Peptide complex having activities of preventing or treating fatty liver disease, and use thereof

Abstract

The present invention relates to a peptide complex having activities of preventing or treating fatty liver disease. The peptide complex of the present invention inhibits, in hepatocytes, the expression of fat synthesis-related genes and proteins and promotes the expression of fatty acid oxidation-related genes and proteins, and thus can inhibit, in hepatocytes, fat accumulation and promote lipolysis. In addition, the peptide complex of the present invention can inhibit the expression of inflammation-related genes in hepatocytes, and inhibits the expression of hepatotoxicity-related proteins, thereby having hepatoprotective activity. Furthermore, the peptide complex of the present invention can inhibit the degree of fat accumulation and liver fibrosis in liver tissue. Therefore, the peptide complex of the present invention is expected to be effectively used for preventing, alleviating or treating fatty liver disease.

Description

Peptide complex having preventive or therapeutic activity for fatty liver disease and use thereof

[0001] [Cross-reference with related applications]

[0002] This application claims the benefit of priority from Korean Patent Application No. 10-2024-0054977, filed April 24, 2024, the entire contents of which are incorporated herein by reference.

[0003] The present invention relates to a peptide complex having preventive or therapeutic activity for fatty liver disease and its use.

[0004]

[0005] The liver not only detoxifies the body, but also synthesizes, metabolizes, stores, and redistributes key nutrients like carbohydrates, proteins, and lipids. Through these processes, it maintains metabolic homeostasis. However, liver function can be damaged by a variety of factors, including viruses, inflammation, excessive alcohol consumption, drugs, and overwork.

[0006] One of the symptoms that is thought to be the main cause of liver function damage and liver disease is fatty liver, the frequency of which has been rapidly increasing recently due to the influence of westernized eating habits, drinking, and stress. Fatty liver is a symptom in which excessive neutral fat accumulates in the liver (more than 5% of the liver weight) due to the influence of drinking, excessive fat intake, and stress. A normal liver is composed of about 5% adipose tissue, and neutral fat, fatty acids, phospholipids, cholesterol, and cholesterol esters are the main components of fat. However, once fatty liver occurs, most of the components are replaced by neutral fat, and if the amount of neutral fat is more than 5% of the liver weight, it is diagnosed as fatty liver disease.

[0007] Fatty liver disease may not develop into any other disease and may remain in a state without significantly affecting health. However, if fatty liver disease worsens and the fat lumps inside the liver cells increase, important components of the cell, including the nucleus, are pushed to one side, which reduces the function of the liver cells. In addition, the swollen liver cells caused by the accumulated fat inside the cells compress the microvessels and lymph nodes between the liver cells, disrupting the circulation of blood and lymph within the liver. This can prevent the liver cells from receiving adequate oxygen and nutrients, which can lead to a decline in liver function. Furthermore, if fatty liver disease worsens further, it can develop into diseases such as hepatitis or cirrhosis, so continuous attention and management are necessary.

[0008] Meanwhile, fatty liver disease can be divided into alcoholic fatty liver disease (ALD) caused by excessive alcohol consumption and non-alcoholic fatty liver disease (NAFLD). Among these, NAFLD is a disease in which neutral fat accumulates in the liver regardless of alcohol consumption. It refers to a group of diseases including simple steatosis, which is only excessive fat accumulation in hepatocytes, non-alcoholic steatohepatitis (NASH), which is accompanied by hepatocyte necrosis, inflammation, and fibrosis, and liver cirrhosis (LC), which is a more advanced form.

[0009] The prevalence of nonalcoholic fatty liver disease (NAFLD) has been increasing not only in several Western countries but also in Korea. While the prevalence varies by country, it is reported to be approximately 20-30% of the general population in developed countries. While NAFLD can be a simple form of steatosis, approximately 10-20% of patients with NAFLD develop nonalcoholic steatohepatitis, and approximately 9-25% of these patients progress to cirrhosis.

[0010] Nonalcoholic fatty liver disease (NAFLD) can usually be reversed with proper nutritional intake and management, but if left untreated, the prognosis can be poor. Therefore, preventing NAFLD from progressing to NAFLD or cirrhosis requires preventing fat accumulation in the liver. However, other than reducing calorie intake or increasing calorie expenditure, no effective treatment has been identified to date.

[0011]

[0012] [Prior Art Literature]

[0013] [Patent Document]

[0014] Republic of Korea Patent No. 10-2486789

[0015]

[0016] The present inventors have conducted research efforts to develop an effective active substance that can be used to prevent, improve, or treat fatty liver disease by inhibiting lipid synthesis and accumulation in hepatocytes and promoting fatty acid oxidation and lipolysis. As a result, the inventors experimentally confirmed that the peptide complex they synthesized exhibits excellent lipid accumulation inhibition and lipolysis promotion activities, as well as hepatocellular protection and liver fibrosis inhibition activities, thereby completing the present invention.

[0017] Accordingly, the purpose of the present invention is to provide a pharmaceutical composition for preventing or treating fatty liver disease or a food composition for preventing or improving fatty liver disease, comprising the peptide complex.

[0018]

[0019] In order to achieve the purpose of the present invention described above,

[0020] One aspect of the present invention provides a pharmaceutical composition for preventing or treating fatty liver disease, comprising a peptide comprising an amino acid sequence of SEQ ID NO: 1 and a peptide comprising an amino acid sequence of SEQ ID NO: 2 as active ingredients.

[0021] Another aspect of the present invention provides a food composition for preventing or improving fatty liver disease, comprising a peptide comprising an amino acid sequence of SEQ ID NO: 1 and a peptide comprising an amino acid sequence of SEQ ID NO: 2 as active ingredients.

[0022]

[0023] The present invention is described in detail below.

[0024]

[0025] 1. Peptides and their activities

[0026] The present invention relates to the use of a complex of a peptide comprising an amino acid sequence of SEQ ID NO: 1 and a peptide comprising an amino acid sequence of SEQ ID NO: 2 for preventing or treating fatty liver.

