CN116407502A

CN116407502A - Oral microemulsion with high EPA content and preparation method and application thereof

Info

Publication number: CN116407502A
Application number: CN202111640996.9A
Authority: CN
Inventors: 甘勇; 章莹; 王晓丽; 周尔芬; 张馨欣; 朱春柳; 周奕含
Original assignee: Shanghai Institute of Materia Medica of CAS
Current assignee: Shanghai Institute of Materia Medica of CAS
Priority date: 2021-12-29
Filing date: 2021-12-29
Publication date: 2023-07-11

Abstract

The invention relates to an oral microemulsion with high EPA content and a preparation method thereof. The oral microemulsion comprises, by weight, 1% -50% of EPA glyceride raw materials, 0.1% -10% of high-iodine-value phospholipids (including PC), 0.01% -10% of other emulsifying agents, 0% -10% of first auxiliary materials for assisting in the enrichment of EPA livers, 0% -40% of other raw auxiliary materials and a proper amount of water which are acceptable in foods and/or pharmacy, wherein the EPA glyceride raw materials provide high EPA content, and the first auxiliary materials provide liver targeting components and/or PEG modified components. The oral microemulsion provided by the invention can realize the synergistic absorption of EPA and phospholipid, further can reduce the hydrolysis of EPA by lipoprotein lipase when the EPA enters the blood circulation by means of the first auxiliary material, and can promote the enrichment of EPA in the liver while improving the bioavailability of EPA, so that the oral microemulsion can be used for preventing and/or treating diseases such as fatty liver and the like.

Description

Oral microemulsion with high EPA content and preparation method and application thereof

Technical Field

The invention 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

Fatty liver refers to a disorder in which there is excessive accumulation of fat in liver cells due to various causes. Fat deposition in liver cells, starting from simple fatty liver, can progress to steatohepatitis with varying degrees of liver fibrosis, eventually leading to cirrhosis and even to primary hepatocellular carcinoma. Fatty liver has become a global problem in recent years with changes in people's lifestyle. At present, the increasing speed of the incidence rate of fatty liver in China is more than twice that of fatty liver in western countries, and the management and diagnosis of fatty liver are very important.

The pathogenesis of fatty liver is very complex, common causes include long-term drinking or overnutrition, obesity and the like, and the medical community considers that the initial development of fatty liver is caused by triglyceride accumulation, and excessive triglyceride in liver cells has various sources including fatty acid excessively ingested in diet, peripheral fat increase caused by Insulin Resistance (IR) of adipose tissue, liver neonatal fat increase caused by hyperinsulinemia and the like. Fat accumulation causes liver cells to be vulnerable to a variety of hepatotoxic damaging factors including lipid peroxidation, oxidative stress in the body, mitochondrial dysfunction, etc., ultimately leading to inflammatory responses in the liver, cell damage and tissue fibrosis.

There is no specific drug or preparation approved for long-term treatment of fatty liver, statin (lipid lowering drug), glitazone (insulin sensitizer), antioxidant and metformin etc. as potential effective drugs for treating fatty liver. However, statins can themselves cause various common types of drug-induced liver injury, and can cause double injury during use. The glitazones can improve steatosis and also cause obvious weight increase of patients, and the long-term use safety of the glitazones is not clear. Random clinical trials of antioxidants (vitamin E and N-acetylcysteine, etc.) have failed to confirm the improvement of liver histological status by drugs and their effects may vary with age, dose and lifestyle changes. The metformin drug treatment of nonalcoholic fatty liver has not yet achieved consistent and clear results clinically.

In addition to drug therapy, lifestyle changes are often considered as the primary clinical recommendation, and are the first step in the management of fatty liver disorders. Physical exercise is generally performed and reducing total fat intake is effective in reducing steatosis. In addition, the components of the diet affect metabolic and endocrine functions and overall energy balance. It is generally recommended to reduce the intake of saturated fatty acids, trans fatty acids and fructose. In contrast, increasing polyunsaturated fatty acid (PUFA) intake has been shown to reduce the risk of fatty liver and this class of substances is more acceptable to humans, without the strong adverse effects of the drug, and can be taken for a long period of time.

Studies have shown that Omega-3 polyunsaturated fatty acids (Omega-3 PUFAs), mainly comprising eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), alpha-linolenic acid (ALA) and the like, are involved in the processes of lipoprotein breakdown, lipid oxidation, lipid metabolism and the like and show a certain beneficial effect in the treatment of liver diseases. Omega-3 PUFAs can be synthesized by ALA in humans, but endogenous conversion efficiency is low and therefore it is necessary to ensure that the in vivo supply is sufficient by regular ingestion from the diet. Common Omega-3 PUFA-rich oils include algae oil, fish oil, krill oil, seal oil, etc., and high levels of Omega-3 PUFA are commercially available by enzymatic transesterification.

Regarding the application report of Omega-3 polyunsaturated fatty acid preparation in liver disease treatment, patent document CN101757497a describes a combined capsule of EPA and traditional Chinese medicine, the oily substance of which is unfavorable for digestion and absorption by human body, patent document CN104856985A describes EPA ethyl ester soft capsule, no positive clinical result has been obtained in fatty liver treatment, patent document CN110312509a describes EPA/DHA composition for treating or preventing liver disease, but provides an injectable fat emulsion formulation. The Omega-3 PUFA type formulations reported so far have the following general problems: (1) The existing products are mainly in the form of capsules, are not easy to be absorbed by human bodies, and can not reach the concentration of active fatty acid in the liver required by treatment; in addition, the digestion and absorption of Omega-3 PUFA in the capsule is affected by food intake, the bioavailability in the fasting state is lower than 50% of that in the satiety state, and the administration method needs to be strictly controlled. (2) Although the bioavailability of the injection fat emulsion reported in some documents is improved by improving the dosage form, the injection fat emulsion cannot realize the enrichment of active fatty acid in the liver at a high concentration, and has poor effect of improving the fatty liver.

Therefore, there is a need to develop a new formulation capable of efficiently enriching active fatty acids in the liver.

Disclosure of Invention

In view of the above, an object of the present invention is to provide an oral microemulsion capable of efficiently enriching eicosapentaenoic acid (EPA) in the liver, which has a high content of EPA and a phospholipid having a high iodine value (including phosphatidylcholine, PC), and in which EPA and PC are controlled to be in a proper ratio, and EPA and PC can be absorbed in a synergistic manner, which contributes to oral absorption and enrichment of EPA in the liver, and which can sufficiently exert a preventive and/or therapeutic effect of EPA on diseases such as fatty liver. It is another object of the present invention to provide applications of the above oral microemulsions including, but not limited to, applications in medical foods, health foods, pharmaceuticals and the like.

The above object can be achieved by the following means.

According to a first aspect of the present invention, there is provided an oral microemulsion comprising, in weight percent, 100% by total weight of the oral microemulsion:

wherein the mass content of EPA in the EPA glyceride raw material is more than or equal to 45%, and the mass ratio of triglyceride in the glyceride component is more than or equal to 58%;

the first emulsifier is phospholipid with iodine value more than 70;

in the phospholipid component of the first emulsifier, the mass ratio of phosphatidylcholine is more than or equal to 50%;

The mass ratio of EPA to phosphatidylcholine is less than or equal to 8:1;

the components of the second emulsifier are different from those of the first emulsifier;

the first auxiliary material comprises at least one of a targeting component and a PEG modified component;

the second auxiliary material is a raw auxiliary material which is acceptable in food and/or pharmacy and is different from the first emulsifier, the second emulsifier and the first auxiliary material.

In some preferred embodiments of the present invention, the oral microemulsion comprises the following components in weight percent:

in some embodiments of the invention, the minimum weight percent of water in the oral microemulsion is 65%; and/or the number of the groups of groups,

the oral microemulsion comprises an oil phase component and a water phase component, wherein the weight percentage of the oil phase component is less than or equal to 30%; and/or the number of the groups of groups,

in the EPA glyceride raw material, the mass content of EPA is more than or equal to 57%; and/or the number of the groups of groups,

in the EPA glyceride raw material, the mass ratio of monoglyceride in the glyceride component is less than 30%, and the mass ratio of diglyceride in the glyceride component is less than 30%; and/or the number of the groups of groups,

the first emulsifier is phospholipid with iodine value more than or equal to 90; and/or the number of the groups of groups,

of all phospholipid components of the oral emulsion, the mass ratio of phospholipids with iodine value >70 is greater than 90%; and/or the number of the groups of groups,

In the phospholipid component of the first emulsifier, the mass ratio of phosphatidylcholine is more than or equal to 70 percent; and/or the number of the groups of groups,

the mass ratio of EPA to phosphatidylcholine is less than or equal to 5:1; and/or the number of the groups of groups,

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 lipid modification unit without PEG modification; and/or the number of the groups of groups,

the oral microemulsion is PEG modified microemulsion, and the weight percentage of the PEG modified raw material in the oral microemulsion is 0.01% -10%; and/or the number of the groups of groups,

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 number of the groups of groups,

the oral microemulsion is of an oil-in-water structure; and/or the number of the groups of groups,

the average particle size of the liquid drops in the oral microemulsion is less than 500nm.

In some embodiments of the invention, the EPA glyceride feedstock is selected from one or more of the EPA glycerides having the following composition: 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 number of the groups of groups,

The phospholipid component in the first emulsifier is derived from one or more of soybean phospholipid, sunflower phospholipid, egg yolk phospholipid and synthetic phospholipid; and/or the number of the groups of groups,

the second emulsifier is selected from one or more of phospholipids, sucrose esters, citric acid fatty acid glycerides, polysorbates, fatty acid sorbitan, polyoxyethylene fatty acid esters, span, poloxamers, alginates, and caseinates that are different from the first emulsifier; and/or the number of the groups of groups,

the liver targeting component is selected from one or more of glycyrrhetinic acid and derivatives thereof, galactose and derivatives thereof, mannose and derivatives thereof, hyaluronic acid and derivatives thereof, bile acid and derivatives thereof; and/or the number of the groups of groups,

the PEG modified component is one or more selected from distearyl phosphatidylethanolamine-polyethylene glycol, polyethylene glycol-stearate, vitamin E succinic acid polyethylene glycol ester, soybean phosphatidylethanolamine-polyethylene glycol monomethyl ether, polyethylene glycol oleate and polyethylene glycol laurate; and/or the number of the groups of groups,

the second auxiliary material is selected from one or more of nutritional supplements, antioxidants, auxiliary emulsifying agents, grease, flavoring agents and pH regulators; and/or the number of the groups of groups,

The average particle size of liquid drops in the oral microemulsion is less than or equal to 300nm; and/or the number of the groups of groups,

in the oral microemulsion, the weight percentage of water is 65-95%.

In some embodiments of the invention, the phospholipid component in the first emulsifier is a soybean-derived polyene phospholipid; and/or the number of the groups of groups,

the second emulsifier does not contain phosphatidylcholine with high iodine value; and/or the number of the groups of groups,

the liver targeting component contains glycyrrhetinic acid and derivatives thereof, wherein the glycyrrhetinic acid and derivatives thereof are selected from fat-soluble derivatives of glycyrrhetinic acid; and/or the number of the groups of groups,

the liver targeting ingredient contains 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 phospholipid and PEG modified vitamin E ester; and/or the number of the groups of groups,

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

the oil is one or more selected from soybean oil, medium chain triglyceride, olive oil, linseed oil, walnut oil, sea buckthorn oil, coix seed oil, grape seed oil, ginger oil, coconut oil, camellia oil, rose oil, peppermint oil and lemon oil; and/or the number of the groups of groups,

The antioxidant is selected from one or more of sodium sulfite, sodium bisulphite, sodium metabisulfite, vitamin C and esters thereof, tocopherol and esters thereof.

In some embodiments of the invention, the second emulsifier is free of a phospholipid component; and/or the number of the groups of groups,

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; and/or the number of the groups of groups,

the PEG modified component is one or more selected from distearoyl phosphatidylethanolamine-polyethylene glycol and vitamin E succinic acid polyethylene glycol ester; and/or the number of the groups of groups,

the vitamin E is selected from one or more of alpha-tocopherol, beta-tocopherol, gamma-tocopherol, delta-tocopherol, alpha-tocotrienol, beta-tocotrienol, gamma-tocotrienol and delta-tocotrienol; and/or the number of the groups of groups,

the vitamin B group is one or more selected from vitamin B1, vitamin B2, nicotinic acid, pantothenic acid, vitamin B6, vitamin B12, folic acid and vitamin B7.

In some embodiments of the invention, the oral microemulsion is a PEG-modified microemulsion comprising 0.01% to 10% by total mass of the PEG-modified starting material.

