EPA glyceride can be any one of EPA monoglyceride, EPA diglyceride, EPA triglyceride, or any combination thereof.

[0130] In some embodiments of the present application, the EPA glyceride starting material is from concentrated or re-esterified EPA glyceride.

[0131] In some preferred embodiments of the present application, the EPA glyceride starting material is selected from one or more of the following EPA glycerides having the following compositions: EPA 57%~60% + DHA 0~10%, EPA 57%~60% + DHA 10~20%, EPA 57%~60% + DHA 20~30%, EPA 57%~60% + DHA 30%~40%, EPA 60%~70% + DHA 0~10%, EPA 60%~70% + DHA 10~20%, EPA 60%~70% + DHA 20~30%, EPA 60%~70% + DHA 30% or more, EPA 70%~80% + DHA 0~10%, EPA 70%~80% + DHA 10~20%, EPA 70%~80% + DHA 20% or more, EPA 80%~90%, and EPA >90%. Specific examples include Epax 6015 TGN, KinOmega 7010 TG, KinOmega 6020 TG, KinOmega 6015 TG, KinOmega E80 TG, Incromega TG6015, KinOmega E95 TG. TM TG6015, KinOmega E95 TG.

[0132] First emulsifier (high iodine value phospholipid, iodine value > 70)

[0133] In the present application, "high iodine value phospholipid" refers to a phospholipid having an iodine value >70. The first emulsifier can be a mixture of two or more phospholipids, in which case the iodine value of any one of the phospholipid components satisfies >70.

[0134] The oral microemulsion provided by the present application contains phospholipid with high iodine value (denoted as the first emulsifier, iodine value > 70) for emulsification of the preparation. Among them, the phospholipid in the form of phosphatidylcholine (PC) has better improvement effect on fatty liver, which is presumably because the chemical structure of phosphatidylcholine is consistent with that of important endogenous phospholipid. In some embodiments of the present application, the content of phospholipid with high iodine value in the oral microemulsion is 0.1% to 10% by weight, and further can be 0.5% to 5%, and specific examples are 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.2%, 2.4%, 2.5%, 2.6%, 2.8%, 3%, 3.5%, 4%, 4.2%, 4.5%, 4.6%, 4.8%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, etc.

[0135] The first emulsifier of the present application is phospholipid with high iodine value. In some embodiments, the first emulsifier is phospholipid with iodine value ≥ 90.

[0136] In some embodiments of the present application, the mass fraction of phosphatidylcholine in the phospholipid component of the first emulsifier is ≥ 50%, further can be ≥ 60%, and further can be ≥ 70%. Examples are 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, etc. The inventors have also found that the unsaturation degree of phospholipid also affects the efficacy, and phospholipid with higher unsaturation degree is better for the treatment of fatty liver. The iodine value can be used to represent the unsaturation degree of phospholipid, and the higher the iodine value, the higher the unsaturation degree. When the iodine value is greater than 70, the treatment effect on fatty liver is better. In some embodiments, the iodine value of the first emulsifier is > 70, further can be ≥ 90, and further can be ≥ 100. In some embodiments of the present application, the iodine value of the first emulsifier is 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 92, 94, 95, 96, 98, 99, 100, 101, 102, 103, etc. In some embodiments of the present application, the mass fraction of phosphatidylcholine (PC) in the phospholipid component of the first emulsifier is ≥ 50%, and the iodine value of the first emulsifier is > 70, at which the treatment effect on fatty liver is better. In some preferred embodiments of the present application, the first emulsifier is polyenylphosphatidylcholine.

[0137] The inventors have also found that the mass ratio of EPA and PC also affects the efficacy, and a relatively low ratio is more beneficial for treating fatty liver. In some embodiments of the present application, the mass ratio of EPA to phosphatidylcholine is ≤8:1, more preferably, the mass ratio of EPA to phosphatidylcholine is ≤5:1. In some embodiments of the present application, the mass ratio of EPA to phosphatidylcholine is 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 5.5:1, etc.

[0138] In some embodiments of the present application, the iodine value of the first emulsifier (high-iodine-value phospholipid) is ≥80, and the mass ratio of EPA to phosphatidylcholine is ≤8:1, at which point, there is a good lipid-lowering effect and a good fatty liver treatment effect. Further, the mass fraction of phosphatidylcholine in the phospholipid component of the first emulsifier is ≥50%, and more preferably ≥60%, and more preferably ≥70%.

[0139] In some embodiments of the present application, the high-iodine-value phospholipid (first emulsifier) used is derived from soybeans, egg yolk, or synthetic phospholipids. In some embodiments, the phospholipid component of the first emulsifier is derived from one or more of soybean phospholipids, sunflower phospholipids, egg yolk phospholipids, and synthetic phospholipids.

[0140] In some preferred embodiments of the present application, the high-iodine-value phospholipid (first emulsifier) used is polyene phosphatidylcholine derived from soybeans.

[0141] In some embodiments of the present application, the phospholipid component of the first emulsifier can be a modified phospholipid, such as a phospholipid modified with a hydrophilic component such as PEG. In some embodiments of the present application, the PEG-modified phospholipid is selected from one or more of DSPE-PEG-galactose, DSPE-PEG-mannose, DSPE-PEG, etc.

[0142] Any of the phospholipid components in the present application can be an independent phospholipid molecule, or a derivative or modified phospholipid of a phospholipid molecule.

[0143] The phospholipid component in the oral microemulsion is not limited to being provided by the first emulsifier, but can also be provided by the second emulsifier. However, the phospholipid component provided by the second emulsifier is not a phospholipid with an iodine value >70. The phospholipid component in the second emulsifier can also be a modified phospholipid as described above.

[0144] The first emulsifier in the oral microemulsion can also simultaneously perform other functions, such as also serving as a first auxiliary material, such as a high-iodine-value phospholipid modified liver targeting molecule, or a PEG-modified high-iodine-value phospholipid. Specific examples include DSPE-glycyrrhizic acid, DSPE-hyaluronic acid, DSPE-PEG-galactose, DSPE-PEG-mannose, DSPE-PEG-glycyrrhizic acid, etc.

[0145] In some embodiments, the mass percentage of phospholipids with iodine value > 70 is greater than 90% of all phospholipid components in the oral emulsion.

[0146] In some embodiments, the mass percentage of phospholipids with iodine value > 90 is greater than 90% of all phospholipid components in the oral emulsion.

[0147] Second emulsifier

[0148] The second emulsifier in the present application does not contain the same component as the first emulsifier. That is, the second emulsifier is composed of a component different from the first emulsifier.

[0149] In some embodiments of the present application, the emulsifier component in the oral microemulsion can include other emulsifiers (denoted as the second emulsifier) in addition to the first emulsifier (high-iodine-value phospholipid), which plays a role in flexible control of emulsification. In some embodiments of the present application, the second emulsifier is selected from one or more of other phospholipids (different from the phospholipid in the first emulsifier), sucrose esters, citric acid fatty acid glycerides, fatty acid glycerides, polysorbates, fatty acid sorbitans, polyoxyethylene fatty acid esters, spans, poloxamers, alginates (independently preferably sodium alginate), caseinates (independently can be sodium caseinate), etc. In some embodiments of the present application, the content of the second emulsifier in the oral microemulsion is 0.01% to 10% by weight, and further can be 0.1% to 5%, and specific examples are 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.2%, 2.4%, 2.5%, 2.6%, 2.8%, 3%, 3.5%, 4%, 4.2%, 4.5%, 4.6%, 4.8%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, etc.

[0150] In some embodiments, the second emulsifier is an emulsifier that does not contain high-iodine-value phospholipids.

[0151] In some embodiments, the second emulsifier does not contain high-iodine-value phosphatidylcholine.

[0152] In some embodiments, the second emulsifier contains phospholipid components, but these phospholipid components are different from the first emulsifier, that is, they are not high-iodine-value phospholipids.

[0153] In some embodiments, the second emulsifier contains phospholipid components, but all are saturated phospholipids.

[0154] In some embodiments, the second emulsifier does not contain a phosphatidylcholine component.

[0155] In some embodiments, the second emulsifier does not contain a phospholipid component.

[0156] First adjuvant

[0157] The present application optionally comprises a first adjuvant. The first adjuvant is used to provide an adjuvant for assisting the enrichment of EPA in the liver. The inventors have found that increasing the enrichment of EPA in the liver is beneficial for the full exertion of the preventive and therapeutic effects of EPA on fatty liver and other diseases. EPA is transported into the body by lymph with phospholipids, cholesterol and apolipoprotein to form chylomicrons. The addition of an adjuvant with liver targeting effect can form chylomicrons with EPA and phospholipids during the absorption process, and selectively transport EPA to the liver. An adjuvant that reduces enzymatic action can indirectly achieve liver enrichment of EPA by reducing the uptake of EPA by other tissues during the transport process.

[0158] In some embodiments of the present application, the content of the first adjuvant in the oral microemulsion is 0-10% by weight, and can further be 0.05%-5%. Specific examples include 0%, 0.01%, 0.02%, 0.05%, 0.06%, 0.08%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.2%, 2.4%, 2.5%, 2.6%, 2.8%, 3%, 3.5%, 4%, 4.2%, 4.5%, 4.6%, 4.8%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, etc.

[0159] In some embodiments of the present application, the first adjuvant is at least one of an adjuvant with a liver targeting effect, an adjuvant that reduces enzymatic action, etc. In some embodiments of the present application, the first adjuvant is at least one of a liver targeting component, a PEG modified component (e.g. a PEG modified lipid component).