[0027] [Amino acid sequence of sequence number 1]

[0028] LKTRN

[0029] [Amino acid sequence of sequence number 2]

[0030] KGSATGWMA

[0031] The term "peptide" as used herein means a linear molecule formed by amino acid residues being linked to each other by peptide bonds.

[0032] The peptide comprising the amino acid sequence of SEQ ID NO: 1 of the present invention or the peptide comprising the amino acid sequence of SEQ ID NO: 2 may be used without modification, but a variant or fragment of an amino acid having a different sequence by deletion, insertion, substitution, or a combination thereof of amino acid residues may be used within a range that does not affect the original activity of the peptide, for example, the activity of preventing or treating fatty liver.

[0033] The peptide of the present invention can be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, etc., within a range that does not change its activity.

[0034] The peptide of the present invention includes a peptide comprising an amino acid sequence substantially identical to a peptide comprising an amino acid sequence of SEQ ID NO: 1 or a peptide comprising an amino acid sequence of SEQ ID NO: 2, and a variant or active fragment thereof. The substantially identical amino acid sequence refers to an amino acid sequence having a sequence identity of 75% or more, for example, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, or 98% or more, with the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 2, respectively. In addition, the peptide may additionally include a targeting sequence, a tag, a labeled residue, or an amino acid sequence manufactured for a specific purpose to increase half-life or peptide stability.

[0035] The peptide of the present invention may be modified at the N-terminus and / or C-terminus to select a portion of the amino acid sequence and increase its activity. Such N-terminus and / or C-terminus modifications can significantly improve the stability of the peptide of the present invention, for example, increasing the half-life of the peptide when administered in vivo. The term "stability" encompasses not only in vivo stability, which protects the peptide of the present invention from attack by in vivo protein-cleaving enzymes, but also storage stability (e.g., room temperature storage stability).

[0036] The above N-terminal modification may be a modification in which a protecting group selected from the group consisting of an acetyl group, a fluoreonylmethoxycarbonyl group, a formyl group, a palmitoyl group, a myristyl group, a stearyl group, and polyethylene glycol (PEG) is bonded to the N-terminus of the peptide. The above C-terminal modification may be a modification in which a hydroxyl group (-OH), an amino group (-NH2), a hydrazino group (-NHNH2), or the like is bonded to the C-terminus of the peptide, but is not limited thereto.

[0037] The peptide of the present invention can be manufactured using various methods widely known in the art. For example, the peptide of the present invention can be manufactured using chemical synthesis methods known in the art, particularly solid-phase synthesis techniques or liquid-phase synthesis techniques.

[0038] In addition, the peptide of the present invention is artificially synthesized, or non-naturally occurring or engineered, wherein "non-naturally occurring or engineered" means a state that is not a state that occurs in nature, but is created by artificial modification. Here, the artificial modification may include artificially synthesizing an amino acid sequence by mimicking a plurality of amino acid structures, or engineering to obtain chemical stability, enhanced pharmacological properties, altered specificity, or reduced antigenicity.

[0039] The peptide complex of the present invention comprises a peptide comprising an amino acid sequence of SEQ ID NO: 1 and a peptide comprising an amino acid sequence of SEQ ID NO: 2.

[0040] The peptide complex of the present invention may mean a mixture of a peptide including the amino acid sequence of SEQ ID NO: 1 and a peptide including the amino acid sequence of SEQ ID NO: 2.

[0041] In the peptide complex of the present invention, the ratio of the peptide including the amino acid sequence of SEQ ID NO. 1 and the peptide including the amino acid sequence of SEQ ID NO. 2 is not limited to a specific range, and for example, the ratio may be used by selecting an appropriate range within the range of 100:1 to 1:100 in weight ratio.

[0042] The peptide complex of the present invention has the activity of inhibiting fat synthesis and accumulation by suppressing the expression of genes and proteins related to fat synthesis in hepatocytes. The fat synthesis-related genes may be one or more selected from the group consisting of SREBP1 (Sterol regulatory element-binding protein 1), FAS (FS-7-associated surface antigen), and ELOVL6 (ELOVL Fatty Acid Elongase 6). The fat synthesis-related proteins may be one or more selected from the group consisting of SREBP1 and FAS.

[0043] The peptide complex of the present invention has the activity of inhibiting fat synthesis by increasing the expression of pACCα (phosphorylated Acetyl-CoA carboxylase α) protein in hepatocytes.

[0044] The peptide complex of the present invention has an activity of promoting lipolysis by promoting the expression of genes and proteins related to fatty acid oxidation in hepatocytes. The fatty acid oxidation-related genes may be at least one selected from the group consisting of PGC1α (Peroxisome proliferator-activated receptor-γ coactivator 1-α), CPT1 (Carnitine palmitoyltransferase I), Acox (Acyl-coenzyme A oxidase), PPARα (Peroxisome proliferator-activated receptor α), and ACADL (Acyl-CoA Dehydrogenase Long Chain). The fatty acid oxidation-related proteins may be at least one selected from the group consisting of pAMPKα (phosphorylated AMP-activated protein kinase α), PGC1α, CPT1, and GLUT4 (Glucose transporter type 4).

[0045] The peptide complex of the present invention has the activity of suppressing the expression of inflammation-related genes in hepatocytes. The inflammation-related genes may be one or more selected from the group consisting of IL-1β (Interleukin-1 beta), TNFα (Tumor necrosis factor α), and IL-6 (Interleukin-6).

[0046] The peptide complex of the present invention exhibits hepatoprotective activity by suppressing the expression of toxicity-related proteins in hepatocytes. The toxicity-related proteins may be one or more selected from the group consisting of ALT (Alanine transaminase) and AST (aspartate aminotransferase).

[0047] The peptide complex of the present invention has an activity of inhibiting liver fibrosis in liver tissue.

[0048] The peptide complex of the present invention described above can exhibit excellent efficacy in preventing, improving, or treating fatty liver disease by having the above-described activity.

[0049]

[0050] 2. Composition for preventing, improving or treating fatty liver disease

[0051] pharmaceutical composition

[0052] According to another aspect of the present invention, a pharmaceutical composition for preventing or treating fatty liver disease is provided, comprising as an active ingredient a peptide complex comprising (i) a peptide comprising the amino acid sequence of SEQ ID NO: 1; and (ii) a peptide comprising the amino acid sequence of SEQ ID NO: 2.