According to a second aspect of the present invention, there is provided a method of preparing an oral microemulsion according to the first aspect of the present invention, comprising the steps of:

preparing an oil phase under heating from an oil phase component comprising the EPA glyceride feedstock;

mixing an aqueous phase component comprising a phospholipid with an aqueous solvent, preparing an aqueous phase under heating;

mixing the oil phase and the water phase, shearing and stirring to prepare oil-in-water microemulsion;

carrying out high-pressure homogenization treatment on the oil-in-water microemulsion to prepare nanoemulsion with the droplet size smaller than 500 nm;

optionally, the nanoemulsion is filtered, packaged and sterilized.

According to a third aspect of the present invention there is provided the use of an oral microemulsion according to the first aspect of the present invention, or the oral microemulsion obtained by the method of preparation according to the second aspect of the present invention.

In some embodiments of the invention, the use of the oral microemulsion in the manufacture of a medicament for preventing and/or treating a disease associated with fat accumulation, or in medical foods, health foods, is provided.

In some preferred embodiments of the present invention, the fat accumulation-related disease is selected from one or more of fatty liver, liver injury, hepatitis, preferably, the fatty liver is a non-alcoholic fatty liver.

The inventor also discovers through experimental exploration that EPA active ingredients play an important role in PUFA, and the curative effect of treating fatty liver is obviously related to EPA purity in fish oil raw materials. In addition, the glycerol ester type EPA microemulsion can make the enrichment of EPA in liver better than the EPA EE microemulsion when being used in combination with other components in the invention. Thus, the present invention provides an oral microemulsion prepared from a glycerol-type starting material having a high EPA content.

The oral microemulsion provided by the invention can make up the blank that no specific medicine or preparation for long-term treatment of fatty liver is approved at present. The oral microemulsion provided by the invention adopts EPA glyceride raw materials with high EPA purity, so that high-content active EPA is provided, phospholipid with high iodine value (comprising phosphatidylcholine and PC) is provided by phospholipid raw materials, wherein the content of EPA in the EPA glyceride raw materials is more than or equal to 45%, the mass ratio of EPA triglyceride in glyceride components is more than or equal to 58%, the iodine value of phospholipid in the first emulsifier is more than 70, the mass ratio of EPA to PC is controlled to be less than or equal to 8:1, the synergistic absorption of EPA and phospholipid can be realized, the EPA can enter the liver, the phospholipid content in the oral microemulsion with the administration dosage is far lower than the administration dosage of polyene phosphatidylcholine capsules in the market, and the polyene phosphatidylcholine in the market products is about 1.8-2.3 times of the oral microemulsion. According to the oral microemulsion provided by the invention, the first auxiliary material (liver targeting component and/or PEG modified component) is further added, so that the EPA can be enriched in the liver in a targeting effect and/or enzymolysis inhibition mode, and compared with the first auxiliary material with high water solubility, the first auxiliary material with high fat solubility is easier to combine with the EPA microemulsion and chylomicron formed after entering the body, so that the effects of improving the bioavailability of EPA and simultaneously promoting the EPA to be enriched in the liver are better exerted. The oral microemulsion provided by the invention can be used for treating fat accumulation related diseases, especially nonalcoholic fatty liver disease (NAFLD), in a 4-6 week or longer administration period, and has good effects on reducing body weight, blood plasma triglyceride level, body insulin resistance, oxidative stress and inflammatory reaction.

Compared with EPA soft capsules, the oral microemulsion provided by the invention can improve the bioavailability of EPA by about 3 times.

According to the invention, the PEG modified raw material can be provided by the first emulsifier, the second emulsifier and/or the first auxiliary material, so that the PEG modified oral microemulsion can be obtained, and the hydrolysis of EPA by lipoprotein lipase when the EPA enters the blood circulation can be reduced, thereby promoting the EPA to gather in the liver. Further combined with liver targeting components, the method not only can greatly improve the enrichment of EPA in the liver, but also can effectively improve the bioavailability.

The inventor discovers through experimental exploration that the content of phosphatidylcholine and the selection of phospholipid unsaturation degree have influence on the drug effect. In the invention, when the iodine value of the phospholipid is more than or equal to 70, the weight ratio of EPA to phosphatidylcholine is less than or equal to 8:1, the blood lipid reducing effect is better, and the treatment effect on fatty liver is better.

The fish oil oral microemulsion provided by the invention can reduce ALT and AST levels formed by inducing NASH by high-fat diet, alleviate inflammatory reaction, prevent lipid peroxidation and reduce serum TG and TC levels, so that the oral microemulsion provided by the invention can be used as a potential medicament for preventing and/or treating fatty liver, in particular nonalcoholic fatty liver disease (NAFLD).

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more fully understand the present application and its advantageous effects, the following brief description will be given with reference to the accompanying drawings, which are required to be used in the description of the embodiments.

FIG. 1 shows the NAS scoring results for rat fatty liver of different formulations according to one embodiment of the present invention;

FIG. 2 is a graph of change in blood glucose levels versus time for rats four weeks after administration of various formulations in one embodiment of the invention;

FIG. 3 is a graph showing the results of AUC calculations of blood glucose levels in rats four weeks after administration of various formulations in one embodiment of the invention;

FIG. 4 shows the NAS scoring results for rat fatty liver for various formulations according to one embodiment of the present invention;

FIG. 5 shows HE staining of rat liver tissue morphology with different formulations according to one embodiment of the invention.

Detailed Description

The present invention will be described in further detail with reference to the drawings, embodiments and examples. It should be understood that these embodiments and examples are provided solely for the purpose of illustrating the invention and are not intended to limit the scope of the invention in order that the present disclosure may be more thorough and complete. It will also be appreciated that the present invention may be embodied in many different forms and is not limited to the embodiments and examples described herein, but may be modified or altered by those skilled in the art without departing from the spirit of the invention, and equivalents thereof fall within the scope of the present application. Furthermore, in the following description, numerous specific details are set forth in order to provide a more thorough understanding of the invention, it being understood that the invention may be practiced without one or more of these details.

Unless defined otherwise, 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 invention belongs. The terminology used herein in the description of the invention is for the purpose of describing the embodiments and examples only and is not intended to be limiting of the invention.

Terminology

Unless otherwise indicated or contradicted, terms or phrases used herein have the following meanings:

the term "and/or," "and/or," as used herein, includes any one of two or more of the listed items in relation to each other, as well as any and all combinations of the listed items in relation to each other, including any two of the listed items in relation to each other, any more of the listed items in relation to each other, or all combinations of the listed items in relation to each other. It should be noted that, when at least three items are connected by a combination of at least two conjunctions selected from "and/or", "or/and", "and/or", it should be understood that, in this application, the technical solutions certainly include technical solutions that all use "logical and" connection, and also certainly include technical solutions that all use "logical or" connection. For example, "a and/or B" includes three parallel schemes A, B and a+b. For another example, the technical schemes of "a, and/or B, and/or C, and/or D" include any one of A, B, C, D (i.e., the technical scheme of "logical or" connection), and also include any and all combinations of A, B, C, D, i.e., any two or three of A, B, C, D, and also include four combinations of A, B, C, D (i.e., the technical scheme of "logical and" connection).

The term "plurality" as used herein refers to "plurality" and "plurality" as used herein, unless otherwise specified, refers to 2 or more in number. For example, "one or more" means one kind or two or more kinds.

As used herein, "a combination thereof," "any combination thereof," and the like include all suitable combinations of any two or more of the listed items.

The "suitable" in the "suitable combination manner", "suitable manner", "any suitable manner" and the like herein refers to the fact that the technical scheme of the present invention can be implemented, the technical problem of the present invention is solved, and the technical effect expected by the present invention is achieved.

Herein, "preferred", "better", "preferred" are merely to describe better embodiments or examples, and it should be understood that they do not limit the scope of the invention.

In the present invention, "further", "still further", "particularly" and the like are used for descriptive purposes to indicate differences in content but should not be construed as limiting the scope of the invention.

In the present invention, "optional" means optional or not, that is, means any one selected from two parallel schemes of "with" or "without". If multiple "alternatives" occur in a technical solution, if no particular description exists and there is no contradiction or mutual constraint, then each "alternative" is independent. In the present invention, "optionally containing", and the like are described, meaning "containing or not containing". "optional component X" means that component X is present or absent.

In the present invention, the terms "first", "second", "third", "fourth", etc. are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or quantity, nor as implying an importance or quantity of a technical feature being indicated. Moreover, the terms "first," "second," "third," "fourth," and the like are used for non-exhaustive list description purposes only, and are not to be construed as limiting the number of closed forms.

In the invention, the technical characteristics described in an open mode comprise a closed technical scheme composed of the listed characteristics and also comprise an open technical scheme comprising the listed characteristics.

In the present invention, a numerical range (i.e., a numerical range) is referred to, and optional numerical distributions are considered to be continuous within the numerical range and include two numerical endpoints (i.e., a minimum value and a maximum value) of the numerical range and each numerical value between the two numerical endpoints unless otherwise specified. When a numerical range merely points to integers within the numerical range, both end integers of the numerical range are included, as well as each integer between the two ends, unless expressly stated otherwise. Further, when a plurality of range description features or characteristics are provided, these ranges may be combined. In other words, unless otherwise indicated, the ranges disclosed herein are to be understood to include any and all subranges subsumed therein.

In the present invention, the terms mean the divisor, and the fluctuation range is usually.+ -. 10% and may further mean.+ -. 8%,.+ -. 5%,.+ -. 3%, etc., unless otherwise specified. The divisor in the present invention provides both the listed values and the interval of values represented by the divisor. For example, about 200nm provides a technical scheme of 200nm and a technical scheme of a numerical interval formed by 200nm + -fluctuation range.

The temperature parameter in the present invention is not particularly limited, and may be a constant temperature treatment or may vary within a predetermined temperature range. It should be appreciated that the constant temperature process described allows the temperature to fluctuate within the accuracy of the instrument control. Allows for fluctuations within a range such as + -5 ℃, + -4 ℃, + -3 ℃, + -2 ℃, + -1 ℃.

In the present invention,% (w/w) and wt% each represent weight percent.

All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Unless otherwise contradicted by purpose and/or technical solution of the present application, the cited documents related to the present invention are incorporated by reference in their entirety for all purposes. When reference is made to a cited document in the present invention, the definitions of the relevant technical features, terms, nouns, phrases, etc. in the cited document are also incorporated. In the case of the cited documents, examples and preferred modes of the cited relevant technical features are incorporated into the present application by reference, but are not limited to the embodiments that can be implemented. It should be understood that when a reference is made to the description herein, it is intended to control or adapt the present application in light of the description herein.

Abbreviations herein, unless otherwise specified, mean the following: PUFA means polyunsaturated fatty acids, omega-3 PUFA means Omega-3 polyunsaturated fatty acids, EPA means eicosapentaenoic acid, DHA means docosahexaenoic acid, ALA means alpha-linolenic acid, DSPE means distearoyl phosphatidylethanolamine, PEG means polyethylene glycol, TG means triglycerol, TPGS means vitamin E polyethylene glycol succinate, DSPE means distearoyl phosphatidylethanolamine, PC means phosphatidylcholine, ALT means glutamic pyruvic transaminase, AST means glutamic oxaloacetic transaminase, SOD means superoxide dismutase, MDA means malondialdehyde, NAFLD means non-alcoholic fatty liver disease.

In the present invention, "EPA" alone refers to EPA molecules or EPA units in a molecule, unless otherwise specified. For example, the mass content of EPA (EPA units) in the EPA glyceride raw material, the mass ratio of EPA (PEA units) to phosphatidylcholine in the first emulsifier.

In the present invention, the degree of unsaturation characterizing the phospholipid is mainly iodine value, and the term "average iodine value" is used without any particular limitation.

As used herein, the "phospholipid having a high iodine value", such as a phospholipid having an iodine value of >70, may be a phospholipid having a higher iodine value, such as a phospholipid having an iodine value of 90 or more, without particular limitation.

The term "enzymatic hydrolysis" as used herein refers to, without limitation, hydrolysis of fat by pancreatic lipase in the gastrointestinal tract, and hydrolysis by lipoprotein lipase in the blood after reconstitution of the fat by EPA. Is hydrolyzed by lipoprotein lipase when entering blood circulation.

The medium chain triglyceride used in the invention is also called as medium chain triglyceride, and is called medium chain triglycerides in English and is called MCT for short. In the national food safety standards, medium chain triglycerides may be used as food materials or emulsifiers.

As used herein, a "branched chain amino acid" is selected from 3 amino acids having a branched side chain; the branched-chain amino acids used in the present invention may be a combination of amino acids capable of promoting metabolism in the body, for example, selected from leucine, valine and isoleucine.

In the present invention, polyethylene glycol (PEG) and Polyethylene Oxide (POE) have the same meaning and may be used interchangeably. The molecular weight of PEG is not particularly limited, and may be a number average molecular weight or a weight average molecular weight.