[0160] The auxiliary material with liver targeting effect comprises a liver targeting component. The liver targeting component can target the cell surface at the lesion of fatty liver and other diseases, bind to the corresponding receptors on the liver cell membrane, etc. The liver targeting component can be an independent molecule (such as glycyrrhetic acid, bile acid, etc.), or a part of a certain molecule (such as DSPE-glycyrrhetic acid, etc.). In some embodiments, the liver targeting component is selected from one or more of a liver targeting molecule and a modified liver targeting molecule, and further, the modified liver targeting molecule can contain one or more of a PEG modified unit and a lipid modified unit (including but not limited to a phospholipid modified unit). In some embodiments of the present application, the liver targeting component is selected from one or more of glycyrrhetic acid and its derivatives, galactose and its derivatives, mannose and its derivatives, hyaluronic acid and its derivatives, bile acid and its derivatives, etc. In some embodiments of the present application, the liver targeting component contains one or more of DSPE-PEG-galactose, DSPE-PEG-mannose, DSPE-glycyrrhetic acid, DSPE-PEG-glycyrrhetic acid, DSPE-hyaluronic acid, etc. In some embodiments of the present application, the derivative of glycyrrhetic acid, the derivative of bile acid, and the derivative of hyaluronic acid are each independently preferably a corresponding phospholipid derivative, and further can be a phosphatidylethanolamine derivative. The derivative of glycyrrhetic acid is exemplified by DSPE-glycyrrhetic acid, glycyrrhetic acid succinate, etc. The derivative of hyaluronic acid is exemplified by DSPE-hyaluronic acid, etc. In some embodiments of the present application, the liver targeting component contains glycyrrhetic acid and its derivatives, and further, the glycyrrhetic acid and its derivatives are selected from lipid-soluble derivatives of glycyrrhetic acid. In some embodiments of the present application, the liver targeting component is selected from glycyrrhetic acid and its derivatives, and further, the glycyrrhetic acid and its derivatives are selected from one or more of glycyrrhetic acid, DSPE-glycyrrhetic acid, DSPE-PEG-glycyrrhetic acid, glycyrrhetic acid succinate, etc.

[0161] In some embodiments, the liver targeting component is selected from one or more of a liver targeting molecule and a modified liver targeting molecule, and further, the modified liver targeting molecule contains one or more of a PEG modified lipid unit and a lipid modified unit without PEG modification; and further, the lipid modified unit without PEG modification is exemplified by a general lipid modified unit, and further, such as a fatty acid ester modified unit.

[0162] In some embodiments of the present application, the PEG-modified component is selected from one or more of the following: DSPE-PEG-galactose, DSPE-PEG-mannose, DSPE-PEG, PEG-stearate, and the like.

[0163] In some embodiments of the present application, the PEG-modified component is selected from one or more of the following: DSPE-PEG-galactose, DSPE-PEG-mannose, DSPE-PEG, PEG-stearate, and the like.

[0164] In some embodiments of the present application, the PEG-modified component is selected from one or more of the following: DSPE-PEG-galactose, DSPE-PEG-mannose, DSPE-PEG, PEG-stearate, and the like.

[0165] In some embodiments of the present application, the PEG-modified component is selected from one or more of the following: DSPE-PEG-galactose, DSPE-PEG-mannose, DSPE-PEG, PEG-stearate, and the like.

[0166] In some embodiments of the present application, the PEG-modified component is selected from one or more of the following: DSPE-PEG-galactose, DSPE-PEG-mannose, DSPE-PEG, PEG-stearate, and the like.

[0167] In some embodiments of the present application, the PEG-modified component is selected from one or more of the following: DSPE-PEG-galactose, DSPE-PEG-mannose, DSPE-PEG, PEG-stearate, and the like.

[0168] It should be understood that a component of the present application can not only perform one function, for example, the PEG-modified component in an oral microemulsion (for example, vitamin E polyethylene glycol succinate, PEG stearate, DSPE-PEG) can also serve as an emulsifier (first emulsifier or second emulsifier) and a first adjuvant. For example, a phospholipid modified with PEG and a liver targeting molecule (for example, DSPE-PEG-galactose, DSPE-PEG-galactose) can serve as a liver targeting adjuvant and a component that reduces enzymatic degradation.

[0169] The PEG component in the oral microemulsion is not limited to being provided by the PEG-modified component in the first adjuvant, but can also be provided by the first emulsifier, the second emulsifier, the liver targeting component, etc. For example, the PEG-modified high iodine value phospholipid (iodine value > 70) can simultaneously serve as the first emulsifier and the PEG-modified component. For example, the PEG-modified common phospholipid (non-iodine value phospholipid) can simultaneously serve as the second emulsifier and the PEG-modified component. For example, the PEG-modified liver targeting molecule can simultaneously serve as the liver targeting component and the PEG-modified component. For example, the phospholipid-PEG-liver targeting molecule can simultaneously serve as the emulsifier, the PEG-modified component and the liver targeting component. In some embodiments, the oral microemulsion comprises a phospholipid-PEG-liver targeting molecule, wherein the phospholipid unit can or can not be a high iodine value phospholipid.

[0170] When the PEG unit provides a terminal group in the components of the oral microemulsion, the terminal group of the PEG unit can be OH or monomethyl ether.

[0171] In some embodiments, the oral microemulsion is a PEG-modified microemulsion, and further, the PEG-modified raw material can be present in the oral microemulsion in a weight percentage of 0.01% to 10%, for example, 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, etc.

[0172] Any PEG-modified component in the first adjuvant that plays a role in EPA enrichment, wherein the molecular weight of the PEG unit is each independently preferably 500 Da to 5000 Da. For example, 1000 Da, 2000 Da, etc.

[0173] Second adjuvant

[0174] The second adjuvant in the present application is different from the first emulsifier, the second emulsifier and the first adjuvant.

[0175] The second adjuvant is optionally included in the present application. The second adjuvant can be selected from one or more of a nutritional supplement, an antioxidant, an emulsifying aid, an oil, a flavoring agent, a pH adjusting agent, etc.

[0176] In some embodiments of the present application, the second adjuvant is present in the oral microemulsion in an amount of 0-40% by weight, and further can be 1-20%. Specific examples include 0%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, and the like.

[0177] In some embodiments, the nutritional supplement can be selected from one or more of vitamin A, vitamin E (such as alpha-tocopherol, beta-tocopherol, gamma-tocopherol, delta-tocopherol, alpha-tocotrienol, beta-tocotrienol, gamma-tocotrienol, delta-tocotrienol, and the like), vitamin B complex (such as vitamin B1, vitamin B2, niacin, pantothenic acid, vitamin B6, vitamin B12, folic acid, vitamin B7, and the like), vitamin D, silymarin, glucomannan, branched chain amino acids, and the like.

[0178] In some embodiments, the antioxidant can be selected from one or more of sodium sulfite, sodium bisulfite, sodium metabisulfite, vitamin C and its esters, tocopherol and its esters, and the like.

[0179] In some embodiments, the co-emulsifier can be selected from one or more of glycerol, propylene glycol, mannitol, oleic acid, sodium oleate, cholesterol, and the like.

[0180] In some embodiments, the oil can be selected from one or more of soybean oil, medium-chain triglycerides, olive oil, flaxseed oil, walnut oil, sea buckthorn oil, Job's tears oil, grape seed oil, ginger oil, coconut oil, camellia oil, rose oil, peppermint oil, lemon oil, and the like.

[0181] In some embodiments, the flavoring agent can be selected from one or more of fruit flavoring essence, erythritol, natural plant flavoring, sweetener, and the like.

[0182] In some embodiments, the pH regulator can be selected from one or more of various buffer salt systems (such as citric acid-sodium citrate, acetic acid-sodium acetate, phosphate agents, and the like), bases (such as NaOH and the like), acids (such as HC1 and the like), and the like. The pH regulator is mainly used to adjust the pH environment of the water phase when preparing the oral microemulsion.

[0183] In the present application, water in the oral microemulsion as a solvent can be purified water, distilled water, etc., as long as it is suitable for the preparation of oral preparations. The amount of water in the oral microemulsion is an appropriate amount of water, as long as it can produce the oral microemulsion with the particle size required by the present application. The appropriate amount of water makes the oral microemulsion have a suitable ratio of oil phase and water phase, and can form an oil-in-water structure. The oral microemulsion provided by the present application contains oil phase components and water phase components. In some embodiments, the weight percentage of the oil phase components is ≤30%. In some embodiments of the present application, the minimum weight percentage of water in the oral microemulsion is 65%, but the total weight percentage of each component does not exceed 100%. In some embodiments of the present application, the weight percentage of water in the oral emulsion is 65%-95%, preferably 70%-94%, for example 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%.

[0184] Some embodiments of the oral microemulsion

[0185] In some preferred embodiments of the present application, the oral microemulsion contains the following components by weight percentage:

[0186]

[0187] The oral microemulsion provided by the present application has an oil-in-water structure, in which the oil phase components such as EPA glyceride raw materials form the oil phase, and the water phase components such as emulsifiers form the water phase.

[0188] The following are some embodiments with 1g as 1 mass part. It should be understood that other mass values can also be used as 1 mass part.

[0189] In some embodiments of the present application, the oral microemulsion is a PEG-modified microemulsion, which can further contain PEG-modified raw materials with a total mass percentage of 0.01%-10%, and further can be 0.05%-5%, for example 0.01%, 0.02%, 0.04%, 0.05%, 0.08%, 0.1%, 0.2%, 0.4%, 0.5%, 0.8%, 1.0%, 1.2%, 1.4%, 1.5%, 1.8%, 2%, 2.2%, 2.5%, 2.8%, 3.0%, 3.3%, 3.5%, 3.8%, 4.0%, 4.2%, 4.5%, 4.8%, 5.0%. The PEG-modified components can be provided by one or more of the first emulsifier (such as PEG-modified phospholipids), the second emulsifier (such as polyoxyethylene fatty acid ester), and the first excipient (such as PEG-modified components therein).

[0190] In some embodiments of the present application, the oral microemulsion comprises fish oil (providing EPA glyceride raw material), high-iodine-value phospholipid (e.g., soybean phospholipid, etc.), sucrose ester, antioxidant (e.g., alpha-tocopherol), nutritional supplement (optional), NaOH and water, further, the oral microemulsion comprises 10 g to 400 g fish oil, 1 g to 60 g high-iodine-value phospholipid, 0.1 g to 6 g sucrose ester, 0.5 g to 6 g antioxidant, 0 to 6 g nutritional supplement, NaOH and water. The type, model / specification, amount of each component can further refer to the part of Example 1.1.

[0191] In some embodiments of the present application, the oral microemulsion comprises fish oil (providing EPA glyceride raw material), high-iodine-value phospholipid (e.g., egg yolk phospholipid, soybean phospholipid, polyenyl phosphatidylcholine, etc.), tocopherol, NaOH and water, further, the oral microemulsion comprises 10 g to 200 g fish oil (further, 20 g to 100 g), 3 g to 25 g high-iodine-value phospholipid (further, 4 g to 20 g), 0.5 g to 5 g tocopherol (further, 0.6 g to 3 g), NaOH and water. The type, model / specification, amount of each component can further refer to the part of Example 1.2.