[0053] The peptide comprising the amino acid sequence of the above sequence number 1 and the peptide comprising the amino acid sequence of the above sequence number 2 are identical to the peptides described in the above section “1. Peptide and its activity”, and the specific description thereof is cited and not described repeatedly.

[0054] As described above, the peptide complex of the present invention has fat synthesis inhibitory activity, fat decomposition promoting activity, and hepatocyte protective activity in hepatocytes, and therefore has excellent fatty liver prevention or treatment activity.

[0055] In one embodiment, the peptide complex of the present invention can inhibit fat synthesis and accumulation in hepatocytes and promote lipolysis. Specifically, the peptide complex of the present invention can inhibit fat accumulation in hepatocytes by inhibiting the expression of genes and proteins related to fat synthesis and promoting the expression of genes and proteins related to fatty acid oxidation in hepatocytes, thereby promoting lipolysis. Furthermore, the peptide complex of the present invention can exert hepatoprotective activity by inhibiting the expression of inflammation-related genes and inhibiting the expression of hepatotoxicity-related proteins in hepatocytes. Furthermore, the peptide complex of the present invention can inhibit hepatic fibrosis in liver tissue.

[0056] Therefore, the peptide complex of the present invention can be effectively utilized as an active ingredient of a pharmaceutical composition for preventing or treating fatty liver disease, and the pharmaceutical composition of the present invention can be effectively utilized for preventing or treating fatty liver disease.

[0057] In the present invention, the fatty liver disease may be non-alcoholic fatty liver disease. The non-alcoholic fatty liver disease (NAFLD) may include simple steatosis, non-alcoholic steatohepatitis (NASH) accompanied by hepatocyte necrosis, inflammation, and fibrosis, or liver cirrhosis (LC), which is a more advanced form.

[0058] As used herein, the term “prevention” means any act of inhibiting or delaying the onset of a disease by administering the composition.

[0059] As used herein, the term "treatment" refers to any form of treatment that provides a benefit to a subject suffering from a disease or at risk of developing a disease, including improving the condition of the subject (e.g., one or more symptoms), delaying the progression of the disease, delaying the onset of symptoms, or slowing the progression of symptoms. Accordingly, the terms "treatment" and "prevention" are not intended to imply a cure or complete elimination of symptoms.

[0060] As used herein, the term "administration" means introducing a given substance into an individual by any appropriate method, and the pharmaceutical composition of the present invention may be administered via any general route capable of reaching an in vivo target. For example, the route of administration of the pharmaceutical composition of the present invention is not particularly limited, but may be administered orally or parenterally, and in the case of parenteral administration, may be administered by intramuscular injection, intravenous injection, subcutaneous injection, intraperitoneal injection, topical administration, transdermal administration, etc., but is not limited thereto.

[0061] The dosage of the pharmaceutical composition may be, but is not limited to, 0.0001 to 1000 μg, 0.001 to 1000 μg, 0.01 to 1000 μg, 0.1 to 1000 μg, or 1.0 to 1000 μg per day, and may be administered in various amounts depending on factors such as the formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity. In addition, the frequency of administration of the pharmaceutical composition may be 1 to 4 times, 2 to 3 times, or 2 times per day, and the administration period may be, but is not limited to, 4 weeks or more, 8 weeks or more, 4 to 12 weeks, or 8 to 12 weeks.

[0062] The pharmaceutical composition of the present invention may additionally comprise a therapeutically effective amount of the peptide complex described above and / or an appropriate carrier, excipient or diluent commonly used in the preparation of pharmaceutical compositions.

[0063] The term "therapeutically effective amount" above means an amount sufficient for the peptide complex, which is an active ingredient of the pharmaceutical composition of the present invention, to achieve its activity or efficacy, for example, an amount sufficient to achieve the efficacy of preventing or treating fatty liver disease.

[0064] Examples of carriers, excipients or diluents usable in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil.

[0065] The pharmaceutical composition of the present invention can be formulated in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injection solutions according to conventional methods, and can be manufactured in the form of unit doses or by being placed in multi-dose containers.

[0066] When formulating, it is prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants that are commonly used.

[0067] Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations can be prepared by mixing the pharmaceutical composition of the present invention with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium, styrax, and talc can also be used.

[0068] Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, they may contain various excipients such as wetting agents, sweeteners, fragrances, and preservatives.

[0069] Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solutions and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include withepsol, macrogol, Tween 61, cacao butter, laurin, and glycerogelatin.

[0070] Preparations for transdermal administration may include dusting powders, emulsions, suspensions, oils, sprays, ointments, cream pastes, gels, foams, or solutions. The pharmaceutical composition of the present invention may be an anhydrous ointment, containing paraffin, especially low viscosity paraffin, which is suitable for topical use and is liquid at body temperature, or may contain natural or partially synthetic fats, for example coconut fatty acid triglycerides, hydrogenated oils, for example hydrogenated peanut oil or castor oil, partial fatty acid esters of glycerol, for example glycerol monostearate or distearate, silicones, for example polymethylsiloxanes, such as hexamethyldisiloxane or octamethyltrisiloxane, fatty alcohols, for example those associated with aqueous creams and which increase the water absorption capacity, and sterols, wool wax, other emulsifiers and / or other additives.

[0071] The dosage of the peptide complex comprising the peptide comprising the amino acid sequence of SEQ ID NO: 1 and the peptide comprising the amino acid sequence of SEQ ID NO: 2 contained in the pharmaceutical composition of the present invention varies depending on the patient's condition and weight, the severity of the disease, the drug form, the route and period of administration, and may be appropriately selected depending on the case. For example, the peptide complex may be administered at a dosage of 0.0001 to 1000 mg / kg per day, specifically 0.1 to 1000 mg / kg, and the application may be applied once a day or divided into several times. The dosage of the peptide complex of the present invention may be increased or decreased depending on the route of administration, the severity of the disease, gender, weight, age, etc. Therefore, the dosage does not limit the scope of the present invention in any way.