In the present invention, unless otherwise specified, "above" and "below" each independently include the present number.

In the present invention, ". Gtoreq." and "greater than or equal to" have the same meaning, and are used interchangeably, and each represents greater than or equal to. "less than or equal to" and "less than or equal to" have the same meaning, and are used interchangeably, and each means less than or equal to.

First aspect of the invention

According to a first aspect of the present invention, there is provided an oral microemulsion capable of efficiently enriching eicosapentaenoic acid (EPA) in the liver, which has a high content of EPA (for example, EPA content in EPA glyceride raw material is not less than 45%) and a high iodine value phospholipid (for example, iodine value > 70), EPA and PC are controlled to be in a proper ratio (for example, not more than 8:1), EPA and PC can be synergistically absorbed, which is helpful for EPA oral absorption and enrichment in the liver, and can fully exert the preventive and/or therapeutic effects of EPA on fatty liver and other diseases.

In some embodiments of the present invention, there is provided an oral microemulsion having a total weight percent of 100%, the oral microemulsion comprising the following components:

the first emulsifier is phospholipid with iodine value more than 70;

the mass ratio of EPA to phosphatidylcholine is less than or equal to 8:1;

the first auxiliary material comprises at least one of liver targeting components and PEG modified components;

the second auxiliary material is an auxiliary material which is acceptable in food and/or pharmacy and is different from the first emulsifier, the second emulsifier and the first auxiliary material.

The first and second adjuvants may be optional components.

Preferably, the mean particle size of the droplets in the oral microemulsion is less than 500nm.

In some embodiments of the present invention, the oral microemulsion comprises the following specific components: EPA glyceride raw material, phospholipid, other emulsifier (non-phospholipid), a first auxiliary material (auxiliary material for assisting EPA to enrich in liver), water and a second auxiliary material (other food and/or pharmaceutically acceptable raw auxiliary materials).

In some embodiments, the sum of the weight percentages of EPA glyceride feedstock, first emulsifier, second emulsifier, first adjunct, second adjunct and water does not exceed 100%, preferably one of them is 100%.

In some embodiments of the invention, the oral microemulsion comprises, in weight percent, 1% to 50% EPA glyceride raw material, 0.1% to 10% high iodine value phospholipid (including PC), 0.01% to 10% other emulsifier (noted as second emulsifier), 0% to 10% first auxiliary material to aid in EPA liver enrichment, 0% to 40% other food and/or pharmaceutically acceptable raw auxiliary material, and an appropriate amount of water, wherein EPA glyceride raw material provides high levels of EPA, and the first auxiliary material provides liver targeting ingredients and/or PEG modified ingredients.

The oral microemulsion provided by the invention adopts EPA glyceride raw materials with high EPA purity, thereby providing high-content active EPA, providing phospholipid with high iodine value (comprising phosphatidylcholine and PC) through phospholipid raw materials, controlling the mass ratio of EPA and PC to be proper, realizing the cooperative absorption of EPA and phospholipid, further adding a first auxiliary material (liver targeting component and/or PEG modified component), promoting the enrichment of EPA in liver through a targeting effect and/or enzymolysis inhibition mode, and promoting the enrichment of EPA in liver while improving the bioavailability of EPA. The oral microemulsion provided by the invention can realize better effects on body weight and liver index in a 4-6 week or even longer administration period, aiming at fat accumulation related diseases, in particular to nonalcoholic fatty liver disease (NAFLD).

The inventor discovers through experimental exploration that EPA is a key active fatty acid for treating diseases such as fatty liver among various components of PUFA, and the curative effect for treating fatty liver has a relatively high correlation with EPA purity in fish oil raw materials. Thus, the present invention employs Omega-3 polyunsaturated fatty acid feedstock with high EPA content. Further, the inventors have found that the use of a glyceride type EPA microemulsion in combination with other ingredients of the present invention results in a better enrichment of EPA in the liver than EPA EE microemulsions. Accordingly, the inventors have conducted extensive experimental investigation and have screened EPA glyceride type raw materials as raw materials for providing active fatty acid components. Further, even at high concentrations, the formulations of the present invention made with high concentrations of EPA glycerides more readily reach the liver sites than the usual formulations made with high concentrations of EPA ethyl esters.

The formulation provided by the invention is a microemulsion formulation suitable for oral administration, preferably in an oil-in-water configuration. In the invention, EPA exists in a microemulsified form, the solubility in water is greatly improved, and liquid drops with the average particle diameter smaller than 500nm are beneficial to digestion and absorption of indissolvable component EPA by small intestine, improve bioavailability and assist EPA to reach liver parts. In addition, the present invention has found that microemulsified formulations help reduce the bioavailability differences of EPA in empty satiety states. In some embodiments of the present invention, the average particle size of the droplets in the oral microemulsion is less than 500nm, further, the average particle size may be less than or equal to 300nm, further still less than or equal to 250nm, still further still about 150nm. In some specific embodiments of the invention, the average particle size of the droplets in the oral microemulsion is about 150nm to 300nm.

EPA glyceride raw materials

The oral microemulsion provided by the invention contains EPA glyceride raw materials. In some embodiments of the present invention, the EPA glyceride source is present in an amount of 1% to 30% by weight, and may further be present in an amount of 4% to 20% by weight, for example, 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% by weight, and the like.

The main component of the EPA glyceride raw material used in the present invention is a polyunsaturated fatty acid ester including a large amount of EPA esters, in which a high content of EPA components can be provided. In the present invention, the mass content of EPA in the EPA glyceride raw material is preferably not less than 45%, more preferably not less than 50%, still more preferably not less than 57%. If the EPA content in the EPA glyceride source is low (e.g., < 20%), sufficient exposure to the active substance is not achieved after administration, and the fat metabolism burden of the patient is increased by the high content of inactive fatty acids in the product. In the present invention, the EPA glyceride type raw material having a high EPA content can reduce the intake of inactive fatty acids and prevent the burden of fat metabolism in the body. The method can effectively reduce the adverse effect on other indexes of the organism while effectively reducing the synthesis of glyceride by the liver. In some embodiments of the invention, the EPA is present in the EPA glyceride feedstock at a mass content of greater than or equal to 60%. In some embodiments of the invention, the mass content of EPA in the EPA glyceride raw material is 60% -70%. The mass ratio of EPA in the EPA glyceride raw material is, for example, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, or the like.

The EPA in the EPA glyceride raw material of the present invention is mainly in the form of EPA glyceride, and can provide a large amount of active EPA, and the glyceride component in the EPA glyceride raw material may be any one or any combination of monoglyceride, diglyceride and triglyceride. Preferably, the mass ratio of the triglyceride in the glyceride component is not less than 58%, further preferably, the mass ratio of the monoglyceride in the glyceride component is less than 30%, and the mass ratio of the diglyceride in the glyceride component is less than 30%. In some preferred embodiments, the mass fraction of triglycerides in the glyceride composition is greater than or equal to 70%. In some preferred embodiments, the mass ratio of triglycerides in the glyceride composition is 70%, 71%, 72%, 73%, 74%, 75%, 78%, 79%, 80%, 82%, 85%, 88%, 90%, 92%, 95%, etc.

In the present invention, the EPA glyceride raw material may be synthesized by hydrolyzing natural EPA triglyceride to release free fatty acid, separating and purifying, and then re-attaching EPA to the glycerol backbone, and the obtained EPA glyceride may be any one of EPA monoglyceride, EPA diglyceride, and EPA triglyceride, or any combination thereof.

In some embodiments of the invention, the EPA glyceride feedstock is from a concentrated or re-esterified EPA glyceride.

In some preferred embodiments of the invention, the EPA glyceride feedstock is selected from one or more of the EPA glycerides having the following composition: EPA 57% -60% + DHA 0-10%, EPA 57% -60% + DHA 10-ultra20%, 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%. Specific examples are Epax6015 TGN, kinOmega 7010TG, kinOmega 6020 TG, kinOmega 6015 TG, kinOmega E80 TG, incromega ^TM TG6015、KinOmega E95 TG。

First emulsifier (high iodine number phospholipid, iodine number)>70)

In the present invention, "high iodine number phospholipid" refers to a phospholipid having an iodine number >70. The first emulsifier may be a mixture of two or more phospholipids, in which case the iodine value of either phospholipid component satisfies >70.

The oral microemulsions provided by the present invention comprise phospholipids of high iodine value (denoted as first emulsifier, iodine value > 70) for achieving emulsification of the formulation. Among them, the phospholipid in the form of Phosphatidylcholine (PC) has a better effect of improving fatty liver, presumably because phosphatidylcholine is chemically consistent with important endogenous phospholipids. In some embodiments of the present invention, the high iodine value phospholipids are present in the oral microemulsion in an amount of 0.1% to 10% by weight, and may further be present in an amount of 0.5% to 5% by weight, such as, for example, 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%, and the like.

The first emulsifier of the present invention is a high iodine number phospholipid. In some embodiments, the first emulsifier is a phospholipid having an iodine value of 90 or greater.

In some embodiments of the invention, the first emulsifier comprises a phospholipid component having a mass ratio of phosphatidylcholine of greater than or equal to 50%, more preferably greater than or equal to 60%, and still more preferably greater than or equal to 70%. Examples thereof include 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%, and the like. The inventor also found that the unsaturation degree of the phospholipid can also have influence on the drug effect, and the phospholipid with higher unsaturation degree is better for treating the fatty liver. The unsaturation of a phospholipid may be characterized by an iodine value, with higher iodine values indicating higher unsaturation. When the iodine value is more than 70, the therapeutic effect on fatty liver is better. In some embodiments, the first emulsifier has an iodine value >70, further may be > 90, still further may be > 100. In some embodiments of the invention, the first emulsifier has an iodine value of 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 invention, the phospholipid component of the first emulsifier comprises Phosphatidylcholine (PC) in an amount of 50% or more by mass, and the iodine value of the first emulsifier is >70, which is preferable for treating fatty liver. In some preferred embodiments of the invention, the first emulsifier is a polyene phosphatidylcholine.

The inventors have also found that the mass ratio of EPA to PC also affects the efficacy and that it is more beneficial to treat fatty liver when the ratio is relatively low. In some embodiments of the invention, the mass ratio of EPA to phosphatidylcholine is less than or equal to 8:1, more preferably the mass ratio of EPA to phosphatidylcholine is less than or equal to 5:1. In some embodiments of the invention 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.

In some embodiments of the invention, the first emulsifier (high iodine value phospholipid) has an iodine value of 80 or more and a mass ratio of EPA to phosphatidylcholine of 8:1 or less, and has good blood lipid reducing effect and good fatty liver treatment effect. Further, the mass ratio of phosphatidylcholine in the phospholipid component of the first emulsifier is not less than 50%, more preferably not less than 60%, still more preferably not less than 70%.

In some embodiments of the invention, the high iodine number phospholipids (first emulsifier) used are from soy, egg yolk, synthetic phospholipids. In some embodiments, the phospholipid component in the first emulsifier is derived from one or more of soybean phospholipid, sunflower phospholipid, egg yolk phospholipid, and synthetic phospholipid.

In some preferred embodiments of the invention, the high iodine number phospholipid (first emulsifier) used is a soy-derived polyene phosphatidylcholine.

In some embodiments of the invention, the phospholipid component of the first emulsifier may be a modified phospholipid, for example, a phospholipid modified with a hydrophilic component such as PEG. In some embodiments of the invention, the PEG-modified phospholipid is selected from one or more of DSPE-PEG-galactose, DSPE-PEG-mannose, DSPE-PEG, and the like.

Any of the phospholipid components of the present invention may be an independent phospholipid molecule, a derivative of a phospholipid molecule, or a modified phospholipid.

The phospholipid component in the oral microemulsion is not limited to be provided by the first emulsifier, but may be provided by the second emulsifier. The second emulsifier provides a phospholipid component that is not a phospholipid with an iodine value of > 70. The phospholipid component of the second emulsifier may also be a modified phospholipid as described above.

The first emulsifier can also simultaneously perform other functions in the oral microemulsion, for example, the first emulsifier can also be used as a first auxiliary material, for example, the first emulsifier can be a liver targeting molecule modified by phospholipid with high iodine value, and the first emulsifier can also be PEG modified phospholipid with high iodine value. Specific examples thereof include DSPE-glycyrrhetinic acid, DSPE-hyaluronic acid, DSPE-PEG-galactose, DSPE-PEG-mannose, DSPE-PEG-glycyrrhetinic acid, and the like.

In some embodiments, the mass fraction of phospholipids having an iodine value >70 in all phospholipid components of the oral emulsion is greater than 90%.

In some embodiments, the mass fraction of phospholipids having an iodine value of 90 or more is greater than 90% of all phospholipid components of the oral emulsion.