[0192] In some embodiments of the present application, the oral microemulsion comprises fish oil (providing EPA glyceride raw material), high-iodine-value phospholipid (e.g., soybean phospholipid, etc.), bile acid, glycyrrhizic acid, tocopherol, NaOH and water, further, the oral microemulsion comprises 10 g to 200 g fish oil (further, 50 g to 100 g), 1 g to 35 g high-iodine-value phospholipid (further, 2 g to 30 g), 0 to 15 g bile acid (further, 0.1 g to 15 g, further, 0.5 g to 10 g), 0 to 6 g glycyrrhizic acid (further, 1 g to 6 g, further, 1.2 g to 5 g), 0 to 6 g tocopherol (further, 0 to 5 g, further, 0.5 g to 5 g), 0.1 to 6 g nutritional supplement (further, 0.5 to 5 g, further, 1 g to 5 g) NaOH and water. The type, model / specification, amount of each component can further refer to the part of Example 1.3.

[0193] In some embodiments of the present application, the oral microemulsion comprises fish oil (providing the EPA glyceride raw material), high-iodine-value phospholipid (e.g., soybean phospholipid, etc.), DSPE-PEG, PEG-stearate, tocopherol, NaOH and water, further, the oral microemulsion comprises 5g-200g fish oil (further, 10g-100g), 1g-35g high-iodine-value phospholipid (further, 2g-30g), 0-8g DSPE-PEG (further, 0.5g-10g, and further, 0.5g-6g), 0-8g PEG-stearate (further, 0.5g-8g, and further, 0.5g-6g), 0.1g-6g tocopherol (further, 0.1g-5g, and further, 0.5g-5g), NaOH and water. The type, model, amount of each component can further refer to the part of Example 1.4.

[0194] In some embodiments of the present application, the oral microemulsion comprises fish oil (providing the EPA glyceride raw material), high-iodine-value phospholipid (e.g., soybean phospholipid, etc.), glycyrrhetinic acid succinate, DSPE-PEG, DSPE-PEG-galactose, tocopherol, NaOH and water, further, the oral microemulsion comprises 5g-200g fish oil (further, 10g-100g), 1g-35g high-iodine-value phospholipid (further, 2g-30g), 0-12g glycyrrhetinic acid succinate (further, 0-10g, and further, 1g-10g), 0-8g DSPE-PEG (further, 0.5g-8g, and further, 0.5g-6g), DSPE-PEG-galactose, 0.1g-6g tocopherol (further, 0.1g-5g, and further, 0.5g-5g), NaOH and water. The type, model, amount of each component can further refer to the part of Example 1.5.

[0195] Second aspect of the invention

[0196] According to a second aspect of the present application, a method for preparing an oral microemulsion is provided, which can be used to prepare the oral microemulsion of the first aspect of the present application.

[0197] In some embodiments of the present application, the method for preparing the oral microemulsion comprises the following steps:

[0198] S100, preparing an oil phase (preferably under inert gas protection): preparing an oil phase under heating condition (preferably 50-70°C) by mixing the oil phase components comprising the EPA glyceride raw material;

[0199] S200, preparing an aqueous phase (preferably under inert gas protection): preparing an aqueous phase under heating condition (preferably 50-70°C) by mixing the aqueous phase components comprising the phospholipid with an aqueous solvent;

[0200] S300, oil-in-water emulsification: the oil phase and the water phase are mixed (preferably under heating, further preferably at 50-70°C), sheared and stirred to prepare an oil-in-water microemulsion;

[0201] S400, homogeneous emulsification: the oil-in-water microemulsion is subjected to high-pressure homogenization to prepare a nanoemulsion with droplet size less than 500 nm, thereby obtaining an unsterilized oral microemulsion.

[0202] Optionally, after the step S400, a step S500, post-treatment (preferably under inert gas protection) is further performed: the nanoemulsion is filtered, packaged and sterilized to obtain the oral microemulsion (as a sterilized oral microemulsion).

[0203] The inert gas protection used in the preparation of the oral microemulsion can be nitrogen protection.

[0204] It should be understood that the steps of the above preparation method have no order limitation unless otherwise specified. For example, the steps of S100 and S200 have no order limitation, but both are prior to S300. For another example, the order between packaging and sterilization is not limited.

[0205] As used herein, "aqueous solvent" refers to a solvent providing a water phase which is pharmaceutically acceptable, and can be water or a mixed solvent of water and other solvents.

[0206] In some embodiments, a pH regulator is mixed with water (to adjust the pH of the aqueous solution with the pH regulator) to obtain an aqueous solvent for subsequent preparation. The pH of the aqueous solvent is adjusted according to the pH of the final prepared microemulsion, and then a suitable pH regulator is selected. In some embodiments, the pH of the final microemulsion is 7-8.

[0207] In some embodiments of the present application, the preparation method of the oral microemulsion comprises the following steps:

[0208] S100: under inert gas protection, the oil phase components are stirred and mixed until a uniform oil solution is formed, and the water bath is heated to 50-70°C to prepare an oil phase; preferably, the inert gas is nitrogen;

[0209] S200: under inert gas protection, the water phase components in the formula are prepared into an aqueous system, stirred and dissolved until a uniform aqueous solution is formed, and the water bath is heated to 50-70°C to prepare a water phase; preferably, the inert gas is nitrogen;

[0210] S300: the obtained water phase and oil phase are mixed to form an oil-in-water microemulsion by shearing and / or high-pressure homogenization, and the water bath is heated to 50-70°C;

[0211] Preferably, the preparation method further comprises: S400: filtering, sterilizing and packaging the obtained oil-in-water microemulsion, and inert gas protection is adopted during the process.

[0212] In the above preparation method, the homogenization treatment is preferably high-pressure homogenization treatment.

[0213] In some embodiments, the parameters of the homogenization treatment are: one pass at 20 bar, one pass at 200 bar, and six passes at 400 bar.

[0214] In the case of the type and amount of the specified raw materials, those skilled in the art can implement the above preparation method according to the above instructions to obtain the oral microemulsion of the present application.

[0215] The inventors have found that the oral microemulsion with an average particle size of less than 500 nm provided by the present application can contain a material that helps EPA accumulate in the liver, thereby greatly promoting the absorption of intestinal cells and the accumulation of EPA in the liver after absorption, and helping EPA to exert its efficacy to the greatest extent.

[0216] Third aspect of the invention

[0217] According to a third aspect of the present application, the use of the oral microemulsion of the first aspect of the present application or the oral microemulsion obtained by the preparation method of the second aspect of the present application is provided.

[0218] In some embodiments of the present application, the use of the oral microemulsion in the preparation of a medicament is provided, and preferably, the medicament is used for preventing and / or treating fat accumulation-related diseases.

[0219] In some embodiments of the present application, the use of the oral microemulsion in the preparation of a medicament for preventing and / or treating fat accumulation-related diseases is provided.

[0220] In some preferred embodiments of the present application, the fat accumulation-related disease is selected from one or more of fatty liver, liver damage, and hepatitis, and preferably, the fatty liver is non-alcoholic fatty liver.

[0221] In some embodiments of the present application, the use of the oral microemulsion in medical food and health food is provided.

[0222] One aspect of the invention

[0223] Another object of the present application is to provide the use of the above-mentioned oral microemulsion, including but not limited to the use in medical food, health food, and pharmaceuticals.

[0224] In some embodiments of the present application, the use of the oral microemulsion in the preparation of health food, medicine for the prevention and / or treatment of fatty liver is provided.

[0225] Fourth aspect of the invention

[0226] According to a fourth aspect of the present application, a method for preventing and / or treating a fatty accumulation related disease is provided, comprising administering to a subject in need thereof a therapeutically effective amount of the oral microemulsion of the present application.

[0227] As used herein, "therapeutically effective amount" means an amount of the oral microemulsion of the present application (or an amount of EPA) that will elicit the biological or medical response of a subject, for example, an amount of the oral microemulsion of the present application (or an amount of EPA) that will bring physiologically and / or pharmacologically positive effects to a subject, including but not limited to reducing or inhibiting enzyme or protein activity or improving symptoms, relieving conditions, slowing or delaying disease progression, or preventing diseases, etc.

[0228] As used herein, "pharmaceutically acceptable" means those agents, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for administration to a patient and are commensurate with a reasonable benefit / risk ratio.

[0229] As used herein, "patient" means an animal, preferably a mammal, and more preferably a human. The term "mammal" primarily refers to warm-blooded vertebrate class of mammals, including but not limited to: cats, dogs, rabbits, bears, foxes, wolves, monkeys, deer, mice, pigs, cows, sheep, horses, and humans. Specific embodiments

[0231] The embodiments of the present application will be described in detail below with reference to the examples. It should be understood that these examples are only used to illustrate the present application and not intended to limit the scope of the present application, and these examples are only used to illustrate the present application, and are intended to illustrate the specific formula composition, preparation method, and functions and effects thereof, and should not be understood as any form of limitation on the scope of the present application. The experimental methods in the following examples are not specified, and the priority is given to the guidance given in the present application, and can also be carried out according to the experimental manual or conventional conditions in the art, or according to the conditions suggested by the manufacturer, or according to the known experimental methods in the art.

[0232] In the following specific examples, the measurement parameters of the raw material components are as follows: there may be slight deviations within the weighing accuracy range if not otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operation accuracy are allowed.

[0233] In the following examples, the particle size was tested by Zetasizer Nano ZS 90 (Malvern) laser particle size analyzer.

[0234] The information of the EPA raw material and phospholipid raw material used in the following examples and comparative examples is shown in Table 1.

[0235] Table 1. Information of the EPA raw material and phospholipid raw material used in the examples and comparative examples of the present application.

[0236]

[0237]

[0238] The EPA content in fish oil refers to the amount of EPA units, and the triglyceride content refers to the total content of triglycerides of various fatty acids in fish oil. The percentage content involved in Table 1 is mass percentage.

[0239] In the following examples, EPA TG represents EPA glyceride, EPA EE represents EPA ethyl ester, PC represents phosphatidylcholine, MeOH represents methanol, BHT represents 2,6-di-tert-butyl-p-cresol (antioxidant), TG represents triglyceride, TC represents total cholesterol, ALT represents glutamic-pyruvic transaminase, and AST represents glutamic-oxaloacetic transaminase.