[0072] The pharmaceutical composition of the present invention may include a peptide complex comprising a peptide comprising the amino acid sequence of SEQ ID NO: 1 of the present invention and a peptide comprising the amino acid sequence of SEQ ID NO: 2 at a concentration of 0.01 μM to 1000 μM, specifically, the peptide complex may be present at a concentration of 0.01 μM to 1000 μM; 0.05 μM to 800 μM, 0.05 μM to 700 μM, 0.05 μM to 600 μM, 0.05 μM to 500 μM, 0.05 μM to 300 μM, 0.05 μM to 200 μM; It may be included at a concentration of, but is not limited to, 0.1 μM to 800 μM, 0.1 μM to 700 μM, 0.1 μM to 600 μM, 0.1 μM to 500 μM, 0.1 μM to 300 μM, 0.1 μM to 200 μM; 1 μM to 800 μM, 1 μM to 700 μM, 1 μM to 600 μM, 1 μM to 500 μM, 1 μM to 300 μM, 1 μM to 200 μM; 5 μM to 800 μM, 5 μM to 700 μM, 5 μM to 600 μM, 5 μM to 500 μM, 5 μM to 300 μM, or 5 μM to 200 μM.

[0073] The weight ratio between the peptide complex and the pharmaceutically acceptable carrier may be, for example, 500:1 to 1:500, and as an example, the weight ratio may be 450:1 to 1:450, 400:1 to 1:400, 350:1 to 1:350, 300:1 to 1:300, 250:1 to 1:250, 200:1 to 1:200, 150:1 to 1:150, 100:1 to 1:100, 80:1 to 1:80, 60:1 to 1:60, 40:1 to 1:40, 20:1 to 1:20, 10:1 to 1:10, 8:1 to 1:8, 6:1 to 1:6, 4:1 to It can be, but is not limited to, 1:4, or 2:1 or 1:2.

[0074] Additionally, the peptide complex may be incorporated into nanosomes or nanoparticles, for example, to further improve cell penetration or stability. For example, the nanosomes may be manufactured using a microfluidizer using lecithin as a raw material, and may be incorporated into lecithin particles, for example. Any known method for manufacturing the nanosomes may be used. The size of the nanosome particles may be from 10 to 200 nm, for example, from 10 to 180 nm, from 10 to 160 nm, from 10 to 140 nm, from 10 to 120 nm, from 10 to 100 nm, from 10 to 80 nm, from 10 to 60 nm, from 10 to 40 nm, from 10 to 20 nm, from 50 to 200 nm, from 50 to 180 nm, from 50 to 160 nm, from 50 to 140 nm, from 50 to 120 nm, from 50 to 100 nm, from 50 to 80 nm, or from 50 to 60 nm.

[0075] Meanwhile, the pharmaceutical composition of the present invention may additionally contain at least one component exhibiting activity for improving, alleviating, treating or preventing fatty liver disease, in addition to the peptide complex including the peptide including the amino acid sequence of SEQ ID NO: 1 and the peptide including the amino acid sequence of SEQ ID NO: 2. The component may be, for example, any one component selected from vitamin C, vitamin B1, vitamin B2, vitamin B6, vitamin H, vitamin PP, pro-vitamin B5, vitamin A, vitamin D, vitamin E, vitamin K1 or carotene or a mixture thereof, but is not limited thereto.

[0076] Another aspect of the present invention provides a method for preventing or treating fatty liver disease, comprising the step of administering the pharmaceutical composition to a subject.

[0077] The term "subject" above refers to a human or non-human animal, such as humans, primates, mammals and vertebrates.

[0078] In addition, another aspect of the present invention provides a use of the pharmaceutical composition for the prevention or treatment of fatty liver disease.

[0079]

[0080] Food composition

[0081] According to another aspect of the present invention, a food composition for preventing or improving fatty liver disease is provided, comprising as an active ingredient a peptide complex comprising (i) a peptide comprising the amino acid sequence of SEQ ID NO: 1; and (ii) a peptide comprising the amino acid sequence of SEQ ID NO: 2.

[0082] Among the terms or elements mentioned in the description of the above peptide and pharmaceutical composition, those already mentioned are as described above.

[0083] In one embodiment, the peptide complex of the present invention inhibits fat synthesis and accumulation in hepatocytes, promotes lipolysis, and exhibits hepatocellular protective activity. Therefore, the peptide complex of the present invention can be effectively utilized as an active ingredient in a food composition for preventing or ameliorating fatty liver disease, and the food composition of the present invention can be effectively utilized for the prevention or amelioration of fatty liver disease.

[0084] As used herein, the term "improvement" may mean any action that at least reduces the severity of a symptom, for example, a parameter associated with alleviating or treating a condition.

[0085] The above food composition may be used by adding the peptide complex as is or in combination with other foods or food ingredients, and may be used appropriately according to a conventional method. For example, it may contain various flavoring agents or natural carbohydrates as additional ingredients, as in conventional beverages. Examples of the above-mentioned natural carbohydrates may include conventional sugars such as monosaccharides, such as glucose, fructose, etc.; disaccharides, such as maltose, sucrose, etc.; and polysaccharides, such as dextrin, cyclodextrin, etc.; and sugar alcohols such as xylitol, sorbitol, erythritol, etc. In addition to the above-mentioned flavoring agents, natural flavoring agents (thaumatin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) may be advantageously used. The proportion of the above-mentioned natural carbohydrates may be appropriately determined by a person skilled in the art.

[0086] In addition to the above, the food composition may contain various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. These ingredients may be used independently or in combination, and the ratio of these additives may also be appropriately selected by those skilled in the art.

[0087] In addition, the food composition of the present invention may additionally contain at least one component that exhibits improvement, alleviation, treatment or prevention of fatty liver disease, in addition to the peptide complex comprising the peptide comprising the amino acid sequence of SEQ ID NO: 1 and the peptide comprising the amino acid sequence of SEQ ID NO: 2. The component may be, for example, any one component selected from vitamin C, vitamin B1, vitamin B2, vitamin B6, vitamin H, vitamin PP, pro-vitamin B5, vitamin A, vitamin D, vitamin E, vitamin K1 or carotene or a mixture thereof, but is not limited thereto.