Second emulsifier

The second emulsifier in the present invention does not contain the same components as the first emulsifier. I.e. the second emulsifier consists of a different composition than the first emulsifier.

In some embodiments of the present invention, the emulsifier component of the oral microemulsion may include other emulsifiers (noted as second emulsifiers) in addition to the first emulsifier (high iodine number phospholipids) to provide a flexible control of emulsification. In some embodiments of the invention, the second emulsifier is selected from one or more of other phospholipids (different from the phospholipids in the first emulsifier), sucrose esters, citric acid fatty acid glycerides, polysorbates, sorbitan fatty acid esters, polyoxyethylene fatty acid esters, span, poloxamers, alginates (sodium alginate is preferred independently), caseinates (sodium caseinate may be independently), and the like. In some embodiments of the present invention, the second emulsifier is present in the oral microemulsion in an amount of 0.01% to 10% by weight, and further may be 0.1% to 5% by weight, such as 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%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10% by weight, and the like.

In some embodiments, the second emulsifier is an emulsifier that does not comprise a high iodine number phospholipid.

In some embodiments, the second emulsifier does not comprise phosphatidylcholine of high iodine value.

In some embodiments, the second emulsifier comprises a phospholipid component, but these phospholipid components are different from the first emulsifier, i.e., are not high iodine number phospholipids.

In some embodiments, the second emulsifier comprises a phospholipid component, but are both saturated phospholipids.

In some embodiments, the second emulsifier is free of a phosphatidylcholine component.

In some embodiments, the second emulsifier is free of a phospholipid component.

First auxiliary material

The invention optionally comprises a first auxiliary material. The first adjuvant is used to provide an adjuvant that aids in the enrichment of EPA in the liver. The inventor researches find that improving the enrichment of EPA in the liver is beneficial to fully exerting the prevention and treatment effects of EPA on fatty liver and other diseases. EPA and phospholipids, cholesterol and apolipoproteins constitute chylomicrons which are transported into the body via lymph. The auxiliary materials with liver targeting effect are added, and the EPA and the phospholipid can be formed into chylomicron together in the absorption process, so that the EPA can be selectively delivered to liver parts. The auxiliary materials for reducing enzymolysis can indirectly realize liver enrichment of EPA by reducing the ingestion of EPA by other tissue parts in the transportation process.

In some embodiments of the present invention, the content of the first auxiliary material in the oral microemulsion is 0-10% by weight, and further may be 0.05% -5%. Specific examples thereof 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%.

In some embodiments of the present invention, the first auxiliary material is at least one of an auxiliary material having a liver targeting effect, an auxiliary material for reducing enzymolysis, and the like. In some embodiments of the invention, the first adjunct is at least one of a liver targeting ingredient, a PEG-modified ingredient (e.g., a PEG-modified lipid ingredient).

Wherein the auxiliary material with liver targeting effect comprises a liver targeting component. The liver targeting component can target the cell surface of disease focus such as fatty liver, bind corresponding receptor on liver cell membrane, etc. The liver targeting component may be a separate molecule (e.g., glycyrrhetinic acid, bile acid, etc.), or may be part of a molecule (e.g., DSPE-glycyrrhetinic acid, etc.). In some embodiments, the liver targeting ingredient is selected from one or more of a liver targeting molecule and a modified liver targeting molecule, further, the modified liver targeting molecule may contain one or more of a PEG modification unit and a lipid modification unit (including but not limited to a phospholipid modification unit). In some embodiments of the invention, the liver targeting ingredient is selected from one or more of glycyrrhetinic acid and derivatives thereof, galactose and derivatives thereof, mannose and derivatives thereof, hyaluronic acid and derivatives thereof, bile acid and derivatives thereof, and the like. In some embodiments of the invention, the liver targeting ingredient comprises one or more of DSPE-PEG-galactose, DSPE-PEG-mannose, DSPE-glycyrrhetinic acid, DSPE-PEG-glycyrrhetinic acid, DSPE-hyaluronic acid, and the like. In some embodiments of the present invention, the glycyrrhetinic acid derivative, the bile acid derivative, and the hyaluronic acid derivative are each independently preferably a corresponding phospholipid derivative, and further may be a phosphatidylethanolamine derivative. Examples of the derivatives of glycyrrhetinic acid include DSPE-glycyrrhetinic acid, glycyrrhetinic acid succinate, and the like. Examples of the hyaluronic acid derivative include DSPE-hyaluronic acid. In some embodiments of the invention, the liver targeting ingredient comprises glycyrrhetinic acid and derivatives thereof, further wherein the glycyrrhetinic acid and derivatives thereof are selected from the group consisting of fat-soluble derivatives of glycyrrhetinic acid. In some embodiments of the invention, the liver targeting ingredient is selected from glycyrrhetinic acid and derivatives thereof, further, glycyrrhetinic acid and derivatives thereof are selected from one or more of glycyrrhetinic acid, DSPE-PEG-glycyrrhetinic acid, glycyrrhetinic succinate, and the like.

In some embodiments, the liver targeting ingredient is selected from one or more of a liver targeting molecule and a modified liver targeting molecule, further comprising one or more of a PEG modified lipid unit and a lipid modified unit without PEG modification; further, examples of the lipid modification unit without PEG modification include a common lipid modification unit, and further include a fatty acid ester modification unit.

Wherein, the auxiliary materials for reducing enzymolysis refer to reducing the degradation of EPA by fatty acid enzymes. In some embodiments, the enzymolysis reducing adjuvant comprises a PEG-modified component, and a PEG protective layer may be formed on the surface of the microemulsion droplet. In some preferred embodiments, the PEG-modified component is a PEG-modified lipid component and/or a polyethylene glycol monomethyl ether-modified lipid component. The PEG modified lipid component is an amphipathic molecule, can participate in emulsification, is dissolved in a water phase as a hydrophilic end, and is coated in an oil phase, so that a protective layer of PEG can be formed for the surface of emulsified liquid drops, and the effect of preventing enzymolysis is achieved. The PEG modified lipid molecules may contain only PEG and lipid (such as polyethylene glycol fatty acid ester, polyethylene glycol phospholipid, etc.), and may also contain other components (such as DSPE-PEG-galactose).

In some embodiments of the invention, the PEG-modified component is selected from one or more of distearoyl phosphatidylethanolamine-polyethylene glycol, polyethylene glycol-stearate, vitamin E polyethylene glycol succinate, soybean phosphatidylethanolamine-polyethylene glycol monomethyl ether, polyethylene glycol oleate, polyethylene glycol laurate, and the like.

In some embodiments of the invention, the PEG-modified component is selected from one or more of PEG-modified phospholipids, PEG-modified vitamin E esters.

In some embodiments of the invention, the PEG-modified component is selected from one or more of DSPE-PEG-galactose, DSPE-PEG-mannose, DSPE-PEG, PEG-stearate, and the like.

In some embodiments of the invention, the PEG-modified component is vitamin E polyethylene glycol succinate (TPGS) PEG stearate and/or DSPE-PEG.

In some embodiments of the invention, the adjuvant having liver targeting effect is glycyrrhetinic acid and its derivatives, hyaluronic acid and its derivatives and/or bile acid.

It will be appreciated that the components of the present invention may perform more than one function, such as PEG modified ingredients in oral microemulsions (e.g. vitamin E polyethylene glycol succinate, PEG stearate, DSPE-PEG in particular) and may act as both an emulsifier (either the first emulsifier or the second emulsifier) and a first adjuvant. Taking PEG and phospholipid modified by liver targeting molecule as examples (specifically, for example, DSPE-PEG-galactose and DSPE-PEG-galactose), the phospholipid can be used as liver targeting auxiliary material and auxiliary material for reducing enzymolysis.

The PEG component in the oral microemulsion is not limited to be provided by the PEG-modified component in the first adjuvant, but may be provided simultaneously by the first emulsifier, the second emulsifier, the liver targeting component, and the like. For example, a PEG-modified high iodine number phospholipid (iodine number > 70) may serve as both the first emulsifier and the PEG-modified component. For example, PEG-modified common phospholipids (non-iodine value phospholipids) can be used as both the secondary emulsifier and PEG-modified component. For example, a PEG-modified liver targeting molecule can serve as both a liver targeting moiety and a PEG-modified moiety. Also for example, phospholipid-PEG-liver targeting molecules can act as both an emulsifier, a PEG-modified component, and a liver targeting component. In some embodiments, the oral microemulsion comprises a phospholipid-PEG-liver targeting molecule, wherein the phospholipid units may or may not be high iodine phospholipids.

When the PEG units provide end groups in the components of the oral microemulsion, the end groups of the PEG units may be OH or monomethyl ether.

In some embodiments, the oral microemulsion is a PEG-modified microemulsion, further, the weight percentage of PEG-modified starting material in the oral microemulsion may be 0.01% -10%, such as, 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.

Any one of PEG modified components which play a role in enriching EPA in the first auxiliary materials, wherein the molecular weight of the PEG units is preferably 500 Da-5000 Da independently. Examples thereof include 1000Da and 2000 Da.

Second auxiliary material

The second auxiliary material in the invention is different from the first emulsifier, the second emulsifier and the first auxiliary material.

The invention optionally comprises a second adjuvant. The second adjuvant may be selected from one or more of nutritional supplements, antioxidants, co-emulsifiers, oils, flavoring agents, pH modifiers, etc.

In some embodiments of the present invention, the content of the second auxiliary material in the oral microemulsion is 0-40% by weight, and further may be 1% -20%. Specific examples thereof 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%.

In some embodiments, the nutritional supplement may be selected from one or more of vitamin a, vitamin E (e.g., alpha-tocopherol, beta-tocopherol, gamma-tocopherol, delta-tocopherol, alpha-tocotrienol, beta-tocotrienol, gamma-tocotrienol, delta-tocotrienol, etc.), vitamin B group (e.g., vitamin B1, vitamin B2, niacin, pantothenic acid, vitamin B6, vitamin B12, folic acid, vitamin B7, etc.), vitamin D, silymarin, glucomannan, branched-chain amino acids, etc.

In some embodiments, the antioxidant may be selected from one or more of sodium sulfite, sodium bisulfite, sodium metabisulfite, vitamin C and esters thereof, tocopherols and esters thereof, and the like.

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

In some embodiments, the lipid is selected from one or more of soybean oil, medium chain triglycerides, olive oil, linseed oil, walnut oil, sea buckthorn oil, coix seed oil, grape seed oil, ginger oil, coconut oil, camellia oil, rose oil, peppermint oil, lemon oil, and the like.

In some embodiments, the flavoring agent may be selected from one or more of fruit flavored essence, erythritol, natural plant flavors, sweeteners, and the like.

In some embodiments, the pH adjuster may be selected from one or more of various buffer salt systems (e.g., citric acid-sodium citrate, acetic acid-sodium acetate, phosphate agents, etc.), bases (e.g., naOH, etc.), acids (e.g., HCl, etc.), and the like. The pH regulator is mainly used for regulating the pH environment of the water phase when the oral microemulsion is prepared.

In the present invention, the water in the oral microemulsion may be purified water, distilled water, or the like as a solvent, as long as it is suitable for preparing an oral preparation. The amount of water used in the oral microemulsion is an appropriate amount of water, so long as the oral microemulsion of the desired particle size of the present invention can be prepared. The proper amount of water makes the oral microemulsion have proper oil phase and water phase ratio, and can form an oil-in-water structure. The oral microemulsions provided herein comprise an oil phase component and a water phase component, in some embodiments, the weight percent of the oil phase component is less than or equal to 30%. In some embodiments of the invention, the minimum weight percent of water in the oral microemulsion is 65%, but the sum of the weight percentages of the components does not exceed 100%. In some embodiments of the invention, the weight percentage of water in the oral emulsion is 65% to 95%, preferably 70% to 94%, such as 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%.

Some examples of oral microemulsions

the oral microemulsion provided by the invention has an oil-in-water structure, wherein oil phase components such as EPA glyceride raw materials are formed in an oil phase, and water phase components such as an emulsifier are formed in a water phase.

In the following, 1g is used as 1 part by mass of some embodiments, it should be understood that other mass values may be used as 1 part by mass.

In some embodiments of the invention, the oral microemulsion is a PEG-modified microemulsion, further may comprise a PEG-modified starting material in an overall mass ratio of 0.01% to 10%, further may be 0.05% to 5%, such as, 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% of the PEG-modified component may be provided by one or more of the first emulsifier (e.g., PEG-modified phospholipid), the second emulsifier (e.g., polyoxyethylene fatty acid ester), the first adjuvant (e.g., PEG-modified component therein).