[0240] In the following examples, "high EPA TG content microemulsion" and "high content EPA TG microemulsion" have the same meaning and can be used interchangeably; "high EPA content microemulsion" and "high content EPA microemulsion" have the same meaning and can be used interchangeably.

[0241] 1. Formulation Examples

[0242] In the following examples, an oral microemulsion is prepared by a method comprising the following steps (in each comparative example, the microemulsion is prepared by referring to the following method).

[0243] S1, preparing an oil phase: the oil phase ingredients including glyceride-type EPA raw material, tocopherol, etc. are mixed under nitrogen gas protection until a uniform oil solution is formed, and heated to 65°C in a water bath for standby;

[0244] S2, preparing an aqueous phase: the purified water is adjusted to the desired pH with a pH adjuster to prepare an aqueous solvent of the desired pH, and the phospholipid (taken as an example of soybean phospholipid) and other water-soluble raw material ingredients are added to the prepared aqueous solvent, stirred and dissolved until a uniform aqueous solution is formed, and heated to 65°C in a water bath for standby;

[0245] S3, preparing a microemulsion: under nitrogen protection, the aqueous phase is added to the oil phase under shearing to form an oil-in-water microemulsion by shearing stirring;

[0246] S4, homogenization and emulsification: a nanoemulsion with a droplet size of less than 500 nm is prepared by high-pressure homogenization, with one pass at 20 bar, one pass at 200 bar, and six passes at 400 bar.

[0247] S5, filtering and packaging: the nanoemulsion is filtered through a filter membrane with a pore size of 0.5-1.0 μm, filled, nitrogen-filled, sealed, and a filled nanoemulsion is obtained;

[0248] S6, high-pressure sterilization: the filled nanoemulsion is sterilized in a high-pressure sterilizer, and an oral microemulsion is obtained, which can be used as a pharmaceutical preparation.

[0249] In the above-mentioned embodiments, the glyceride-type EPA raw material is a polyunsaturated fatty acid glyceride (PUFA glyceride), and some of the commercial names are fish oil. The EPA in the glyceride-type EPA raw material has a high content, and the mass percentage is at least 40%, and in some embodiments, it is specifically about 40%, 60%, or 70%.

[0250] In the following examples and comparative examples, the oil phase components other than the glyceride-type EPA raw material and tocopherol include glycyrrhetic acid succinate, PEG-stearate, ascorbic acid palmitate, glycerol monooleate, silymarin, α-tocopherol acetate, vitamin A; the other water-soluble raw material components include sucrose ester, bile acid, glycyrrhetic acid, vitamin E succinate polyethylene glycol ester, DSPE-PEG, DSPE-PEG-galactose, hyaluronic acid, poloxamer, branched chain amino acid, sodium caseinate, vitamin B complex, vitamin C, vitamin D, glucomannan, and sodium alginate. In Comparative Example 2, EPA ethyl ester is used as a substitute for the glyceride-type EPA raw material in the preparation of the oil phase in step S1.

[0251] In the following examples, iodine value is used to represent the unsaturation degree of phospholipids.

[0252] Example 1.1 Preparation of a microemulsion containing different proportions of EPA glyceride (EPATG) and phosphatidylcholine (PC)

[0253] Oral microemulsions containing different proportions of EPA TG and phosphatidylcholine were prepared according to the various prescriptions in Table 2. The fish oil in formulations 1-2, 1-8, and 1-10 was EPAX 4030TGN from Epax Company, which contained 40 g of EPA per 100 g and had a triglyceride content of greater than or equal to 90% according to the product specification. The fish oil in formulations 1-4, 1-9, and 1-13 was KinOmega 7010TG, which contained about 70% of EPA and had a triglyceride content of about 70% according to the product specification. The fish oil in formulations 1-1 and 1-11 was KinOmega E80TG, which contained about 80% of EPA and had a triglyceride content of about 70% according to the product specification. The fish oil in the other formulations was EPAX 6015TGN from Epax Company, which contained about 60% of EPA and had a triglyceride content of greater than or equal to 90% according to the product specification. The phospholipid in formulations 1-2 and 1-12 was soybean phospholipid from Shanghai Taiwei Pharmaceutical Co., Ltd., which had a phosphatidylcholine content of about 50% and an iodine value of greater than 90. The phospholipid in formulations 1-10 and 1-13 was polyenylphosphatidylcholine EPIKURON 200 from Cargill, which had an iodine value of about 94-97. The phospholipid in the other formulations was soybean phospholipid S75 from Lipoid, Germany, which had a phosphatidylcholine content of 70% and an iodine value of 85-95. The second emulsifier in formulations 1-1, 1-3, and 1-12 was sodium alginate, the antioxidant was vitamin C, and the nutritional supplement was a branched-chain amino acid composition. The second emulsifier in formulations 1-10 and 1-13 was sodium caseinate, the antioxidant was ascorbyl palmitate, and the nutritional supplement was silymarin. The second emulsifier in the other formulations was sucrose ester, the antioxidant was α-tocopherol, and the nutritional supplement was a vitamin B complex.

[0254] Table 2. Various prescriptions and particle sizes of microemulsions with high EPA TG content

[0255]

[0256]

[0257] In the above step S2, NaOH was used to adjust the pH of the final microemulsion to the corresponding range, and purified water was added to the entire system to 1 kg. The same meanings apply in the following examples.

[0258] The "particle size" refers to the average particle size of the microemulsion in different batches prepared under the same prescription and preparation process. The same meaning is indicated in each of the following examples. According to the prescription in Table 2, microemulsions with different proportions of EPA TG and phosphatidylcholine can be prepared, and the droplet particle size of the microemulsion is less than 500 nm. Among them, the oil phase proportion of the four groups of formulations 1-10 to 1-13 in the example is higher, and the obtained microemulsion is more viscous, which produces discomfort when drinking, and is not suitable as the prescription for subsequent production.

[0259] Example 1.2. Preparation of EPA TG microemulsion containing phospholipids with different iodine values

[0260] According to the types and amounts of each component in Table 3, microemulsions containing phospholipids with different iodine values are prepared. Among them, in the four groups of formulations 2-1 to 2-5, the fish oil comes from EPAX 6015TGN of Epax Company; in the five groups of formulations 2-6 to 2-9, the fish oil comes from KinOmega 7010TG. Egg yolk lecithin E80 (specifically egg yolk phosphatidylcholine, iodine value of 60-70), soybean phospholipid S75 (specifically soybean phosphatidylcholine, iodine value of about 85-95), and soybean phospholipid S100 (specifically soybean phosphatidylcholine, iodine value of 97-107) come from Lipoid in Germany. Polyene phosphatidylcholine EPIKURON200 (iodine value of about 94-97) comes from Cargill in the United States. The antioxidant in formulations 2-1, 2-2, 2-3, and 2-4 is α-tocopherol; the antioxidant in formulations 2-5, 2-6, 2-7, and 2-8 is ascorbyl palmitate.

[0261] Table 3. EPA TG microemulsion prescription containing phospholipids with different iodine values and microemulsion particle size

[0262]

[0263] Wherein "-" indicates the amount is 0.

[0264] According to the prescription in Table 3, microemulsions with good stability can be prepared, and the droplet particle size of the microemulsion is less than 500 nm, which can be used as an oral microemulsion, wherein the emulsification effect of phospholipid S75 is better, and the emulsification effect of phospholipid S100 is poorer.

[0265] Example 1.3. Preparation of EPA TG microemulsion containing liver targeting auxiliary materials

[0266] EPA TG microemulsions containing liver targeting effect adjuvants (specifically, bile acids, glycyrrhizinic acid) were prepared according to the types and amounts of each component in Table 4. Fish oil in seven groups of formulations 3-1 to 3-7 was EPAX 6015TGN from Epax Company, and fish oil in three groups of formulations 3-8 to 3-10 was KinOmega 7010TG. Phospholipids in formulations 3-2, 3-8, and 3-9 were derived from polyenylphosphatidylcholine EPIKURON 200 from Cargill, with an iodine value of about 94-97; phospholipids in other formulations were derived from soybean phospholipid S75 from Lipoid, Germany, with a phosphatidylcholine content of 70% and an iodine value of 85-95. The second emulsifier in formulations 3-1, 3-2, and 3-3 was poloxamer, the antioxidant was ascorbyl palmitate, and the nutritional supplement was glucomannan; the second emulsifier in formulations 3-4, 3-5, 3-6, and 3-7 was sodium caseinate, the antioxidant was α-tocopherol acetate, and the nutritional supplement was vitamin B complex; the second emulsifier in other formulations was sucrose ester, the antioxidant was α-tocopherol, and the nutritional supplement was vitamin A.

[0267] Table 4. Different formulations of EPA TG microemulsions containing liver targeting effect adjuvants and microemulsion particle sizes

[0268]

[0269] wherein "-" indicates an amount of 0.

[0270] According to the formulations in Table 4, microemulsions with good stability can be prepared, and the microemulsion droplet particle size is less than 500 nm, which can be used as an oral microemulsion. Glycyrrhizinic acid and bile acids have amphiphilic properties, which can assist in emulsification on one hand, and can increase liver targeting after being absorbed with oil and fat wrapped into chylomicrons on the other hand.

[0271] Example 1.4. Preparation of EPA TG microemulsions containing adjuvants with reduced enzymatic action

[0272] The microemulsions containing the excipients with reduced enzymatic action were prepared according to the types and amounts of the components in Table 5. The fish oil in the seven groups of Formulations 4-1 to 4-7 was EPAX 6015TGN from Epax Company, and the fish oil in the three groups of Formulations 4-8 to 4-10 was KinOmega 7010TG. The phospholipid in Formulations 4-4, 4-9 and 4-10 was derived from soybean phospholipid S100 from Lipoid, Germany, with an iodine value of about 94-97; the phospholipid in other formulations was derived from soybean phospholipid S75 from Lipoid, Germany, with a phosphatidylcholine content of 70% and an iodine value of 85-95. The second emulsifier in Formulations 4-1, 4-2 and 4-3 was poloxamer, the antioxidant was ascorbyl palmitate, and the nutritional supplement was branched chain amino acid; the second emulsifier in Formulations 4-4, 4-5, 4-6 and 4-7 was sodium caseinate, the antioxidant was α-tocopherol, and the nutritional supplement was vitamin B complex; the second emulsifier in other formulations was glycerol monooleate, the antioxidant was vitamin C, and the nutritional supplement was vitamin D.