[0088] Meanwhile, the food composition may include a health functional food. As used herein, the term "health functional food" refers to a food manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. using raw materials or ingredients that have functionality useful to the human body. Here, "functionality" means obtaining a beneficial effect for health purposes, such as regulating nutrients for the structure and function of the human body or physiological functions. The health functional food may be manufactured by a method commonly used in the art, and during the manufacturing process, raw materials and ingredients commonly added in the art may be added. In addition, the formulation of the health functional food may be manufactured without limitation as long as it is a formulation recognized as a health functional food. The food composition may be manufactured in various forms, and unlike general drugs, it has the advantage of not having side effects that may occur with long-term use of drugs because it uses food as a raw material, and is highly portable. According to one embodiment, the health functional food can be consumed as a supplement to prevent, improve, or enhance the treatment effect of fatty liver disease.

[0089] In addition, there is no limitation on the type of health functional food to which the food composition according to one embodiment can be used / applied. The food composition containing the peptide complex as an active ingredient can be prepared by mixing other appropriate auxiliary ingredients that can be included in health functional foods and known additives according to the selection of a person skilled in the art. Examples of foods to which the peptide complex can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and in addition, it can be prepared by adding it to juice, tea, jelly, and juice.

[0090]

[0091] The peptide complex of the present invention has activity in preventing, improving, or treating fatty liver disease. Specifically, the peptide complex of the present invention can suppress fat accumulation in hepatocytes by inhibiting the expression of genes and proteins related to fat synthesis and promoting the expression of genes and proteins related to fatty acid oxidation in hepatocytes, thereby promoting lipolysis. Furthermore, the peptide complex of the present invention can suppress the expression of inflammation-related genes in hepatocytes and exert protective activity against hepatocytes by inhibiting the expression of proteins related to hepatotoxicity. Therefore, the peptide complex of the present invention is expected to be effectively utilized for the prevention, improvement, or treatment of fatty liver disease.

[0092] However, the effects of the present invention are not limited to the effects mentioned above, and other effects not mentioned will be clearly understood by those skilled in the art from the description below.

[0093]

[0094] Figure 1 shows that the peptide complex of the present invention reduces the expression of fat synthesis-related genes SREBP1, FAS, and ELOVL6 increased by palmitate treatment in HepG2 hepatocytes.

[0095] Figure 2 shows that the peptide complex of the present invention reduces the expression of fat synthesis-related proteins SREBP1 and FAS, which are increased by palmitate treatment in HepG2 hepatocytes, and increases the expression of pACCα.

[0096] Figure 3a shows that the peptide complex of the present invention reduces the expression of SREBP1, a fat synthesis-related protein increased by oleic acid treatment in HepG2 hepatocytes.

[0097] Figure 3b shows that the peptide complex of the present invention reduces the expression of the fat synthesis-related protein FAS increased by oleic acid treatment in HepG2 hepatocytes.

[0098] Figure 3c shows that the peptide complex of the present invention increases the expression of pACCα, an inhibited form of a lipogenesis-related protein that is reduced by oleic acid treatment in HepG2 hepatocytes.

[0099] Figure 4 shows that the peptide complex of the present invention reduces the level of fat accumulation increased by oleic acid treatment in HepG2 hepatocytes.

[0100] Figure 5 shows that the peptide complex of the present invention increases the amount of glycerol released from HepG2 hepatocytes, which is reduced by palmitate treatment.

[0101] Figure 6 shows that the peptide complex of the present invention increases the expression of fatty acid oxidation-related genes PGC1α, CPT1, Acox, PPARα, and ACADL, which are reduced by palmitate treatment in HepG2 hepatocytes.

[0102] Figure 7 shows that the peptide complex of the present invention increases the expression of fatty acid oxidation-related proteins pAMPKα, PGC1α, CPT1, and GLUT4, which are reduced by palmitate treatment in HepG2 hepatocytes.

[0103] Figure 8 shows that the peptide complex of the present invention reduces the expression of inflammation-related genes IL-1β, TNFα, and IL-6 increased by palmitate treatment in HepG2 hepatocytes.

[0104] Figure 9 shows that the peptide complex of the present invention reduces the expression of hepatotoxicity-related proteins ALT and AST increased by palmitate treatment in HepG2 hepatocytes.

[0105] Figure 10 shows that the peptide complex of the present invention reduces the level of fat accumulation in mouse liver tissue.

[0106] Figure 11 shows that the peptide complex of the present invention suppresses the level of collagen in mouse liver tissue, thereby suppressing the level of liver fibrosis.

[0107]

[0108] Hereinafter, the present invention will be described in detail by way of examples. However, the following examples specifically illustrate the present invention, and the content of the present invention is not limited by the following examples.

[0109]

[0110] Manufacturing Example 1: Manufacturing of peptides and peptide complexes

[0111] Peptides having amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 2 as shown in Table 1 below were synthesized using an automatic peptide synthesizer (Milligen 9050, Millipore, USA), and these synthesized peptides were purified using C18 reverse-phase high-performance liquid chromatography (HPLC) (Waters Associates, USA). The column used was ACQUITY UPLC BEH300 C18 (2.1 mm Х 100 mm, 1.7 μm, Waters Co, USA).

[0112]

[0113] Sequence number Amino acid sequence (N-terminal → C-terminal) 1LKTRN2KGSATGWMA

[0114] A peptide complex was prepared by mixing equal amounts of the peptide of sequence number 1 and the peptide of sequence number 2 manufactured above, and its efficacy was evaluated.

[0115]

[0116] Experimental Example 1: Analysis of gene expression related to fat synthesis

[0117] To confirm whether the peptide complex manufactured in Manufacturing Example 1 can suppress the expression of genes related to fat synthesis, hepatocytes were treated with palmitate to induce fat accumulation, and then the expression levels of markers of fat synthesis-related genes (SREBP1, FAS, and ELOVL6) were evaluated through RT-PCR analysis after treatment with the peptide complex.