In some embodiments of the invention, the oral microemulsion comprises fish oil (providing EPA glyceride feedstock), high iodine value phospholipids (e.g., soy phospholipids, etc.), sucrose esters, antioxidants (e.g., alpha-tocopherol), nutritional supplements (optional), naOH, and water, further, the oral microemulsion comprises 10g to 400g fish oil, 1g to 60g high iodine value phospholipids, 0.1g to 6g sucrose esters, 0.5g to 6g antioxidants, 0 to 6g nutritional supplements, naOH, and water. The kinds, specifications/types and amounts of the components can be further referred to in example 1.1.

In some embodiments of the invention, the oral microemulsion comprises fish oil (providing EPA glyceride feedstock), high iodine value phospholipids (e.g., egg yolk phospholipids, soy phospholipids, polyene phosphatidylcholine, etc.), tocopherols, naOH, and water, further, the oral microemulsion comprises 10g to 200g fish oil (further, e.g., 20g to 100 g), 3g to 25g high iodine value phospholipids (further, e.g., 4g to 20 g), 0.5g to 5g tocopherols (further, e.g., 0.6g to 3 g), naOH, and water. The kinds, specifications/types and amounts of the components can be further referred to in example 1.2.

In some embodiments of the invention, the oral microemulsion comprises fish oil (providing EPA glyceride feedstock), high iodine value phospholipids (e.g., soy lecithin, etc.), bile acid, glycyrrhetinic acid, tocopherol, naOH, and water, further, the oral microemulsion comprises 10g to 200g of fish oil (further, such as 50g to 100 g), 1g to 35g of high iodine value phospholipids (further, such as 2g to 30 g), 0 to 15g of bile acid (further, such as 0.1g to 15g, further, such as 0.5g to 10 g), 0 to 6g of glycyrrhetinic acid (further, such as 1g to 6g, further, such as 1.2g to 5 g), 0 to 6g of tocopherol (further, such as 0 to 5g, further, such as 0.5g to 5 g), 0.1 to 6g of nutritional supplements (further, such as 0.5 to 5g, further, such as 1g to 5 g) NaOH, and water. The kinds, specifications/models and amounts of the components can be further referred to in example 1.3.

In some embodiments of the invention, the oral microemulsion comprises fish oil (providing EPA glyceride stock), high iodine value phospholipids (e.g., soy lecithin, etc.), DSPE-PEG, PEG-stearate, tocopherol, naOH, and water, further, the oral microemulsion comprises 5g to 200g of fish oil (further, e.g., 10g to 100 g), 1g to 35g of high iodine value phospholipids (further, e.g., 2g to 30 g), 0g to 8g of DSPE-PEG (further, e.g., 0.5g to 10g, further, e.g., 0.5g to 6 g), 0g to 8g of PEG-stearate (further, e.g., 0.5g to 8g, further, e.g., 0.5g to 6 g), 0.1g to 6g of tocopherol (further, e.g., 0.1g to 5g, further, e.g to 5 g), naOH, and water. The kinds, specifications/types and amounts of the components can be further referred to in example 1.4.

In some embodiments of the invention, the oral microemulsion comprises fish oil (providing EPA glyceride feedstock), high iodine value phospholipids (e.g., soy lecithin, etc.), glycyrrhetinic succinate, DSPE-PEG-galactose, tocopherol, naOH, and water, further, the oral microemulsion comprises 5 g-200 g fish oil (further, such as 10 g-100 g), 1 g-35 g high iodine value phospholipids (further, such as 2 g-30 g), 0-12 g glycyrrhetinic succinate (further, such as 0-10 g, further, such as 1 g-10 g), 0-8 g DSPE-PEG (further, such as 0.5 g-8 g, further, such as 0.5 g-6 g), DSPE-PEG-galactose, 0.1 g-6 g tocopherol (further, such as 0.1 g-5 g, further, such as 0.5 g-5 g), naOH, and water. The kinds, specifications/types and amounts of the components can be further referred to in example 1.5.

Second aspect of the invention

According to a second aspect of the present invention there is provided a method of preparing an oral microemulsion useful for preparing an oral microemulsion according to the first aspect of the present invention.

In some embodiments of the present invention, the method of preparing an oral microemulsion comprises the steps of:

s100, preparing an oil phase (preferably under the protection of inert gas): preparing an oil phase from the oil phase component comprising the EPA glyceride feedstock under heating conditions (preferably 50 ℃ C. To 70 ℃ C.);

s200, preparing an aqueous phase (preferably under the protection of inert gas), mixing an aqueous phase component comprising the phospholipid with an aqueous solvent, and preparing the aqueous phase under heating conditions (preferably 50-70 ℃);

s300, oil-in-water emulsification: mixing the oil phase and the water phase (preferably mixing under heating, further preferably 50-70 ℃), and shearing and stirring to prepare an oil-in-water microemulsion;

s400, homogenizing and emulsifying: and (3) carrying out high-pressure homogenization treatment on the oil-in-water microemulsion to obtain nanoemulsion with the droplet size smaller than 500nm, thus obtaining the oral microemulsion without sterilization.

Optionally, after said step S400, a step S500 is also performed, the post-treatment (preferably under inert gas protection): filtering, packaging and sterilizing the nanoemulsion to obtain the oral microemulsion (sterilized oral microemulsion);

The oral microemulsion is prepared by using inert gas protection, and can be nitrogen protection.

It should be understood that the steps of the above preparation method, unless otherwise indicated, are not limited in order. For example, the steps of S100 and S200 are not sequential, but both are prior to S300. For example, there is no limitation in order between packaging and sterilization.

As used herein, "aqueous solvent" refers to a solvent that provides a pharmaceutically acceptable aqueous phase, which may be water, or a mixture of water and other solvents.

In some embodiments, the pH adjuster is mixed with water (the pH of the aqueous solution is adjusted with the pH adjuster) to obtain an aqueous solvent for subsequent preparation. The pH value of the aqueous solvent is adjusted according to the pH value of the finally prepared microemulsion, and then a proper pH regulator is selected. In some embodiments, the pH of the final microemulsion is 7 to 8.

s100: stirring and mixing the oil phase components under the protection of inert gas until a uniform oil solution is formed, and heating the oil phase components to 50-70 ℃ in a water bath to prepare an oil phase; preferably, the inert gas is nitrogen;

S200: preparing an aqueous system from aqueous phase components in the formula under the protection of inert gas, stirring and dissolving until a uniform aqueous solution is formed, and heating to 50-70 ℃ in a water bath to prepare an aqueous phase; preferably, the inert gas is nitrogen;

s300: mixing the obtained water phase with the oil phase, shearing and/or homogenizing under high pressure to form oil-in-water microemulsion, and heating to 50-70 ℃ in water bath;

preferably, the preparation method further comprises the following steps: s400: filtering, sterilizing and packaging the obtained oil-in-water microemulsion, wherein inert gas is adopted for protection in the process.

In the above preparation method, the homogenization treatment is preferably performed under high pressure.

In some embodiments, the parameters of the homogenization process are: homogenizing at 20bar for one time, 200bar for one time, 400bar for six times.

In the case of specifying the kind and amount of raw materials, a person skilled in the art can carry out the above-mentioned preparation method according to the above-mentioned guidelines to obtain the oral microemulsion of the present invention.

The inventor finds that the oral microemulsion with the average particle size smaller than 500nm can contain auxiliary materials for assisting EPA in liver enrichment, so that intestinal cell absorption and EPA enrichment after absorption in the liver are greatly promoted, and the EPA is helped to exert efficacy to the greatest extent.

Third aspect of the invention

In some embodiments of the invention there is provided the use of the oral microemulsion in the manufacture of a medicament, preferably for the prevention and/or treatment of a disease associated with fat accumulation.

In some embodiments of the invention, there is provided the use of the oral microemulsion for the manufacture of a medicament for the prevention and/or treatment of a disease associated with fat accumulation.

In some embodiments of the invention, there is provided the use of the oral microemulsion in medical foods, health foods.

One aspect of the invention

It is another object of the present invention to provide applications of the above oral microemulsions including, but not limited to, applications in medical foods, health foods, pharmaceuticals and the like.

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

Fourth aspect of the invention

According to a fourth aspect of the present invention there is provided a method of preventing and/or treating a disease associated with fat accumulation comprising administering to a patient in need thereof a therapeutically effective amount of an oral formulation according to the present invention.

As used herein, a "therapeutically effective amount" refers to an amount of an oral microemulsion of the invention (or an amount of EPA) that will elicit a biological or medical response in an individual, such as an amount of an oral microemulsion of the invention (or an amount of EPA) that will bring about a physiologically and/or pharmacologically positive effect on an individual, including but not limited to reducing or inhibiting enzyme or protein activity or ameliorating symptoms, alleviating a condition, slowing or delaying the progression of a disease, or preventing a disease, etc.

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

As used herein, "patient" refers to an animal, preferably a mammal, more preferably a human. The term "mammal" refers primarily to warm-blooded vertebrates, including but not limited to: such as cats, dogs, rabbits, bears, foxes, wolves, monkeys, deer, mice, pigs, cattle, sheep, horses, and humans.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

Embodiments of the present invention will be described in detail below with reference to examples. It is to be understood that these examples are for the purpose of illustrating the invention and are not to be construed as limiting the scope of the invention, but are merely intended to illustrate the invention in terms of its specific formulation composition, method of preparation, function and effect, and are not to be construed as limiting the scope of the invention in any way. The experimental methods in the following examples, in which specific conditions are not noted, are preferably referred to the guidelines given in the present invention, and may be according to the experimental manual or conventional conditions in the art, the conditions suggested by the manufacturer, or the experimental methods known in the art.

In the specific examples described below, the measurement parameters relating to the raw material components, unless otherwise specified, may have fine deviations within the accuracy of weighing. Temperature and time parameters are involved, allowing acceptable deviations from instrument testing accuracy or operational accuracy.

In the examples described below, the particle size was measured using a Zetasizer Nano ZS (Markov) laser particle sizer.

Information on EPA raw materials and phospholipid raw materials used in the following examples and comparative examples are shown in Table 1.

Table 1 information on EPA raw materials and phospholipid raw materials used in examples and comparative examples of the present invention.

Wherein EPA content in fish oil refers to the amount of EPA units and triglyceride content refers to the total content of triglycerides of various fatty acids in fish oil. The percentages referred to in Table 1 are all percentages by mass.

In the following examples, EPA TG represents EPA glyceride, EPAEE represents EPA ethyl ester, PC represents phosphatidylcholine, meOH represents methanol, BHT represents 2, 6-di-t-butyl-p-cresol (antioxidant), TG represents triglyceride, TC represents total cholesterol, ALT represents glutamic pyruvic transaminase, and AST represents glutamic pyruvic transaminase.

In the following examples, "high EPA TG-content microemulsion" and "high EPA TG-content microemulsion" have the same meaning and are used interchangeably; the terms "high EPA content microemulsion" and "high EPA content microemulsion" have the same meaning and are used interchangeably.

1. Formulation examples

In the following examples, oral microemulsions were prepared by a method comprising the following steps (in each comparative example, microemulsions were prepared by the following method).

S1, preparing an oil phase: stirring and mixing oil phase components including glyceride type EPA raw materials, tocopherol and the like under the protection of nitrogen gas until a uniform oil solution is formed, and heating the oil phase components in a water bath to 65 ℃ for standby;

S2, preparing a water phase: regulating pH of purified water with pH regulator to obtain water-based solvent with required pH, adding phospholipid (soybean phospholipid as example) and other water-soluble raw materials into the prepared water-based solvent, stirring for dissolving to form uniform water solution, and heating in water bath to 65deg.C for use;

s3, preparing microemulsion: adding the water phase into the oil phase under the protection of nitrogen under the shearing condition, and forming oil-in-water microemulsion through shearing and stirring;

s4, homogenizing and emulsifying: homogenizing under high pressure for one time under 20bar pressure, homogenizing under 200bar pressure for one time, and homogenizing under 400bar pressure for six times to obtain nanoemulsion with droplet size smaller than 500 nm;

s5, filtering and packaging: filtering the nanoemulsion by a filter membrane with the diameter of 0.5-1.0 μm, filling nitrogen, and sealing to obtain a filled nanoemulsion;

s6, autoclaving: sterilizing the filled nanoemulsion in an autoclave to obtain oral microemulsion which can be used as a medicinal preparation.

Wherein the glyceride type EPA feedstock is polyunsaturated fatty acid glycerides (PUFA glycerides), some of which are known under the trade name fish oil, the glyceride type EPA feedstock is employed having a high content of EPA of at least 40% by mass, in some embodiments about 40%, 60%, 70%.

In the following examples and comparative examples, oil phase components other than glyceride type EPA raw materials, tocopherols, include glycyrrhetinic acid succinate, PEG-stearate, ascorbyl palmitate, glycerol monooleate, silymarin, alpha-tocopherol acetate, and vitamin A; other water-soluble raw and auxiliary materials comprise sucrose ester, bile acid, glycyrrhetinic acid, vitamin E succinic acid polyethylene glycol ester, 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 was used as a substitute for glyceride type EPA raw material in the S1 step to prepare an oil phase.