[0273] The excipient with reduced enzymatic action is vitamin E succinate PEG and / or PEG-stearate, wherein the weight average molecular weight of PEG is 1000 Da and 2000 Da, respectively.

[0274] Table 5. Different formulations of EPA TG microemulsion containing excipients with reduced enzymatic action and particle size of microemulsion

[0275]

[0276] wherein "-" represents an amount of 0.

[0277] The microemulsion with good stability can be prepared according to the formulations in Table 5, and the particle size of the microemulsion droplets is less than 500 nm, which can be used as an oral microemulsion. The PEG-modified vitamin E succinate and / or stearate is added to the aqueous phase, and the surface of the microemulsion droplets is wrapped by PEG, thereby playing a certain role in preventing the hydrolysis and utilization of EPA by other tissues.

[0278] Example 1.5. Preparation of EPA TG microemulsion containing liver targeting excipient and excipient with reduced enzymatic action

[0279] EPA TG microemulsions containing liver targeting adjuvants and adjuvants with reduced enzymatic action were prepared according to the types and amounts of components in Table 6. In the seven groups of Formulations 5-1 to 5-7, the fish oil was EPAX 6015TGN from Epax Company, and in the three groups of Formulations 5-8 to 5-10, the fish oil was KinOmega 7010TG. The phospholipids in Formulations 4-4, 4-9 and 4-10 were derived from soybean phospholipids S100 from Lipoid, Germany, with an iodine value of about 94 to 97; the phospholipids in other formulations were derived from soybean phospholipids S75 from Lipoid, Germany, with a phosphatidylcholine content of 70% and an iodine value of 85 to 95. The antioxidant in Formulations 4-1, 4-2 and 4-3 was ascorbyl palmitate; the antioxidant in Formulations 4-4, 4-5, 4-6 and 4-7 was α-tocopherol; and the antioxidant in other formulations was vitamin C.

[0280] In the above formula, the liver targeting adjuvant was glycyrrhizin succinate. The adjuvant with reduced enzymatic action was DSPE-PEG and DSPE-PEG-galactose, both of which had a weight average molecular weight of 2000 Da.

[0281] Table 6. Different formulations of EPA TG microemulsions containing liver targeting adjuvants and adjuvants with reduced enzymatic action and particle sizes of the microemulsions

[0282]

[0283]

[0284] In the above formula, "-" means the amount is 0.

[0285] According to the formulations in Table 6, microemulsions with good stability can be prepared, and the particle sizes of the microemulsion droplets are less than 500 nm, which can be used as oral microemulsions. The surfaces of the prepared microemulsion droplets are coated with PEG, which provides protection against degradation for the microemulsion droplets.

[0286] Formulation Control Examples

[0287] The microemulsions in the following control examples were prepared according to the method for preparing oral microemulsions in "1. Formulation Examples". In Control Example 1, commercially available ordinary fish oil was used instead of the fish oil with high EPA content (high-EPA fish oil) in the Formulation Examples, and in Control Example 2, EPA ethyl ester (high-EPA ethyl ester) was used instead of the high-EPA fish oil in the Formulation Examples.

[0288] Control Example 1. Preparation of a microemulsion containing commercially available ordinary fish oil

[0289] The microemulsions were prepared according to the ingredients and amounts in Table 7 using commercially available ordinary fish oil. The fish oil used in the four groups of preparations D1-1 to D1-4 was commercially available fish oil from the fish oil soft capsules (Shanghai Chembiokejin Co. Ltd.) which used imported fish oil from Norway / Iceland, according to the product specification, each 100 g of fish oil contained EPA 18.3 g, DHA 12.9 g, and the content of triglyceride was 98%. The soybean phospholipid in the four groups of preparations D1-1 to D1-4 was from Lipoid S75 in Germany, the content of phosphatidylcholine was 70%, and the iodine value was 85-95.

[0290] Table 7. Different formulations of microemulsions containing commercially available ordinary fish oil, glycyrrhetinic acid and DSPE-PEG

[0291]

[0292] Example 2. Preparation of microemulsions containing different proportions of EPA ethyl ester

[0293] The microemulsions containing EPA ethyl ester were prepared according to the formulations in Table 8. The EPA ethyl ester was used as a substitute for fish oil in the four groups of preparations D2-1 to D2-4, and the EPA ethyl ester used was from the raw material used in , according to the product specification, each 100 g of oil contained more than 96 g of EPA ethyl ester. The soybean phospholipid in the four groups of preparations was from Lipoid S75 in Germany, the content of phosphatidylcholine was 70%, and the iodine value was 85-95.

[0294] Table 8. Different formulations of microemulsions containing EPA ethyl ester, hyaluronic acid and DSPE-PEG and the particle size of the microemulsions

[0295]

[0296]

[0297] 2. Evaluation in vivo of the preparation animals

[0298] Example 2.1. Evaluation of the effect of microemulsion preparations on improving bioavailability

[0299] 2.1.1. Experimental animals

[0300] Male beagle dogs (Shanghai Experimental Animal Research Center), weighing about 10 kg.

[0301] 2.1.2. This example is divided into 4 control groups and 4 experimental groups, wherein the control groups include: ordinary fish oil microemulsion (formulation D1-2), EPAX 6015TGN raw material (raw material group), EPAX 6015TGN raw material (raw material group, fasting), EPA EE microemulsion (formulation D2-2); the experimental groups include: formulation 1-6, formulation 5-7, formulation 1-6 (fasting), formulation 5-7 (fasting). The equivalent EPA administration dose of each group is 120 mg / kg.

[0302] 2.1.3. Experimental method: the beagle dogs were randomly divided into 8 groups, 6 in each group, and were not fasted before the experiment except for the fasting group, and were not water-restricted. The fasting group was fasted for 12 h and not water-restricted. The beagle dogs were respectively given corresponding test substances by gavage, and blood samples of 2 mL were collected at 0.5 h, 1 h, 2 h, 3 h, 4 h, 6 h, 8 h, 12 h, and 24 h after administration, respectively, and were centrifuged at 4℃ and 3000 rpm for 10 min, and the plasma was separated and stored at -20℃ for determination of the drug concentration.

[0303] 2.1.4. The total EPA content in the plasma was detected by methyl esterification-GC method. The GC chromatographic conditions were as follows: (88%-cyanopropyl) aryl-polysiloxane capillary column (60 m x 0.25 mm x 0.2 μm), programmed temperature, 0 min 170℃, 3.5℃ / min to 240℃, maintained for 10 min, injector temperature 250℃, detector temperature 270℃. The carrier gas was helium, and the flow rate was 1.0 mL / min. Split ratio: 10:1. Injection volume 1 μL.

[0304] Step one: a clean dry glass test tube with a stopper was taken, 100 μL of 300 μg / mL heneicosanoic acid methyl ester (internal standard) was added, and after blowing dry with N2, 200 μL of plasma, 2 mL of 0.5 mol / L KOH-MeOH solution, and 0.5 mL of BHT isooctane solution were added. The tube was sealed, vortexed for 60 s, mixed uniformly, and then left to stand for 10 min to 15 min. The isooctane layer was collected and placed in a clean sample vial with a small amount of anhydrous sodium sulfate added.

[0305] Step two: 2 mL of 5% (v / v) H2SO4-MeOH solution was added to the lower solution of step one, the solution was sealed after being slightly charged with N2, and was simply vortexed and mixed. The solution was reacted at 70℃ for 30 min. After being taken out, it was cooled to about 40℃, 0.5 mL of isooctane was added, vortexed for 30 s, 0.5 mL of saturated sodium chloride solution was added, vortexed for 15 s, and the isooctane layer was collected and combined with the organic layer of step one. After being dried with anhydrous sodium sulfate, it was transferred to a sample vial with a sample sleeve for injection as a test sample solution.

[0306] The relative bioavailability F (such as AUC 微乳AUC 原料 The evaluation was performed by taking the AUC of the raw material group as 100%.

[0307] 2.1.5. Experimental results

[0308] The pharmacokinetic parameters of beagle dogs after administration of different formulations are shown in Table 9. The results show that, compared with the EPAX 6015 TGN raw material group, the bioavailability of the microemulsion prepared by using ordinary fish oil raw material (formulation D1-2) does not significantly increase at the same dose of EPA, and the oral microemulsion prepared by using high content EPA fish oil (four experimental groups) can increase the bioavailability of EPA by about 3 times. Compared with the EPAX 6015 TGN raw material group, the bioavailability of the control group 3 administered in the fasting state is significantly decreased. The results of the empty and full stomach comparison of formulations 1-7 and 5-7 show that the bioavailability of the microemulsion in the empty and full stomach states has no significant difference. The oral microemulsion provided by the present application can promote the absorption of fish oil, improve the bioavailability, and has no significant difference in the bioavailability in the empty and full stomach states.

[0309] Table 9. Relative bioavailability of EPA in fish oil oral microemulsion

[0310]

[0311] Example 2.2. Screening of EPA purity, ratio of EPA to phosphatidylcholine (PC), and phosphatidylcholine iodine value

[0312] "EPA purity" refers to the content of EPA in the fish oil raw material.

[0313] 2.2.1. Experimental animals

[0314] Experimental animals: male Sprague Dawley rats (Shanghai Experimental Animal Research Center), weighing 180 g to 200 g.

[0315] 2.2.2. This embodiment is divided into blank group, model group and 9 experimental groups: the blank group: feeding ordinary feed, daily administration of micro-emulsion purified water. Model group: high-fat diet, daily administration of micro-emulsion purified water. Experimental group is respectively: preparation 1-4, the mass ratio of EPA and phosphatidylcholine in the preparation is about 8.7:1; preparation 1-5, the mass ratio of EPA and phosphatidylcholine in the preparation is about 7.2:1; preparation 1-6, the ratio of EPA and phosphatidylcholine in the preparation is about 5.8:1; preparation 1-7, the ratio of EPA and phosphatidylcholine in the preparation is about 4.8:1, fish oil in the preparation is EPAX6015TGN of Epax company; preparation 1-8, fish oil in the preparation is EPAX 4030TGN of Epax company; preparation 1-9, fish oil in the preparation is KinOmega7010TG; preparation 2-1, the phospholipid iodine value in the preparation is about 60-70; preparation 2-2, the phospholipid iodine value in the preparation is about 85-95; preparation 2-3, the phospholipid iodine value in the preparation is about 94-97; preparation 2-4, the phospholipid iodine value in the preparation is about 97-107. The equivalent EPA administration dose of each group is 150mg / kg / day.