[0118] HepG2 cells (human hepatocyte cell line) were cultured in DMEM medium containing 1% penicillin-streptomycin (P / S) and 10% fetal bovine serum (FBS). When the cell confluency reached approximately 70 to 80%, 2 x 10 5 Cells were seeded in 12-well culture plates at a density of 1 ml per well. When the cell confluency in the 12-well culture plates reached approximately 70–80%, the medium was replaced with serum-free DMEM medium. After 4 h, 25 μM palmitate (Sigma, St. Louis, MO, USA) was added to induce lipid accumulation, and the peptide complex was added at a concentration of 0.2, 2, or 20 μM / ml, followed by incubation for 48 h. After incubation, the cells were harvested and RNA was extracted using Trizol (Thermo Fisher Scientific, Pittsburgh, PA, USA). The extracted RNA was TOPScript TMAfter obtaining DNA complementary to RNA using RT DryMIX (enzynomics, Daejeon, Korea), TOPsimple TM Polymerase chain reaction (PCR) was performed on genes related to fat synthesis using DryMIX-nTaq (enzynomics, Daejeon, Korea), and the results were run on a 1.5% agarose gel to compare the mRNA expression levels of the markers under each sample treatment condition. The primer sequences of the genes used in the experiment and the primer sequence of the GAPDH gene as a control are shown in Table 2.

[0119]

[0120] Primer sequence (5'-> 3') SEQ ID NO: SREBP1 Forward(5') GAC CGA CAT CGA AGG TGA AG (3')3SREBP1 Reverse(5') AAG AGA GGA GCT CAA TGT GGC (3')4FAS Forward(5') CGG AAA CTG CAG GAG CTG TC (3')5FAS Reverse(5') CAC GGA GTT GAG CCG CAT (3')6ELOVL6 Forward(5') TGC TCC TGT ACT CCT GGT ACT CC (3')7ELOVL6 Reverse(5') TTC TTC ACT TTG CCG ATG TAG G (3')8GAPDH Forward(5') GAG TCA ACG GAT TTG GTC GT (3')9GAPDH Reverse(5') GAC AAG CTT CCC GTT CTC AG (3')10

[0121] As a result of the experiment, it was confirmed that the expression of SREBP1, FAS, and ELOVL6, which are fat synthesis-related genes, was significantly increased by palmitate treatment in HepG2 hepatocytes, but when the peptide complex was treated, the expression of the increased genes was decreased in a concentration-dependent manner of the peptide complex (Fig. 1).

[0122]

[0123] Experimental Example 2: Analysis of Protein Expression Related to Fat Synthesis

[0124] 2-1: Western blot analysis

[0125] To confirm whether the peptide complex manufactured in Manufacturing Example 1 can inhibit the expression of proteins related to fat synthesis, hepatocytes were treated with palmitate to induce fat accumulation, and then the expression levels of fat synthesis-related proteins (SREBP1, FAS, and pACCα) were evaluated through Western blot analysis after treatment with the peptide complex.

[0126] HepG2 cells (human hepatocyte cell line) were cultured in DMEM medium containing 1% P / S and 10% FBS. When cell confluency reached approximately 70 to 80%, 2 x 10 5Cells were seeded in 12-well culture plates at a density of 1 ml per well. When the cell confluency reached approximately 70–80% in the 12-well culture plates, the medium was replaced with serum-free DMEM. Four hours later, 25 μM palmitate (Sigma, St. Louis, MO, USA) was added to induce lipid accumulation. Simultaneously, the peptide complex was added at concentrations of 0.2, 2, or 20 μM / ml and incubated for 48 hours. After incubation, the cells were dissolved in lysis buffer and centrifuged at 4°C and 12,000 rpm for 30 minutes to obtain proteins, which were then quantified using a BCA kit. SDS-PAGE was performed on the proteins and electrotransferred to membranes. After blocking the protein-electroporated membrane with 5% skim milk, the membrane was reacted overnight at 4°C with anti-SREBP1 primary antibody (Abcam, Cambridge, UK), anti-FAS primary antibody (Cell signaling Technology, Danvers, MA, USA), and anti-pACCα primary antibody (Cell signaling Technology, Danvers, MA, USA), and anti-β-actin primary antibody (Santa Cruz, Dallas, TX, USA) as a loading control. The membrane was washed with PBS-T and incubated with Peroxidase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) secondary antibody (Jackson immunoResearch Laboratories, Inc., PE, USA) and Peroxidase-conjugated AffiniPure Goat Anti-Mouse IgG (H+L) secondary antibody (Jackson immunoResearch Laboratories, Inc., PE, USA) for 1 hour at room temperature.After washing the membrane again with PBS-T, it was visualized using Western detection reagent (Elpis Biotech, Daejeon, Korea) using Gel Doc (Bio-Rad, Hercules, CA, USA).

[0127] As a result of the experiment, it was confirmed that the expression of SREBP1 and FAS, which are fat synthesis-related proteins, was significantly increased by palmitate treatment in HepG2 hepatocytes, but the expression of the increased proteins was significantly decreased when the peptide complex was treated (Fig. 2). Meanwhile, ACCα protein is an enzyme that promotes fat synthesis, and it is known that the activity of the enzyme is inhibited when phosphorylated. The expression of phosphorylated ACCα (pACCα) was significantly decreased by palmitate treatment, but it was confirmed that the decreased protein expression was increased in a peptide complex concentration-dependent manner when the peptide complex was treated.

[0128] 2-2: Immunofluorescence analysis

[0129] To confirm whether the peptide complex manufactured in Manufacturing Example 1 can suppress the expression of proteins related to fat synthesis, hepatocytes were treated with oleic acid to induce fat accumulation, and then the expression levels of fat synthesis-related proteins (SREBP1, FAS, and pACCα) were evaluated using immunofluorescence analysis after treatment with the peptide complex.