In the following examples, the iodine value was used to characterize the unsaturation of the phospholipids.

EXAMPLE 1.1 preparation of microemulsions containing varying proportions of EPA glycerides (EPA TG) and Phosphatidylcholine (PC)

Oral microemulsions containing different proportions of EPA TG and phosphatidylcholine were prepared according to the formulations in table 2. Wherein the fish oil in preparation 1-2, preparation 1-8 and preparation 1-10 is from EPAX 4030TGN of Epax company, and according to the product specification, each 100g contains EPA 40g and triglyceride content is more than or equal to 90%. The fish oils of formulations 1-4, 1-9 and 1-13 were from KinOmega 7010TG, which contained about 70% EPA and about 70% triglycerides according to the product specifications. The fish oils of formulations 1-1 and 1-11 were from KinOmega E80 TG, which according to the product specifications contained about EPA 80% and triglycerides at about 70%. Other formulations contained fish oil from Epax 6015 TGN from Epax corporation, which, according to the product specifications, contained approximately EPA 60% and a triglyceride content of 90% or more. The phospholipids in the preparations 1-2 and 1-12 are derived from soybean phospholipids of Shanghai Taiwei pharmaceutical Co., ltd, and have phosphatidylcholine content of about 50% and iodine value of more than 90; the phospholipids in the preparations 1-10 and 1-13 are from polyene phosphatidylcholine EPIKURON200 of Jiaji corporation in America, and the iodine value is about 94-97; the phospholipid in other preparations is derived from Germany Lipoid soybean phospholipid S75, the content of phosphatidylcholine is 70%, and the iodine value is 85-95. The second emulsifier in the preparation 1-1, 1-3 and 1-12 is sodium alginate, the antioxidant is vitamin C, and the nutritional supplement is branched chain amino acid composition; the second emulsifier in the preparation 1-10 and 1-13 is sodium caseinate, the antioxidant is ascorbyl palmitate, and the nutritional supplement is silymarin; the second emulsifier in other preparations is sucrose ester, the antioxidant is alpha-tocopherol, and the nutritional supplement is vitamin B complex.

TABLE 2 various formulations of EPA TG microemulsions with high content and microemulsion particle size

Wherein, naOH 'pH adjustment' means that the pH of the final microemulsion is adjusted to a corresponding range in the step S2, and purified water 'to 1 kg' means that purified water is added to the whole system of 1kg in the step S2. The following examples show the same meanings.

Wherein, the particle size refers to the range of the average particle size of the microemulsion in different batches under the same prescription and preparation process. The following examples show the same meanings. Microemulsions of different proportions of EPATG and phosphatidylcholine can be prepared according to the formula in table 2, the droplet size of the microemulsions being less than 500nm. The oil phase ratio of the four groups of the preparation 1-10 to the preparation 1-13 in the examples is high, the obtained microemulsion is thick, and the microemulsion is uncomfortable to drink and is not suitable for the prescription of subsequent production.

EXAMPLE 1.2 preparation of EPA TG microemulsions containing phospholipids of different iodine number

Microemulsions containing phospholipids of different iodine values were prepared according to the types and amounts of the components in table 3. Of these, in four groups of formulations 2-1 to 2-5, the fish oil was from Epax 6015TGN from Epax corporation; of the five groups of formulations 2-6 to 2-9, fish oil was from KinOmega 7010TG. Egg yolk lecithin E80 (specifically egg yolk phosphatidylcholine with an iodine value of 60-70), soybean lecithin S75 (specifically soybean phosphatidylcholine with an iodine value of about 85-95), and soybean lecithin S100 (specifically soybean phosphatidylcholine with an iodine value of 97-107) were derived from Germany. Polyene phosphatidylcholine EPIKURON200 (iodine value about 94-97) is available from jia ji company in united states. The antioxidant in the preparations 2-1, 2-2, 2-3 and 2-4 is alpha-tocopherol; the antioxidant in the preparations 2-5, 2-6, 2-7 and 2-8 is ascorbyl palmitate.

TABLE 3 EPA TG microemulsion formulations and microemulsion particle sizes containing phospholipids of different iodine values

Wherein "-" represents an amount of 0.

According to the prescription shown in Table 3, microemulsion with good stability can be prepared, and the droplet size of the microemulsion is less than 500nm, and can be used as oral microemulsion, wherein the phospholipid S75 has good emulsifying effect and the phospholipid S100 has poor emulsifying effect.

EXAMPLE 1.3 preparation of EPA TG micro emulsion containing liver targeting Effect auxiliary materials

EPA TG micro-emulsion containing liver targeting effect auxiliary materials (specifically, bile acid and glycyrrhetinic acid) is prepared according to the types and the amounts of the components in the table 4. Wherein the fish oil in seven groups of formulations 3-1 to 3-7 is from Epax 6015TGN of Epax company and the fish oil in three groups of formulations 3-8 to 3-10 is from KinOmega 7010TG. The phospholipids in the preparations 3-2, 3-8 and 3-9 are derived from polyene phosphatidylcholine EPIKURON200 of Jiaji of America, and have an iodine value of about 94-97; the phospholipid in other preparations is derived from Germany Lipoid soybean phospholipid S75, the content of phosphatidylcholine is 70%, and the iodine value is 85-95. The second emulsifier in the preparation 3-1, 3-2 and 3-3 is poloxamer, the antioxidant is ascorbyl palmitate, and the nutritional supplement is glucomannan; the second emulsifier in the preparation 3-4, 3-5, 3-6 and 3-7 is sodium caseinate, the antioxidant is alpha-tocopherol acetate, and the nutritional supplement is vitamin B complex; the second emulsifier in other preparations is sucrose ester, the antioxidant is alpha-tocopherol, and the nutritional supplement is vitamin A.

TABLE 4 different formulations of EPA TG microemulsions containing liver targeting effect excipients and microemulsion particle sizes

Wherein "-" represents an amount of 0.

According to the prescription shown in Table 4, a microemulsion with good stability can be prepared, and the droplet size of the microemulsion is less than 500nm, so that the microemulsion can be used as oral microemulsion. Glycyrrhetinic acid and bile acid have amphipathy, on one hand, the auxiliary emulsification function is achieved, and on the other hand, after absorption, the glycyrrhetinic acid and the bile acid are wrapped into chylomicron along with grease to increase liver targeting.

EXAMPLE 1.4 preparation of EPA TG micro-emulsion containing auxiliary materials for reducing enzymolysis

Microemulsion containing an auxiliary material having an enzymolysis reducing effect was prepared according to the kinds and amounts of the components in table 5. Wherein the fish oil in seven groups of formulations 4-1 to 4-7 is from Epax 6015TGN of Epax company and the fish oil in three groups of formulations 4-8 to 4-10 is from KinOmega 7010TG. The phospholipids in the preparations 4-4, 4-9 and 4-10 are derived from German Lipoid soybean phospholipid S100, and the iodine value is about 94-97; the phospholipid in other preparations is derived from Germany Lipoid soybean phospholipid S75, the content of phosphatidylcholine is 70%, and the iodine value is 85-95. The second emulsifier in the preparation 4-1, 4-2 and 4-3 is poloxamer, the antioxidant is ascorbyl palmitate, and the nutritional supplement is branched chain amino acid; the second emulsifier in the preparation 4-4, 4-5, 4-6 and 4-7 is sodium caseinate, the antioxidant is alpha-tocopherol, and the nutritional supplement is vitamin B complex; the second emulsifier in other preparations is glycerol monooleate, the antioxidant is vitamin C, and the nutritional supplement is vitamin D.

Wherein, the auxiliary materials with the enzymolysis reducing effect are vitamin E succinic acid polyethylene glycol ester and/or PEG-stearate, and the weight average molecular weight of PEG is 1000Da and 2000Da respectively.

TABLE 5 different formulations of EPA TG microemulsions containing adjuvants for reducing enzymolysis and particle size of the microemulsions

Wherein "-" represents an amount of 0.

According to the prescription shown in Table 5, a microemulsion with good stability can be prepared, and the droplet size of the microemulsion is less than 500nm, so that the microemulsion can be used as an oral microemulsion. PEG modified vitamin E succinate and/or stearate is added into the water phase, and the surface of the prepared microemulsion liquid drop is wrapped by PEG, so that the EPA can be prevented from being hydrolyzed and utilized by other tissues to a certain extent.

EXAMPLE 1.5 preparation of EPA TG micro emulsion containing liver targeting Effect auxiliary Material and auxiliary Material with enzymolysis reducing effect

EPA TG micro-emulsion containing liver targeting auxiliary materials and auxiliary materials for reducing enzymolysis is prepared according to the types and the amounts of the components in the table 6. Wherein, the fish oil in seven groups from preparation 5-1 to preparation 5-7 is from Epax 6015 TGN of Epax company, and the fish oil in three groups from preparation 5-8 to preparation 5-10 is from KinOmega 7010TG. The phospholipids in the preparations 4-4, 4-9 and 4-10 are derived from German Lipoid soybean phospholipid S100, and the iodine value is about 94-97; the phospholipid in other preparations is derived from Germany Lipoid soybean phospholipid S75, the content of phosphatidylcholine is 70%, and the iodine value is 85-95. The antioxidant in the preparations 4-1, 4-2 and 4-3 is ascorbyl palmitate; the antioxidant in the preparations 4-4, 4-5, 4-6 and 4-7 is alpha-tocopherol; the antioxidant in other preparations is vitamin C.

Wherein, the liver targeting auxiliary material is glycyrrhetinic acid succinate. The auxiliary materials with enzymolysis reducing effect are DSPE-PEG and DSPE-PEG-galactose, wherein the weight average molecular weight of PEG is 2000Da.

TABLE 6 different formulations of EPA TG micro-emulsion containing liver targeting effect adjuvant and adjuvant for reducing enzymolysis and micro-emulsion particle size

Wherein "-" represents an amount of 0.

According to the prescription shown in Table 6, a microemulsion with good stability can be prepared, and the droplet size of the microemulsion is less than 500nm, so that the microemulsion can be used as an oral microemulsion. The surface of the prepared microemulsion drop is wrapped by PEG, and the protection effect of degradation prevention is provided for the microemulsion drop.

Comparative examples of formulations

Referring to "1. Method for preparing oral microemulsion in formulation example", the microemulsion in the following comparative example was prepared, and in comparative example 1, the fish oil having a high EPA content (high EPA fish oil) in formulation example was replaced with commercially available ordinary fish oil, and in comparative example 2, the high EPA fish oil in formulation example was replaced with EPA ethyl ester (high EPA ethyl ester).

Comparative example 1. Preparation of microemulsion containing commercially available common fish oil

The microemulsion was prepared using commercially available common fish oils according to the ingredients and amounts listed in Table 7. The fish oils used in four groups D1-1 to D1-4 were all commercial fish oils from the norway/iceland import fish oils used in fish oil soft capsules (Shang Chen times the health stock company, ltd.) and contained 18.3g EPA, 12.9g dha and 98% triglycerides per 100g fish oil according to the product specifications. The soybean phospholipids in four groups from the preparation D1-1 to the preparation D1-4 are derived from German Lipoid S75, the content of phosphatidylcholine is 70%, and the iodine value is 85-95.

TABLE 7 different formulations of microemulsions containing commercially available common fish oil, glycyrrhetinic acid and DSPE-PEG

Comparative example 2 preparation of microemulsions containing EPA ethyl esters in different proportions

Microemulsions containing EPA ethyl ester were prepared according to the formulation formulations in table 8. Four groups of formulations D2-1 to D2-4 used EPA ethyl esters as a substitute for fish oils, the EPA ethyl esters used being from

According to the product specification, the raw materials adopted in the process contain more than 96g of EPA ethyl ester per 100g of oil. Four groups of preparationsThe soybean phospholipids are all from Germany Lipoid S75, the phosphatidylcholine content is 70%, and the iodine value is 85-95.

TABLE 8 different formulations of microemulsions comprising EPA Ethyl ester, hyaluronic acid and DSPE-PEG and microemulsion particle sizes

2. Formulation animal in vivo evaluation

EXAMPLE 2.1 evaluation of the Effect of microemulsion formulations on improving bioavailability

2.1.1. Experimental animal

Male beagle dogs (Shanghai laboratory animal research center) weighing about 10 kg.

2.1.2. The present example is divided into 4 control groups and 4 experimental groups, wherein the control groups include: common fish oil microemulsion (formulation D1-2), EPAX 6015 TGN raw material (raw material group, fasting), EPA EE microemulsion (formulation D2-2); the experimental group included: formulations 1-6, 5-7, 1-6 (fasting), 5-7 (fasting). Each group was administered 120mg/kg of reduced EPA.