[0316] 2.2.3. Experimental method: 90 SD rats were taken, 10 rats in each group, and special non-alcoholic fatty liver feed was given to model. After the modeling was completed, according to the fasting blood glucose, body weight, plasma TC(total cholesterol), TG(triglyceride) level, the groups were divided. The mice in the blank control group and the model control group were given purified water by gavage every day, and the preparation group was given the corresponding preparation. Continuous administration for 6 weeks, during the administration period, the feed of each group was unchanged. Before starting administration and after 6 weeks of administration, the animals were anesthetized after fasting for 4h, and at least 0.5mL of whole blood sample was collected in a heparinized centrifuge tube, centrifuged at 4℃, 6500rpm for 15min, and the supernatant serum was taken. Immediately after the last blood sampling, the animals were sacrificed, the liver was dissected and weighed, the right lobe was taken and fixed with 10% formalin, routinely paraffin-embedded section, HE staining, and observed under optical microscope. The rest of the tissue was stored at-80℃ for later use.

[0317] 2.2.4. Indicators to be tested:

[0318] After 6 weeks of administration, the body weight and liver weight of the rats were measured, and the liver index of the rats was calculated (liver index = liver weight of rats / last body weight of rats), and the liver index was also recorded as liver index, wherein "last body weight of rats" refers to the weight immediately after the last administration of blood sampling. The expression of triglyceride (TG), total cholesterol (TC), glutamic-pyruvic transaminase (ALT), and glutamic-oxalacetic transaminase (AST) was detected. The total cholesterol in the liver was determined by CHOD-PAP method, the liver triglyceride was determined by GPO-PAP method, and the tissue morphology was observed by HE staining.

[0319] 2.2.5. Experimental results

[0320] The experimental results are shown in Table 10. Compared with the blank group, the body weight and liver index of the rats in the model group increased significantly, while the body weight and liver index of the rats in each preparation group (9 groups in total) decreased. The body weight and liver index of the rats tended to decrease with the decrease of the EPA / phosphatidylcholine ratio and the increase of the phospholipid iodine value. When the EPA / phosphatidylcholine ratio was less than 8:1 (including preparation 1-5, preparation 1-6, and preparation 1-7) and the phospholipid iodine value was greater than 80, the effect was better. The data of preparation 1-7, preparation 1-8, and preparation 1-9 showed that when the fish oil raw material contained 40 g of EPA per 100 g, the effect on the body weight and liver index was not obvious, and when the fish oil raw material contained 60% or more of EPA, the effect on the body weight and liver index was better.

[0321] Table 10. Body weight change and liver index of rats in each group (n=10)

[0322]

[0323] In the formula, EPA / PC represents the mass ratio of EPA to phosphatidylcholine, and the iodine value represents the iodine value of phospholipid.

[0324] The specific data of the TG and TC levels in the serum of rats in each group are shown in Table 11, which reflects the blood lipid content of rats. The TC and TG levels in the serum of rats tended to decrease with the decrease of the EPA / phosphatidylcholine ratio and the increase of the phospholipid iodine value, but the decrease was not significant. The TC level in the serum of rats did not change significantly with the increase of the EPA purity, but the TG level tended to decrease with the increase of the EPA purity.

[0325] Table 11. TG and TC levels in the serum of rats in each group (n=10)

[0326]

[0327] The specific data of the ALT and AST levels in the serum of rats in each group are shown in Table 12. The ALT level in the serum of rats did not change significantly with the decrease of the EPA / phosphatidylcholine ratio, but tended to decrease with the increase of the phospholipid iodine value. The AST level in the serum of rats tended to decrease with the decrease of the EPA / phosphatidylcholine ratio and the increase of the phospholipid iodine value, and the effect was better when the EPA / phosphatidylcholine ratio was less than 8:1 (preparation 1-5, preparation 1-6, and preparation 1-7) and the phospholipid iodine value was greater than 80. The ALT and AST levels did not change significantly with the change of the EPA purity.

[0328] Table 12. ALT and AST levels in the serum of rats in each group (n=10)

[0329]

[0330] The TC and TG levels in the rat liver of each group are shown in Table 13. The TC level in the rat liver did not change significantly with the decrease of the EPA / phosphatidylcholine ratio and the increase of the phospholipid iodine value. The TG level in the rat liver slightly increased when the EPA / phosphatidylcholine ratio was 7:1, but there was no significant difference. The TG level slightly decreased when the EPA / phosphatidylcholine ratio was 6:1, but the decrease was not significant. The TG level began to decrease when the EPA / phosphatidylcholine ratio was less than 5:1 (Formulations 1-7). The TG level in the rat liver showed a decreasing trend with the increase of the phospholipid iodine value. The TC and TG levels in the rat liver did not change significantly with the change of the EPA purity.

[0331] Table 13. Liver lipid content of rats in each group (n=10)

[0332]

[0333] The results of the fatty liver NAS score are shown in Table 14. Formulations 1-6 (about 6:1), 1-7 (about 5:1) with a lower EPA / phosphatidylcholine ratio and Formulations 2-3 (iodine value about 95) and 2-4 (iodine value about 103) with a higher phospholipid iodine value had better score results. Formulations 1-7 (EPA purity about 60%) and 1-9 (EPA purity about 70%) had lower scores than Formulation 1-8 (EPA purity about 40%). Figure 1

[0334] In summary, ① the microemulsion prepared from fish oil raw material with a higher EPA content (i.e., higher purity) had a more obvious effect on reducing the body weight and liver index of the fatty liver model rats than the microemulsion prepared from fish oil raw material with an EPA purity of 40%, and the blood TG level showed a decreasing trend with the increase of the EPA content of the fish oil raw material. ② The oral microemulsion had a better effect on reducing the body weight, liver index, blood lipids and liver damage of the fatty liver model rats when the EPA / phosphatidylcholine ratio was less than or equal to 8:1 and the phospholipid iodine value was greater than 70. When the EPA / phosphatidylcholine ratio was less than or equal to 5:1 and the phospholipid iodine value was greater than 90, it was more helpful to the decrease of the liver TG level, and the effect was better.

[0335] Example 2.3. Drug distribution study

[0336] 2.3.1. Experimental animals

[0337] Experimental animals: male Sprague Dawley rats (Shanghai Experimental Animal Research Center), weighing 220-250 g.

[0338] ​2.3.2. This example is divided into 3 control groups and 6 experimental groups: the equivalent EPA administration dose of each group is 500 mg / kg. The control groups include: ordinary fish oil microemulsion (preparation D1-2), EPAX 6015TGN raw material (raw material group, non-microemulsion), EPA EE microemulsion (preparation D2-2). The experimental groups include: high content EPA microemulsion (preparation 1-7), high content EPA microemulsion containing bile acids (preparation 3-5), high content EPA microemulsion containing glycyrrhetinic acid (preparation 3-7); high content EPA microemulsion containing vitamin E succinate polyethylene glycol (preparation 4-6), high content EPA microemulsion containing DSPE-PEG (preparation 4-7), high content EPA microemulsion containing glycyrrhetinic acid succinate and DSPE-PEG (preparation 5-7).

[0339] 2.3.3. Experimental method: 54 SD rats were taken, 6 rats in each group. The experiment was not fasting, and the water was free. Each group was administered by gavage according to the dose of the test substance, EPA 500 mg / kg. After gavage, 3h, the rats were killed immediately, and the heart, liver, spleen, lung, kidney and other tissues were taken out, washed with normal saline, weighed, and stored in a-80℃ refrigerator. Before use, thaw the tissue and mix it with normal saline in proportion to prepare tissue homogenate.

[0340] 2.3.4. Experimental results: The distribution of EPA in different tissues was detected by gas chromatograph, as shown in Table 14, and the detection method was the same as that of plasma EPA detection.

[0341] Table 14. Organ distribution of EPA after administration of different groups (μg·g -1 )

[0342]

[0343] After 3h of administration in different groups, EPA was mainly concentrated in the liver. Among them, the microemulsion significantly increased the concentration of EPA in each organ in vivo, the addition of auxiliary materials with liver targeting effect (preparation 3-5, preparation 3-7, preparation 5-7) or auxiliary materials reducing enzymatic action (preparation 4-6, preparation 4-7, preparation 5-7) can increase the concentration of EPA in the liver to a certain extent, and the auxiliary material with better fat solubility may have better effect (compare preparation 3-5, preparation 3-7 and preparation 4-7, 5-7). The combination of auxiliary materials with liver targeting effect or PEG modified auxiliary materials (preparation 5-7) has better enrichment effect of EPA in the liver. In addition, it is found in this example that compared with the EPA EE microemulsion used in preparation D2-2, the glyceride type EPA microemulsion has higher concentration in the liver.

[0344] Example 2.4. Drug enrichment in liver after multiple administrations of different preparations

[0345] 2.4.1. Experimental animals

[0346] Experimental animals: male Sprague Dawley rats (Shanghai Experimental Animal Research Center), body weight 220g-250g.

[0347] 2.4.2. This example is divided into 9 groups: This example is divided into 3 control groups and 6 experimental groups: the equivalent EPA administration dose of each group is 125mg / kg / time, 2 times / day by gavage. Among them, the control groups include: ordinary fish oil microemulsion (preparation D1-2), EPAX6015TGN raw material (raw material group), EPAEE microemulsion (preparation D2-2). The experimental groups include high content EPA microemulsion (preparation 1-6), high content EPA microemulsion containing bile acids (preparation 3-5), high content EPA microemulsion containing glycyrrhetinic acid (preparation 3-7); high content EPA microemulsion containing vitamin E succinate polyethylene glycol (preparation 4-6), high content EPA microemulsion containing DSPE-PEG (preparation 4-7), high content EPA microemulsion containing glycyrrhetinic acid succinate and DSPE-PEG (preparation 5-7).

[0348] 2.4.3. Experimental method: 135 SD rats were taken, 15 rats in each group. Each group was administered according to the dose of the test substance. No fasting before the experiment, free water, dissection of liver at 7 days, 14 days, 28 days (6h after the last administration), washed clean with normal saline, weighed, and stored in-80℃ refrigerator for testing. Before use, thaw the tissue and mix it with normal saline in proportion to prepare tissue homogenate.

[0349] 2.4.4. Experimental results: The enrichment of EPA in the liver was obtained by gas chromatograph detection, as shown in Table 15, and the detection method was the same as that of plasma EPA detection.