[0130] HepG2 cells (human hepatocyte cell line) were cultured in DMEM medium containing 1% P / S and 10% FBS. When cell confluency reached approximately 70 to 80%, cells were seeded at a density of 3 x 10 5Cells were seeded in 6-well culture plates at a density of 2 ml per well. When the cell confluency in the 6-well culture plates reached approximately 70-80%, the medium was replaced with serum-free DMEM medium. After 4 hours, 350 μM oleic acid (Sigma, St. Louis, MO, USA) was added to induce lipid accumulation, and simultaneously, peptide complexes were added at concentrations of 0.2, 2, or 20 μM / ml, followed by incubation for 48 hours. After incubation, the cells were fixed with 4% paraformaldehyde (PFA) for 10 minutes at room temperature and permeabilized with 0.1% Triton X-100 for 5 minutes. Afterwards, the cells were blocked with phosphate-buffered saline (PBS), 10% FBS, and 0.1% Triton X-100 for 1 hour at room temperature, and then treated with anti-SREBP1 primary antibody (Abcam, Cambridge, UK), anti-FAS primary antibody (Cell signaling Technology, Danvers, MA, USA), and anti-pACCα primary antibody (Cell signaling Technology, Danvers, MA, USA) and reacted for 1 hour. After washing the cells three times, they were treated with goat anti-Rabbit IgG H&L secondary antibody (Abcam, Cambridge, UK) and goat anti-Mouse IgG H&L secondary antibody (Abcam, Cambridge, UK) and reacted for 30 minutes at room temperature. After washing the cells three times, nuclei were counterstained with DAPI (Santa Cruz, Dallas, TX, USA), and images were acquired using a LEICA fluorescence microscope (TCS SP8, Leica, Wetzlar, Germany).

[0131] Experimental results showed that the expression of SREBP1 and FAS, which are fat synthesis-related proteins, was significantly increased by oleic acid treatment in HepG2 hepatocytes, but when the peptide complex was treated, the expression of the increased proteins decreased in a concentration-dependent manner of the peptide complex (Figs. 3a and 3b). In addition, the expression of phosphorylated ACCα (pACCα) was significantly reduced by oleic acid treatment, but when the peptide complex was treated, the decreased protein expression was significantly increased (Fig. 3c).

[0132]

[0133] Experimental Example 3: Analysis of the degree of fat accumulation

[0134] To confirm whether the peptide complex manufactured in Manufacturing Example 1 can inhibit fat accumulation, the level of fat accumulation was evaluated through Nile red staining.

[0135] HepG2 cells (human hepatocyte cell line) were cultured in DMEM medium containing 1% P / S and 10% FBS. When cell confluency reached approximately 70 to 80%, cells were seeded at a density of 3 x 10 5Cells were seeded in 6-well culture plates at a density of 1 ml per well. When the cell confluency reached approximately 70-80% in the 6-well culture plates, the medium was replaced with serum-free DMEM. After 4 hours, 350 μM oleic acid (Sigma, St. Louis, MO, USA) was added to induce lipid accumulation. After 24 hours, the medium was replaced with serum-free DMEM medium supplemented with 350 μM oleic acid, and the peptide complex was added at a concentration of 0.2, 2, or 20 μM / ml, followed by incubation for 48 hours. After incubation, the DMEM was removed and washed with PBS. The cells were replaced with a 10% formalin solution, fixed at room temperature for 30 minutes, and then washed with PBS. Afterwards, Nile red acid (Sigma, St. Louis, MO, USA) solution was diluted in PBS to a concentration of 1 μg / ml and stained at room temperature for 30 minutes. After washing again with PBS, nuclei were stained with DAPI (Santa Cruz, Dallas, TX, USA) for 5 minutes. After washing again with PBS, the coverslips of the stained 6-well plates were attached to slide glasses using an aqueous mounting solution (Biomeda, Foster City, CA, USA) and observed using a fluorescence microscope.

[0136] As a result of the experiment, it was confirmed that the level of fat accumulation in HepG2 hepatocytes was significantly increased by oleic acid treatment, but when the peptide complex was treated, the increased level of fat accumulation was decreased in a concentration-dependent manner of the peptide complex (Fig. 4).

[0137]

[0138] Experimental Example 4: Analysis of the degree of fat decomposition

[0139] To confirm whether the peptide complex manufactured in Manufacturing Example 1 can promote lipolysis, the degree of lipolysis was evaluated using a glycerol colorimetric assay.

[0140] HepG2 cells (human hepatocyte cell line) were cultured in DMEM medium containing 1% P / S and 10% FBS. When cell confluency reached approximately 70 to 80%, 2 x 10 5 Cells were seeded into 12-well culture plates at a density of 1 ml per well. When the cell confluency reached approximately 70–80% in the 12-well culture plates, the medium was replaced with serum-free DMEM. Four hours later, 25 μM palmitate (Sigma, St. Louis, MO, USA) was added to induce lipid accumulation. Simultaneously, the peptide complex was added at concentrations of 0.2, 2, or 20 μM / ml, followed by incubation for 48 hours. After incubation, the supernatant was collected and the amount of glycerol released into the culture medium was measured using a Glycerol colorimetric assay kit (Cayman Chemical, Michigan, USA).

[0141] Experimental results showed that palmitate treatment significantly reduced glycerol release in HepG2 hepatocytes, indicating a reduced level of lipolysis (Fig. 5). However, when treated with the peptide complex, the reduced glycerol release was increased in a concentration-dependent manner, confirming an increased level of lipolysis.

[0142]

[0143] Experimental Example 5: Analysis of Gene Expression Related to Fatty Acid Oxidation

[0144] To confirm whether the peptide complex manufactured in Manufacturing Example 1 can promote the expression of genes related to fatty acid oxidation, hepatocytes were treated with palmitate to induce fat accumulation, and then the expression levels of fatty acid oxidation-related genes (PGC1α, CPT1, Acox, PPARα, and ACADL) were evaluated through RT-PCR analysis after treatment with the peptide complex.