2.1.3. The experimental method comprises the following steps: beagle dogs were randomly divided into 8 groups of 6 animals each, and no fasting, no water, no fasting for 12h, no water, no fasting prior to the experiment. And respectively performing gastric lavage on the beagle, taking 2mL of blood after 0.5h, 1h, 2h, 3h, 4h, 6h, 8h, 12h and 24h of administration, collecting in a heparinized tube, centrifuging at 4 ℃ and 3000rpm for 10min, separating plasma, taking supernatant, and preserving at-20 ℃ to be measured.

2.1.4. The total EPA content in the plasma was measured by methyl esterification-GC method. The GC chromatographic conditions were (88% -cyanopropyl) aryl-polysiloxane capillary column (60 m.times.0.25 mm.times.0.2 μm), temperature programmed, 0min 170 ℃,3.5 ℃ per minute up to 240 ℃, 10min hold, injector temperature 250 ℃, detector temperature 270 ℃. The carrier gas was helium at a flow rate of 1.0mL/min. Split ratio: 10:1. The sample volume was 1. Mu.L.

Step one: clean dry stoppered glass test tube was taken and 300. Mu.g/mL of methyl hendecanoate was added100. Mu.L of ester (internal standard), N ₂ After blow drying, 200 mu L of plasma, 2mL of 0.5mol/L KOH-MeOH solution and 0.5mL of BHT isooctane solution are added, the mixture is sealed by a cover, vortex for 60s, and after uniform mixing, the mixture is kept stand for 10min to 15min, and an isooctane layer is collected and placed in a clean sample injection small bottle to which a small amount of anhydrous sodium sulfate is added.

Step two: 2mL of 5% (v/v) H was added to the lower solution of step one ₂ SO ₄ MeOH solution, surface microbubble N ₂ Sealing, mixing by simple vortex, and reacting in water bath at 70 ℃ for 30min; taking out, cooling to about 40 ℃, adding 0.5mL of isooctane, swirling for 30s, adding 0.5mL of saturated sodium chloride solution, swirling for 15s, collecting an isooctane layer, combining with the organic layer in the step one, drying with anhydrous sodium sulfate, transferring to a sample injection small bottle with a sample injection sleeve, and taking the sample injection small bottle as sample injection solution.

Using relative bioavailability F (e.g. AUC _{Microemulsion (microemulsion)} /AUC _{Raw materials} ) The evaluation was performed, based on AUC of the raw material group, as 100%.

2.1.5. Experimental results

The pharmacokinetic parameters of beagle dogs after administration of the 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 the common fish oil raw material (preparation D1-2) is not obviously improved under the condition of the same EPA dosage, and the bioavailability of EPA can be improved by about 3 times by using the oral microemulsion prepared by using the high-content EPA fish oil (four experimental groups). The bioavailability of control group 3 administered in the fasting state was significantly reduced compared to the EPAX 6015 TGN raw material group. The results of empty satiety comparison of preparations 1-7 and preparations 5-7 show that the bioavailability of the microemulsion is not significantly different in the empty satiety state. The oral microemulsion provided by the application can promote fish oil absorption, improve bioavailability and has no obvious difference in bioavailability when taken under empty satiety.

TABLE 9 relative bioavailability results of EPA in fish oil oral microemulsions

EXAMPLE 2.2 EPA purity, EPA to Phosphatidylcholine (PC) ratio, phosphatidylcholine iodine value Screen

"EPA purity" refers to the EPA content of fish oil feedstock.

2.2.1. Experimental animal

Experimental animals: male Sprague Dawley rats (Shanghai laboratory animal research center) weighing 180 g-200 g.

2.2.2. The present example is divided into a blank group, a model group and 9 experimental groups: wherein the blank group: the normal feed is fed, and the microemulsion is given an equal volume of purified water every day. Model group: the high-fat feed is fed, and the microemulsion is given an equal volume of purified water every day. The experimental groups were: formulations 1-4 having a mass ratio of EPA to phosphatidylcholine of about 8.7:1; formulations 1-5 having a mass ratio of EPA to phosphatidylcholine of about 7.2:1; formulations 1-6, wherein the ratio of EPA to phosphatidylcholine is about 5.8:1; formulations 1-7 having an EPA to phosphatidylcholine ratio of about 4.8:1 and fish oil of Epax 6015 TGN; formulations 1-8 wherein the fish oil is Epax 4030 TGN from Epax corporation; formulations 1-9, wherein the fish oil in the formulation is KinOmega 7010TG; preparation 2-1, wherein the iodine value of the phospholipid in the preparation is about 60-70; 2-2 of a preparation, wherein the iodine value of the phospholipid in the preparation is about 85-95; 2-3 of a preparation, wherein the iodine value of the phospholipid in the preparation is about 94-97; formulations 2-4, wherein the phospholipid iodine value in the formulation is about 97-107. Each group had a dose of 150 mg/kg/day of reduced EPA.

2.2.3. The experimental method comprises the following steps: 90 SD rats were taken and 10 rats per group were given special feed for non-alcoholic fatty liver disease. After the molding was completed, the groups were made according to fasting blood glucose, body weight, plasma TC (total cholesterol), TG (triglyceride) levels. Mice in the blank control group and the model control group are administrated with purified water daily for gastric lavage, and the preparation group is administrated with corresponding preparation. The feed was continuously administered for 6 weeks, and the feed was not changed for each group during the administration period. After 4 hours of anesthesia after fasted animals before starting and after 6 weeks of administration, collecting not less than 0.5mL of whole blood sample, placing in a heparinizing centrifuge tube, centrifuging at 4deg.C and 6500rpm for 15min, and collecting supernatant serum. Immediately after the last dose of blood was taken, the liver was dissected, weighed, the right leaf was cut out and fixed with 10% formalin, the sections were paraffin-embedded conventionally, HE stained and observed under an optical microscope. The rest tissue is frozen at-80 ℃ for standby.

2.2.4. The index to be measured:

after 6 weeks of administration, the rat body weight, liver weight, and rat liver index (liver index = rat liver weight/rat last body weight) was measured, and the liver index was also noted as liver index, where "rat last body weight" means the weight at "sacrifice immediately after the last administration of blood collection". Blood was taken to detect the expression of Triglyceride (TG), total Cholesterol (TC), glutamic pyruvic transaminase (ALT), glutamic oxaloacetic transaminase (AST). Total liver cholesterol was measured by the CHOD-PAP method, liver triglyceride was measured by the GPO-PAP method, and tissue morphology was observed by HE staining.

2.2.5. Experimental results

The experimental results are shown in table 10. Compared with a blank group, the weight and liver index of rats in the model group are obviously increased, and each preparation group (9 groups) can reduce the weight and liver index of rats, the weight gain and liver index of rats have a tendency to be reduced along with the reduction of EPA/phosphatidylcholine ratio, and the EPA/phosphatidylcholine ratio is more preferably lower than 8:1 (including preparations 1-5, preparations 1-6 and preparations 1-7) and the phospholipid iodine value is more than 80. The data of preparations 1-7, preparations 1-8 and preparations 1-9 show that when EPA is contained in 100g of the fish oil raw material by 40g, the effect on the body weight and liver index is not obvious, and when EPA is contained in 100g of the fish oil raw material by 60% or more, the fish oil raw material has better effect on the body weight and liver index.

Table 10 changes in body weight and liver index (n=10) of rats in each group

Wherein EPA/PC represents the mass ratio of EPA to phosphatidylcholine, and iodine value represents the iodine value of the phospholipid.

Specific data of TG and TC levels in the serum of rats in each group are shown in table 11, reflecting the lipid levels of rats. Both the serum TC and TG levels in rats tended to decrease with decreasing EPA/phosphatidylcholine ratio and increasing phospholipid iodine value, but the magnitude of the decrease was not significant. The serum TC levels in rats did not change significantly with the increase in EPA purity, but TG levels showed a decreasing trend with the increase in EPA purity.

Table 11. TG, TC levels in rat serum of each group (n=10)

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Specific data for ALT and AST levels in the serum of rats in each group are shown in Table 12. The ALT level in rats did not change significantly with decreasing EPA/phosphatidylcholine ratio, and tended to decrease with increasing phospholipid iodine value. Rat AST levels tend to decrease with decreasing EPA/phosphatidylcholine ratio and increasing phospholipid iodine number, with EPA/phosphatidylcholine ratios below 8:1 (formulations 1-5, formulations 1-6, formulations 1-7), and phospholipid iodine numbers greater than 80 being more effective. ALT and AST levels do not vary significantly with EPA purity.

Table 12 glutamic pyruvic transaminase (ALT) and glutamic oxaloacetic transaminase (AST) levels (n=10) in rat serum of each group

Specific data for TC and TG levels in the livers of rats in each group are shown in table 13. The decrease in rat liver TC levels with EPA/phosphatidylcholine ratio and the increase in phospholipid iodine value were not significant, with slight increase in liver TG levels at 7:1 EPA/phosphatidylcholine values, but no significant difference, reaching 6: there was a slight decrease at 1 but no apparent trend to below 5:1 (formulations 1-7) starting to appear to decrease. And liver TG levels tended to decrease with increasing phospholipid iodine number. Liver TC, TG levels did not vary significantly with EPA purity.

Table 13 rat liver lipid content assay for each group (n=10)

The fatty liver NAS scoring results are shown in fig. 1. Formulations 1-6 (about 6:1), formulations 1-7 (about 5:1) with lower EPA/phosphatidylcholine ratios and formulations 2-3 (about 95 iodine) with higher phospholipid iodine numbers, and formulations 2-4 (about 103 iodine numbers) gave better scoring results. Formulations 1-7 (EPA purity about 60%) and formulations 1-9 (EPA purity about 70%) were scored lower than formulations 1-8 (EPA purity about 40%).

The method can be summarized as follows: (1) the effect of the microemulsion prepared with the fish oil raw material having higher EPA content (i.e., higher purity) on the reduction of body weight and liver index of fatty liver model rats was more remarkable, and the blood TG level showed a decreasing trend with the increase of EPA content of the fish oil raw material, compared with the microemulsion preparation prepared with the raw material having 40% EPA purity. (2) When the EPA/phosphatidylcholine ratio is less than or equal to 8:1 and the phospholipid iodine value is more than 70, the oral microemulsion has better effects of reducing the weight, liver index, blood fat and liver injury of the fatty liver model rat. Wherein, the EPA/phosphatidylcholine ratio is less than or equal to 5:1, and when the phospholipid iodine value is more than 90, the liver TG is more beneficial to the decrease, and the effect is better.

EXAMPLE 2.3 drug Profile study

2.3.1. Experimental animal

Experimental animals: male Sprague Dawley rats (Shanghai laboratory animal research center) weighing 220 g-250 g.

2.3.2. The present example was divided into 3 control groups and 6 experimental groups: each group had a dose of 500mg/kg of reduced EPA. Wherein the control group comprises: common fish oil microemulsion (formulation D1-2), EPAX 6015 TGN raw material (raw material group, non-microemulsion), EPAEE microemulsion (formulation D2-2). The experimental group included high content EPA microemulsion (formulation 1-7), high content EPA microemulsion containing bile acid (formulation 3-5), high content EPA microemulsion containing glycyrrhetinic acid (formulation 3-7); high content EPA microemulsion containing vitamin E succinic acid polyethylene glycol ester (preparation 4-6), high content EPA microemulsion containing DSPE-PEG (preparation 4-7), and high content EPA microemulsion containing glycyrrhetinic acid succinic acid ester and DSPE-PEG (preparation 5-7).

2.3.3. The experimental method comprises the following steps: 54 SD rats were obtained, 6 per group. The experiment is not fasted and can drink water freely. Each group was administered by gavage at a dose of EPA 500mg/kg for the test subjects. The rats were sacrificed 3h after the stomach irrigation, immediately dissected, and tissues such as heart, liver, spleen, lung, kidney, etc., were cleaned with physiological saline, weighed, and stored in a-80 ℃ refrigerator for later use. Thawing before use, and mixing the tissue with physiological saline in proportion to prepare tissue homogenate.

2.3.4. Experimental results: the distribution of EPA in different tissues was measured by gas chromatograph and shown in Table 14, and the measurement method was the same as that of EPA in blood plasma.

TABLE 14 organ distribution of EPA after different groups of drug administration (μg.g ^-1 )

The EPA is mainly concentrated in the liver after being administrated in different groups, wherein the micro-emulsion remarkably improves EPA concentration in various organs in the body, and the addition of auxiliary materials (preparation 3-5, preparation 3-7 and preparation 5-7) with liver targeting effect or auxiliary materials (preparation 4-6, preparation 4-7 and preparation 5-7) for reducing enzymolysis effect can increase EPA concentration in the liver to a certain extent, wherein the auxiliary materials with better fat solubility can have better effect (comparing preparation 3-5, preparation 3-7 and preparation 4-7 and preparation 5-7). The auxiliary material with liver targeting effect or PEG modified auxiliary material has better enrichment effect of EPA in liver after being used in combination (preparation 5-7). In addition, in this example, it was found that the concentration of the glyceride type EPA microemulsion in the liver was higher than the EPA EE microemulsion used in the preparation D2-2.