[0350] Table 15. Enrichment of EPA in the liver after 7 days / 14 days / 28 days of administration in different groups

[0351]

[0352] The results show that the fish oil oral microemulsion with auxiliary EPA aggregation in the liver (preparation 3-5, preparation 3-7, preparation 4-6, preparation 4-7, preparation 5-7) significantly improves the enrichment rate of EPA in the liver, and makes it reach the steady state concentration in the liver faster. Among them, the microemulsion significantly improves the enrichment rate of EPA in the liver, the addition of auxiliary materials with liver targeting effect (preparation 3-5, preparation 3-7, preparation 5-7) or reducing enzymatic action (preparation 4-6, preparation 4-7, preparation 5-7) can increase the enrichment rate of EPA in the liver, and the combination of the two auxiliary materials (preparation 5-7) has better effect on the enrichment of EPA in the liver. In particular, compared with the EPAEE microemulsion used in preparation D2-2, the glyceride type EPA microemulsion has a faster enrichment rate in the liver.

[0353] Example 2.5. Therapeutic effect of oral microemulsion of high EPA content glyceride type raw material with liver enrichment on non-alcoholic fatty liver

[0354] 2.5.1. Experimental animals

[0355] Experimental animals: male Sprague Dawley rats (Shanghai Experimental Animal Research Center), body weight 180-200 g.

[0356] 2.5.2. This example is divided into blank group, model group, 3 control groups and 4 experimental groups. Among them, the blank group is fed with ordinary feed and given microemulsion purified water of the same volume; the model group is fed with high-fat feed and given microemulsion purified water of the same volume; the control groups include: commercially available pioglitazone hydrochloride tablets (positive control group), the dosage is equivalent to 10 mg / kg of pioglitazone, once a day; preparation D1-4, the equivalent EPA dosage is 300 mg / kg / day, 2 times a day by gavage; EPAX 6015TGN raw material of Epax Company (raw material group), the equivalent EPA dosage is 300 mg / kg / day, 2 times a day by gavage; the experimental groups include: preparation 1-7 medium dose group, the equivalent EPA dosage is 150 mg / kg / day; preparation 5-7 low dose group, the equivalent EPA dosage is 75 mg / kg / day; preparation 5-7 medium dose group, the equivalent EPA dosage is 150 mg / kg / day; preparation 5-7 high dose group, the equivalent EPA dosage is 300 mg / kg / day;

[0357] 2.5.3. Experimental method: 90 SD rats were taken, 10 rats in each group, and non-alcoholic fatty liver special feed was given to make model. After the modeling was completed, the groups were divided according to the fasting blood glucose, body weight, plasma TC and TG levels. The blank control group and the model control group of mice were given purified water by gavage every day, and the preparation group was given the corresponding preparation. Continuous administration for 6 weeks, during the administration period, the feed of each group was unchanged. Before starting administration and 6 weeks after administration, the animals were anesthetized after fasting for 4 h, and at least 0.5 mL of whole blood sample was collected in a heparinized centrifuge tube, centrifuged at 4°C and 6500 rpm for 15 min, and the supernatant serum was taken. Fasting blood glucose was measured at 2, 4 and 6 weeks of administration, and OGTT experiment was performed after 4 weeks of administration. Immediately after the last administration, the animals were sacrificed, the liver was dissected and weighed, the right lobe was fixed with 10% formalin, routinely paraffin-embedded sectioned, HE stained, and observed under an optical microscope. The remaining tissues were stored at -80°C for later use.

[0358] 2.5.4. Indicators to be tested:

[0359] After 6 weeks of administration, the body weight of rats was weighed, the liver weight was weighed, and the liver index of rats (liver index = rat liver weight / rat last body weight) was calculated. The fasting blood glucose was measured by blood glucose meter and test paper. The expression of triglyceride (TG), total cholesterol (TC), glutamic-pyruvic transaminase (ALT), and glutamic-oxalacetic transaminase (AST) was detected by taking blood. The total cholesterol in liver was measured by CHOD-PAP method, and the liver triglyceride was measured by GPO-PAP method. The SOD (superoxide dismutase) and MDA (malondialdehyde) were detected by using the related kits, respectively. The tissue morphology was observed by HE staining.

[0360] 2.5.5. Experimental results

[0361] Compared with the blank control, the body weight and liver index of the model group rats were significantly increased, while different EPA preparations could reduce the body weight and liver index of rats, among which the high-dose group of preparation 5-7 orally administered microemulsion had the best effect on reducing the body weight and liver index. The specific data are shown in Table 16.

[0362] Table 16. Body weight change and liver index of rats in each group (n = 10)

[0363]

[0364] The fasting blood glucose changes of rats in the blank group, model group, control group and experimental group after administration for 2, 4 and 6 weeks are shown in Table 17. Rosiglitazone has a better control on the blood glucose of fatty liver rats, and the fasting blood glucose level is significantly reduced at the 4th week and the 6th week. The medium and high dose groups of preparations 5-7 have the same trend as the positive control, while the medium dose group of preparations 1-7 and the low dose group of preparations 5-7 only show a significant trend of reducing fasting blood glucose at the 6th week.

[0365] Table 17. Fasting blood glucose changes of rats in each group (n = 10)

[0366]

[0367]

[0368] The results of glucose tolerance test of rats are shown in Table 18. Figure 2 , Table 18, Figure 3 The blood glucose AUC of the positive control group and the medium and high dose groups of preparations 5-7 is significantly smaller than that of the model group, indicating that they can improve the insulin resistance of rats to a certain extent.

[0369] Table 18. Glucose tolerance test results of rats in each group (n = 10)

[0370]

[0371] The results of the blood lipid content test in each group of rats are shown in Table 19. Compared with the blank control, the serum TG and TC levels in the model group rats were significantly increased. Compared with the model control group, the serum triglyceride (TG) level in the EPA-treated groups (except for formulations D1-4) was significantly lower than that in the model control group. Among them, the medium and high doses of formulations 5-7 had the most significant effects. For the total cholesterol (TC) level, only the medium and high doses of formulations 5-7 had a significant effect.

[0372] Table 19. Blood lipid levels in rats of each group (n=10)

[0373]

[0374] The results of serum ALT and AST levels in rats from each group are shown in Table 20. Compared with the model control group, oral administration of fish oil microemulsion significantly reduced serum ALT and AST levels, but the differences in the reduction of serum ALT and AST levels among the groups were not significant.

[0375] Table 20. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in rats in each group (n=10)

[0376]

[0377] The results of liver lipid, liver SOD, and MDA content measurements in each group of rats are shown in Table 21. Compared with the blank group, the model group showed a significant increase in lipid deposition and lipid droplet accumulation. The results indicate that different EPA microemulsion formulations had no significant effect on TC levels in the liver, but had a certain effect on reducing TG, increasing liver SOD activity, and decreasing MDA content, reflecting that EPA microemulsion formulations protect the body from lipid peroxidation by regulating free radical metabolic balance.

[0378] Table 21. Determination of liver lipids, liver SOD, and MDA content in rats in each group (n=10)

[0379]

[0380] Fatty liver NAS score results as follows Figure 4 As shown, the HE staining results of rat liver tissue morphology are as follows: Figure 5 As shown. Oral microemulsions containing high-EPA-content glycerides, which have hepatic enrichment effects, can effectively improve liver tissue morphology. Liver sections in the model group showed obvious vacuolated hepatocytes and severe micro and macrovesicular steatosis, with significant inflammatory cell infiltration. In the low, medium, and high-dose groups of formulations 5-7, hepatocyte morphology gradually recovered, swelling gradually decreased, lipid droplet vacuolation gradually decreased, and inflammatory cell infiltration gradually decreased. The high-dose group of formulations 5-7 showed the best improvement in liver tissue damage. Figure 5 ).

[0381] The foregoing experimental results also show that: (1) relative to the EPA soft capsules, the bioavailability of EPA is increased by about 3 times by the microemulsion. (2) The content of phosphatidylcholine and the unsaturation degree selection have an influence on the drug efficacy, and the preferable selection range is that the phosphatidylcholine iodine value is greater than or equal to 80; the ratio of EPA to phosphatidylcholine is less than 8:1. (3) The fish oil oral microemulsion provided by the present application can effectively increase the enrichment of EPA in the liver. (4) The fish oil oral microemulsion provided by the present application can reduce the ALT and AST levels induced by high-fat diet to form NASH, alleviate the inflammatory response, prevent lipid peroxidation, and can reduce the serum TG and TC levels. The oral microemulsion provided by the present application can be used as a potential drug for treating NAFLD.

[0382] Each of the technical features of the above-described embodiments and examples can be combined in any suitable manner. In order to make the description concise, not all possible combinations of the technical features in the above-described embodiments and examples are described, however, as long as the combinations of the technical features do not exist in contradiction, they should be considered within the scope of the present disclosure.

[0383] The above-described embodiments only express several embodiments of the present application, facilitate specific and detailed understanding of the technical solutions of the present application, but should not be understood as a limitation on the scope of patent protection. It should be noted that, for those skilled in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are within the scope of protection of the present application. In addition, it should be understood that after reading the above description of the present application, those skilled in the art can make various changes or modifications to the present application, and the equivalent forms also fall within the scope of the present application. It should also be understood that those skilled in the art can obtain technical solutions based on the technical solutions provided by the present application through logical analysis, reasoning or limited experiments, which are within the scope of protection of the appended claims of the present application. Therefore, the scope of protection of the present patent should be based on the contents of the appended claims, and the description and drawings can be used to explain the contents of the claims.