EXAMPLE 2.4 enrichment of drug in liver after multiple dosing of different formulations

2.4.1. Experimental animal

2.4.2. The present embodiment is divided into 9 groups: the present example was divided into 3 control groups and 6 experimental groups: each group was given a dose of 125 mg/kg/dose of reduced EPA, 2 times/day of gastric lavage. Wherein the control group comprises: common fish oil microemulsion (preparation D1-2), EPAX 6015 TGN raw material (raw material group), EPA EE microemulsion (preparation D2-2). The experimental group included high content EPA microemulsion (formulation 1-6), high content EPA microemulsion containing bile acid (formulation 3-5), high content EPA microemulsion containing glycyrrhetinic acid (formulation 3-7); high content EPA microemulsion containing vitamin E succinic acid polyethylene glycol ester (preparation 4-6), high content EPA microemulsion containing DSPE-PEG (preparation 4-7), and high content EPA microemulsion containing glycyrrhetinic acid succinic acid ester and DSPE-PEG (preparation 5-7).

2.4.3. The experimental method comprises the following steps: 135 SD rats were obtained, 15 per group. Each group was dosed with the test substance. The liver is dissected and taken in 7 days, 14 days and 28 days (6 hours after the last administration) respectively without fasted and free drinking before the experiment, and the liver is cleaned by normal saline, weighed and stored in a refrigerator at the temperature of minus 80 ℃ to be measured. Thawing before use, and mixing the tissue with physiological saline in proportion to prepare tissue homogenate.

2.4.4. Experimental results: the enrichment of EPA in liver is shown in Table 15 by gas chromatograph, and the detection method is the same as that of EPA in blood plasma.

TABLE 15 enrichment of EPA in liver in different groups after 7/14/28 days of dosing

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The results show that the fish oil oral micro-emulsion (preparation 3-5, preparation 3-7, preparation 4-6, preparation 4-7 and preparation 5-7) with the function of assisting the EPA to gather in the liver significantly improves the gathering rate of the EPA in the liver, so that the EPA reaches steady-state concentration in the liver more quickly. The micro-emulsion remarkably improves the EPA enrichment rate in the liver, and the addition of auxiliary materials (preparation 3-5, preparation 3-7 and preparation 5-7) with liver targeting effect or auxiliary materials (preparation 4-6, preparation 4-7 and preparation 5-7) for reducing enzymolysis can increase the EPA enrichment rate in the liver, so that the (preparation 5-7) EPA enrichment effect in the liver is better after the two auxiliary materials are combined. In particular, the rate of enrichment of glyceride type EPA microemulsions in the liver is faster than EPA EE microemulsions employed in formulation D2-2.

EXAMPLE 2.5 therapeutic Effect of oral microemulsions of high EPA-containing glyceride type raw materials with liver-enriching effect on nonalcoholic fatty liver

2.5.1. Experimental animal

2.5.2. The present example was divided into a blank group, a model group, 3 control groups and 4 experimental groups. Wherein the blank group: feeding common feed, and adding purified water with the same volume as that of the microemulsion; model group: feeding high-fat feed, and adding purified water with the same volume as that of the microemulsion; the control group included: pioglitazone hydrochloride tablets (positive control group) are sold in the market, and the administration dosage is converted into pioglitazone 10mg/kg, and the daily dosage is 1 time; formulations D1-4, administered 2 times per day by gastric lavage, with a reduced EPA dose of 300 mg/kg/day; the EPAX 6015TGN raw material (raw material group) of Epax company is converted into EPA administration dosage of 300 mg/kg/day, and 2 times/day gastric lavage administration is carried out; the experimental group included: dosage groups in preparations 1-7, which are converted into EPA administration dosage of 150 mg/kg/day; formulations 5-7 low dose group, reduced EPA dosing at 75 mg/kg/day; dosage groups in preparations 5-7, which are converted into EPA administration dosage of 150 mg/kg/day; formulations 5-7 high dose group, which reduced EPA dosing to 300 mg/kg/day;

2.5.3. The experimental method comprises the following steps: 90 SD rats were taken and 10 rats per group were given special feed for non-alcoholic fatty liver disease. After the molding was completed, the groups were made according to fasting blood glucose, body weight, plasma TC, TG levels. Mice in the blank control group and the model control group are administrated with purified water daily for gastric lavage, and the preparation group is administrated with corresponding preparation. The feed was continuously administered for 6 weeks, and the feed was not changed for each group during the administration period. After 4 hours of anesthesia after fasted animals before starting and after 6 weeks of administration, collecting not less than 0.5mL of whole blood sample, placing in a heparinizing centrifuge tube, centrifuging at 4deg.C and 6500rpm for 15min, and collecting supernatant serum. Fasting blood glucose was measured at 2, 4, 6 weeks of dosing, and OGTT experiments were performed 4 weeks after dosing. Immediately after the last dose of blood was taken, the liver was dissected, weighed, the right leaf was cut out and fixed with 10% formalin, the sections were paraffin-embedded conventionally, HE stained and observed under an optical microscope. The rest tissue is frozen at-80 ℃ for standby.

2.5.4. The index to be measured:

after 6 weeks of administration, the rat body weight, liver weight, and rat liver index (liver index=rat liver weight/rat last body weight) were weighed. The fasting blood glucose was measured using a glucometer and test paper. Blood was taken to examine the expression of Triglyceride (TG), total Cholesterol (TC), glutamic pyruvic transaminase (ALT), glutamic oxaloacetic transaminase (AST). Total liver cholesterol was measured by the CHOD-PAP method, and liver triglyceride was measured by the GPO-PAP method. And detecting SOD (superoxide dismutase) and MDA (malondialdehyde) by adopting the related kit respectively. Tissue morphology was observed by HE staining.

2.5.5. Experimental results

Compared with blank control, the body weight and liver index of the rats in the model group are obviously increased, and different EPA preparations can reduce the body weight and liver index of the rats, wherein the oral administration of the 5-7 oral administration micro-emulsion high dose group has the best effect of reducing the body weight and liver index. The specific data are shown in Table 16.

Table 16, changes in body weight and liver index (n=10) of rats in each group

The changes in fasting blood glucose at 2, 4, and 6 weeks after administration in rats in the blank, model, and control groups are shown in table 17, wherein rosiglitazone has better control over blood glucose in fatty liver rats and the levels of abdominal blood glucose were significantly reduced at weeks 4 and 6. The dose and high dose groups in formulations 5-7 had the same trend as the positive control, whereas the dose groups in formulations 1-7 and the low dose groups in formulations 5-7 had a trend to significantly reduce fasting blood glucose at week 6.

Table 17 changes in fasting blood glucose in rats of each group (n=10)

The results of the rat glucose tolerance experiments are shown in FIG. 2, table 18 and FIG. 3. The AUC of the positive control group and the high dose blood glucose in the formulations 5-7 is significantly smaller than that of the model group, indicating that the positive control group and the high dose blood glucose AUC can improve the insulin resistance of rats to a certain extent.

Table 18. Glucose tolerance test results for rats of each group (n=10)

The results of the lipid level tests for rats in each group are shown in Table 19. The serum TG and TC levels were significantly higher in the rats of the model group compared to the blank control group, and the serum Triglyceride (TG) levels of the EPA-administered group other than formulations D1-4 were significantly lower than those of the model control group, wherein the dose and high dose effects in formulations 5-7 were most pronounced, and only the dose and high dose in formulations 5-7 had significant effects on the Total Cholesterol (TC) level.

Table 19. Rat blood lipid levels in each group (n=10)

The results of ALT and AST levels in the serum of rats in each group are shown in Table 20. Compared with a model control group, the fish oil oral microemulsion can obviously reduce the levels of ALT and AST in serum, and the difference of the levels of ALT and AST in serum is not obvious in each group.

Table 20 levels of glutamic pyruvic transaminase (ALT) and glutamic oxaloacetic transaminase (AST) in rat serum in each group (n=10)

The measurement results of the liver lipid, liver SOD and MDA content of the rats in each group are shown in Table 21. The model group showed a significant increase in lipid deposition and accumulation of lipid droplets by comparison with the blank group. The research results show that. Different EPA microemulsion preparations have no obvious influence on TC levels in livers, but have a certain reduction effect on TG, can raise liver SOD activity, reduce MDA content, and reflect that the EPA microemulsion preparations can protect body lipid peroxidation by regulating free radical metabolic balance.

Table 21. Rat liver fat, liver SOD, MDA content assay in each group (n=10)

The fatty liver NAS scoring results are shown in fig. 4, and the HE staining results of rat liver tissue morphology are shown in fig. 5. Wherein the oral microemulsion of the glyceride type raw material with high EPA content and liver enrichment effect can effectively improve the liver tissue morphology. Liver sections of the model group showed pronounced vacuolated hepatocytes and severe micro-and macrobleb steatosis with pronounced inflammatory response cell infiltration. The liver cell morphology of the low-dose, medium-dose and high-dose preparation 5-7 groups is gradually recovered, the swelling degree is gradually reduced, the lipid drop cavitation phenomenon is gradually reduced, and the inflammatory cell infiltration is gradually reduced, wherein the liver tissue injury improvement effect of the high-dose preparation 5-7 groups is better (figure 5).

The foregoing experimental results also demonstrate that: (1) Compared with EPA soft capsules, the micro-emulsion improves the bioavailability of EPA by about 3 times. (2) The content and unsaturation degree of the phosphatidylcholine have influence on the drug effect, and the preferred selection range is that the iodine value of the phosphatidylcholine is more 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 invention can effectively improve the enrichment of EPA in liver. (4) The fish oil oral microemulsion provided by the invention can reduce ALT and AST levels formed by high-fat diet induced NASH, relieve inflammatory reaction, prevent lipid peroxidation, and reduce serum TG and TC levels. The oral microemulsion provided by the invention can be used as a potential drug for treating NAFLD.

The technical features of the above-described embodiments and examples may be combined in any suitable manner, and for brevity of description, all of the possible combinations of the technical features of the above-described embodiments and examples are not described, however, as long as there is no contradiction between the combinations of the technical features, they should be considered to be within the scope described in the present specification.

The above examples merely represent a few embodiments of the present invention, which facilitate a specific and detailed understanding of the technical solutions of the present invention, but are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Further, it is understood that various changes and modifications of the present invention may be made by those skilled in the art after reading the above teachings, and equivalents thereof fall within the scope of the present application. It should also be understood that, based on the technical solutions provided by the present invention, those skilled in the art obtain technical solutions through logical analysis, reasoning or limited experiments, all of which are within the scope of protection of the appended claims. The scope of the patent is therefore intended to be covered by the appended claims, and the description and drawings may be interpreted as illustrative of the contents of the claims.

Claims

1. An oral microemulsion, characterized in that the total weight percentage of the oral microemulsion is 100%, and the oral microemulsion comprises the following components in percentage by weight:

water;

the first emulsifier is phospholipid with iodine value more than 70;

the mass ratio of EPA to phosphatidylcholine in the first emulsifier is less than or equal to 8:1;

2. The oral microemulsion of claim 1, comprising the following components in weight percent:

and (3) water.

3. The oral microemulsion according to any one of claim 1 to 2, wherein,

in the oral microemulsion, the minimum weight percentage of water is 65%; and/or the number of the groups of groups,

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 number of the groups of groups,

the EPA glyceride feedstock is selected from one or more of the EPA glycerides having the following composition: 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 number of the groups of groups,

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 polyene phospholipid of soybean origin; and/or the number of the groups of groups,

6. The oral microemulsion of claim 5, wherein,

the second emulsifier does not contain phospholipid component; and/or the number of the groups of groups,

7. The method for producing an oral microemulsion according to any one of claims 1 to 6, comprising the steps of: preparing an oil phase under heating from an oil phase component comprising the EPA glyceride feedstock;

optionally, the nanoemulsion is filtered, packaged and sterilized.

8. Use of an oral microemulsion according to any one of claims 1 to 6, or obtained according to the preparation method of claim 7, in the preparation of a medicament for preventing and/or treating a disease associated with fat accumulation, or use of an oral microemulsion according to any one of claims 1 to 6, or obtained according to the preparation method of claim 7, in medical foods, health foods.

9. The use according to claim 8, wherein the liver disease associated with fat accumulation is one or more of fatty liver, liver injury, hepatitis.

10. The use according to claim 9, wherein the fatty liver is a non-alcoholic fatty liver.