Claims

1. An oral microemulsion characterized in that, The total weight percentage of the oral microemulsion is 100% by weight percentage, and the oral microemulsion comprises the following components: EPA glyceride raw material 1%~30%; First emulsifier 0.1%~10%; Second emulsifier 0%~10%; First auxiliary material 0.05%~10%; Second auxiliary material 0~40%; and Water; wherein the mass content of EPA in the EPA glyceride raw material is ≥45%, and the mass percentage of triglyceride in the glyceride component is ≥58%; The first emulsifier is a phospholipid with an iodine value ≥80; In the phospholipid component of the first emulsifier, the mass percentage of phosphatidylcholine is ≥50%; The mass ratio of EPA to phosphatidylcholine in the first emulsifier is ≤8:1; The components of the second emulsifier are all different from those of the first emulsifier; The first auxiliary material comprises at least one of a liver targeting component and a PEG modified component; the liver targeting component is selected from one or more of glycyrrhetinic acid and its derivatives, galactose, mannose, hyaluronic acid, and bile acid; the liver targeting component is selected from one or more of a liver targeting molecule and a modified liver targeting molecule, wherein the modified liver targeting molecule contains one or more of a PEG modified lipid unit and a non-PEG modified lipid modification unit; the PEG modified component is selected from one or more of distearoylphosphatidylethanolamine-polyethylene glycol, polyethylene glycol-stearate, vitamin E succinate polyethylene glycol ester, soybean phosphatidyl ethanolamine-polyethylene glycol monomethyl ether, polyethylene glycol oleate, and polyethylene glycol laurate; The glycyrrhetinic acid derivative is selected from a fat-soluble derivative of glycyrrhetinic acid, and the fat-soluble derivative of glycyrrhetinic acid is selected from one or more of DSPE-glycyrrhetinic acid, DSPE-PEG-glycyrrhetinic acid, glycyrrhetinic acid fatty acid ester, and glycyrrhetinic acid succinate; The second auxiliary material is a food and / or pharmaceutically acceptable raw and auxiliary material, and is different from the first emulsifier, the second emulsifier, and the first auxiliary material.

2. The oral microemulsion according to claim 1, characterized in that, The total weight percentage of the oral microemulsion is 100% by weight percentage, and the oral microemulsion comprises the following components: EPA glyceride raw material 1%~30%; First emulsifier 0.1%~10%; Second emulsifier 0%~10%; First auxiliary material 0.05%~10%; Second auxiliary material 0~40%; and Water; wherein the mass content of EPA in the EPA glyceride raw material is ≥45%, and the mass percentage of triglyceride in the glyceride component is ≥58%; The first emulsifier is a phospholipid with an iodine value ≥80; In the phospholipid component of the first emulsifier, the mass percentage of phosphatidylcholine is ≥50%; The mass ratio of EPA to phosphatidylcholine in the first emulsifier is ≤8:1; The components of the second emulsifier are all different from those of the first emulsifier; The first auxiliary material comprises at least one of a liver targeting component and a PEG modified component; the liver targeting component is selected from one or more of glycyrrhetinic acid and its derivatives, galactose, mannose, hyaluronic acid, and bile acid; the liver targeting component is selected from one or more of a liver targeting molecule and a modified liver targeting molecule, wherein the modified liver targeting molecule contains one or more of a PEG modified lipid unit and a non-PEG modified lipid modification unit; the PEG modified component is selected from one or more of distearoylphosphatidylethanolamine-polyethylene glycol, polyethylene glycol-stearate, vitamin E succinate polyethylene glycol ester, soybean phosphatidyl ethanolamine-polyethylene glycol monomethyl ether, polyethylene glycol oleate, and polyethylene glycol laurate; The glycyrrhetinic acid derivative is selected from a fat-soluble derivative of glycyrrhetinic acid, and the fat-soluble derivative of glycyrrhetinic acid is selected from one or more of DSPE-glycyrrhetinic acid, DSPE-PEG-glycyrrhetinic acid, glycyrrhetinic acid fatty acid ester, and glycyrrhetinic acid succinate; The second auxiliary material is a food and / or pharmaceutically acceptable raw and auxiliary material, and is different from the first emulsifier, the second emulsifier, and the first auxiliary material. The total weight percentage of the oral microemulsion is 100% by weight percentage, and the oral microemulsion comprises the following components: EPA glyceride raw material 1%~30%; First emulsifier 0.1%~10%; Second emulsifier 0%~10%; First auxiliary material 0.05%~10%; Second auxiliary material 0~40%; and Water; wherein the mass content of EPA in the EPA glyceride raw material is ≥45%, and the mass percentage of triglyceride in the glyceride component is ≥58%; The first emulsifier is a phospholipid with an iodine value ≥80; In the phospholipid component of the first emulsifier, the mass percentage of phosphatidylcholine is ≥50%; The mass ratio of EPA to phosphatidylcholine in the first emulsifier is ≤8:1; The components of the second emulsifier are all different from those of the first emulsifier; The first auxiliary material comprises at least one of a liver targeting component and a PEG modified component; the liver targeting component is selected from one or more of glycyrrhetinic acid and its derivatives, galactose, mannose, hyaluronic acid, and bile acid; the liver targeting component is selected from one or more of a liver targeting molecule and a modified liver targeting molecule, wherein the modified liver targeting molecule contains one or more of a PEG modified lipid unit and a non-PEG modified lipid modification unit; the PEG modified component is selected from one or more of distearoylphosphatidylethanolamine-polyethylene glycol, polyethylene glycol-stearate, vitamin E succinate polyethylene glycol ester, soybean phosphatidyl ethanolamine-polyethylene glycol monomethyl ether, polyethylene glycol oleate, and polyethylene glycol laurate; The glycyrrhetinic acid derivative is selected from a fat-soluble derivative of glycyrrhetinic acid, and the fat-soluble derivative of glycyrrhetinic acid is selected from one or more of DSPE-glycyrrhetinic acid, DSPE-PEG-glycyrrhetinic acid, glycyrrhetinic acid fatty acid ester, and glycyrrhetinic acid succinate; The second auxiliary material is a food and / or pharmaceutically acceptable raw and auxiliary material, and is different from the first emulsifier, the second emulsifier, and the first auxiliary material. The oral microemulsion is a PEG-modified microemulsion, the PEG-modified raw material in the oral microemulsion is 0.01%~10% by weight; and / or, The PEG-modified component is selected from one or more of polyethylene glycol-modified lipid components and polyethylene glycol monomethyl ether-modified lipid components; and / or, The oral microemulsion is an oil-in-water structure; and / or, The average droplet size in the oral microemulsion is less than 500 nm.

4. The oral microemulsion of claim 1, wherein, The EPA glyceride raw material is selected from one or more of concentrated EPA glyceride and re-esterified EPA glyceride; and / or, The EPA glyceride raw material is selected from one or more of EPA glycerides having the following compositions: EPA 57%~60% + DHA 0~10%, EPA 57%~60% + DHA 10~20%, EPA 57%~60% + DHA 20~30%, EPA 57%~60% + DHA 30%~40%, EPA 60%~70% + DHA 0~10%, EPA 60%~70% + DHA 10~20%, EPA 60%~70% + DHA 20~30%, EPA 60%~70% + DHA 30% above, EPA 70%~80% + DHA 0~10%, EPA 70%~80% + DHA 10~20%, EPA 70%~80% + DHA 20% above, EPA 80%~90%, and EPA >90%; and / or, The phospholipid component in the first emulsifier is from one or more of soybean phospholipids, sunflower phospholipids, and synthetic phospholipids; and / or, The second emulsifier is selected from one or more of phospholipids different from the first emulsifier, sucrose esters, citric acid fatty acid glycerides, fatty acid glycerides, polysorbates, fatty acid sorbitans, polyoxyethylene fatty acid esters, poloxamers, alginates, and caseinate; and / or, The liver targeting component is selected from one or more of glycyrrhetic acid, galactose, mannose, hyaluronic acid, and bile acid; and / or, The PEG-modified component is selected from one or more of polyethylene glycol-stearate and vitamin E succinate polyethylene glycol ester; and / or, The second auxiliary material is selected from one or more of nutritional supplements, antioxidants, co-emulsifiers, oils, flavorings, and pH adjusters; and / or, The average droplet size in the oral microemulsion is ≤300 nm; and / or, In the oral microemulsion, the weight percentage of water is 65%~95%.

5. The oral microemulsion of claim 4, wherein, The phospholipid component in the first emulsifier is a polyene phospholipid of soybean origin; and / or, The second emulsifier does not contain high-iodine-value phosphatidylcholine; and / or, The liver targeting component contains glycyrrhetic acid and its derivatives; and / or, the liver targeting component comprises one or more of DSPE-PEG-galactose, DSPE-PEG-mannose, DSPE-glycyrrhetinic acid, DSPE-PEG-glycyrrhetinic acid and DSPE-hyaluronic acid; the PEG-modified component is selected from one or more of PEG-modified phospholipids and PEG-modified vitamin E esters; and / or, the nutritional supplement is selected from one or more of vitamin A, vitamin E, vitamin B complex, vitamin D, silymarin, glucomannan and branched chain amino acids; and / or, the oil is selected from one or more of soybean oil, medium-chain triglyceride, olive oil, flaxseed oil, walnut oil, sea buckthorn oil, fructus coicis oil, grape seed oil, ginger oil, coconut oil, camellia oil, rose oil, peppermint oil and lemon oil; and / or, the antioxidant is selected from one or more of sodium sulfite, sodium bisulfite, sodium metabisulfite, vitamin C and esters thereof, tocopherol and esters thereof.

6. The oral microemulsion according to claim 5, wherein, the second emulsifier does not comprise a phospholipid component; and / or, the PEG-modified component is selected from one or more of distearoylphosphatidylethanolamine-polyethylene glycol and vitamin E succinate polyethylene glycol ester; and / or, the vitamin E is selected from one or more of α-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol, α-tocotrienol, β-tocotrienol, γ-tocotrienol and δ-tocotrienol; and / or, the vitamin B complex is selected from one or more of vitamin B1, vitamin B2, niacin, pantothenic acid, vitamin B6, vitamin B12, folic acid and vitamin B7.

7. A process for the preparation of an oral microemulsion according to any one of claims 1 to 6, characterized in that, comprising the steps of: preparing an oil phase under heating conditions, the oil phase comprising oil phase components including the EPA glyceride raw material; preparing an aqueous phase under heating conditions, the aqueous phase comprising aqueous phase components including phospholipids and an aqueous solvent; mixing the oil phase and the aqueous phase, and shearing to prepare an oil-in-water microemulsion; subjecting the oil-in-water microemulsion to high-pressure homogenization to obtain a nanoemulsion with a droplet particle size of less than 500 nm; optionally, filtering, packaging and sterilizing the nanoemulsion.

8. Use of the oral microemulsion according to any one of claims 1-6 or obtained by the preparation method of claim 7 in the preparation of a medicament for preventing and / or treating a fat accumulation-related disease, the fat accumulation-related disease is one or more of fatty liver, liver damage and hepatitis.

9. Use according to claim 8, characterized in that, the fatty liver is non-alcoholic fatty liver.

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