[0145] HepG2 cells (human hepatocyte cell line) were cultured in DMEM medium containing 1% P / S and 10% FBS. When cell confluency reached approximately 70 to 80%, 2 x 10 5 Cells were seeded in 12-well culture plates at a density of 1 ml per well. When the cell confluency in the 12-well culture plates reached approximately 70–80%, the medium was replaced with serum-free DMEM medium. After 4 h, 25 μM palmitate (Sigma, St. Louis, MO, USA) was added to induce lipid accumulation, and the peptide complex was added at a concentration of 0.2, 2, or 20 μM / ml, followed by incubation for 48 h. After incubation, the cells were harvested and RNA was extracted using Trizol (Thermo Fisher Scientific, Pittsburgh, PA, USA). The extracted RNA was TOPScript TM After obtaining DNA complementary to RNA using RT DryMIX (enzynomics, Daejeon, Korea), TOPsimple TMPolymerase chain reaction (PCR) was performed on genes related to fatty acid oxidation using DryMIX-nTaq (enzynomics, Daejeon, Korea), and the results were run on a 1.5% agarose gel to compare the mRNA expression levels of the markers under each sample treatment condition. The primer sequences of the genes used in the experiment and the primer sequence of the GAPDH gene as a control are shown in Table 3.

[0146]

[0147] Primer sequence (5'-> 3') SEQ ID NO:PGC1α Forward(5') AGT CTG TAT GGA GTG ACA TCG AG (3')11PGC1α Reverse(5') GGC AAT CCG TCT TCA TCC AC (3')12CPT1 Forward(5') CCT CCA GTT GGC TTA TCG TG (3')13CPT1 Reverse(5') TTC TTC GTC TGG CTG GAC AT (3')14Acox Forward(5') CCG CCG AGA GAT CGA GAA C (3')15Acox Reverse(5') CAG TTG CCT GGT GAA GCA AG (3')16PPARα Forward(5') AAG GGC TTC TTT CGG CGA AC (3')17PPARα Reverse(5') TGA CCT TGT TCA TGT TGA AGT TCT TCA (3')18ACADL Forward(5') TGC AAT AGC AAT GAC AGA GCC (3')19ACADL Reverse(5') CGC AAC TAC AAT CAC AAC ATC AC (3')20GAPDH Forward(5') GAG TCA ACG GAT TTG GTC GT (3')9GAPDH Reverse(5') GAC AAG CTT CCC GTT CTC AG (3')10

[0148] As a result of the experiment, it was confirmed that the expression of fatty acid oxidation-related genes PGC1α, CPT1, Acox, PPARα, and ACADL was significantly reduced by palmitate treatment in HepG2 hepatocytes, but the expression of the reduced genes was significantly increased when the peptide complex was treated (Fig. 6).

[0149]

[0150] Experimental Example 6: Analysis of Protein Expression Related to Fatty Acid Oxidation

[0151] To confirm whether the peptide complex manufactured in Manufacturing Example 1 can suppress the expression of proteins related to fat synthesis, hepatocytes were treated with palmitate to induce fat accumulation, and then the expression levels of fatty acid oxidation-related proteins (pAMPKα, PGC1α, CPT1, and GLUT4) were evaluated through Western blot analysis after treatment with the peptide complex.

[0152] HepG2 cells (human hepatocyte cell line) were cultured in DMEM medium containing 1% P / S and 10% FBS. When cell confluency reached approximately 70 to 80%, 2 x 10 5Cells were seeded in 12-well culture plates at a density of 1 ml per well. When the cell confluency reached approximately 70–80% in the 12-well culture plates, the medium was replaced with serum-free DMEM. Four hours later, 25 μM palmitate (Sigma, St. Louis, MO, USA) was added to induce lipid accumulation. Simultaneously, the peptide complex was added at concentrations of 0.2, 2, or 20 μM / ml and incubated for 48 hours. After incubation, the cells were dissolved in lysis buffer and centrifuged at 4°C and 12,000 rpm for 30 minutes to obtain proteins, which were then quantified using a BCA kit. SDS-PAGE was performed on the proteins and electrotransferred to membranes. After blocking the protein-electroporated membrane with 5% skim milk, the membrane was incubated overnight at 4°C with anti-pAMPKα primary antibody (Cell signaling Technology, Danvers, MA, USA), anti-PGC1α primary antibody (Abcam, Cambridge, UK), anti-CPT1 primary antibody (Abcam, Cambridge, UK), and anti-GLUT4 primary antibody (Invitrogen, Carlsbad, California, USA), and anti-β-actin primary antibody (Santa Cruz, Dallas, TX, USA) as a loading control. The membrane was washed with PBS-T and incubated with Peroxidase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) secondary antibody (Jackson immunoResearch Laboratories, Inc., PE, USA) and Peroxidase-conjugated AffiniPure Goat Anti-Mouse IgG (H+L) secondary antibody (Jackson immunoResearch Laboratories, Inc., PE, USA) for 1 hour at room temperature.After washing the membrane again with PBS-T, it was visualized using Western detection reagent (Elpis Biotech, Daejeon, Korea) using Gel Doc (Bio-Rad, Hercules, CA, USA).

[0153] As a result of the experiment, it was confirmed that the expression of fatty acid oxidation-related proteins pAMPKα, PGC1α, CPT1, and GLUT4 was significantly reduced by palmitate treatment in HepG2 hepatocytes, but when the peptide complex was treated, the expression of the reduced proteins was significantly increased (Fig. 7).

[0154]

[0155] Experimental Example 7: Analysis of Inflammation-Related Gene Expression

[0156] To confirm whether the peptide complex manufactured in Manufacturing Example 1 can suppress the expression of inflammation-related genes, hepatocytes were treated with palmitate to induce fat accumulation, and then the expression levels of inflammation-related genes (IL-1β, TNFα, and IL-6) were evaluated through RT-PCR analysis after treatment with the peptide complex.

[0157] HepG2 cells (human hepatocyte cell line) were cultured in DMEM medium containing 1% P / S and 10% FBS. When cell confluency reached approximately 70 to 80%, 2 x 10 5Cells were seeded in 12-well culture plates at a density of 1 ml per well. When the cell confluency in the 12-well culture plates reached approximately 70–80%, the medium was replaced with serum-free DMEM medium. After 4 h, 25 μM palmitate (Sigma, St. Louis, MO, USA) was added to induce lipid accumulation, and the peptide complex was added at a concentration of 0.2, 2, or 20 μM / ml, followed by incubation for 48 h. After incubation, the cells were harvested and RNA was extracted using Trizol (Thermo Fisher Scientific, Pittsburgh, PA, USA). The extracted RNA was TOPScript TM After obtaining DNA complementary to RNA using RT DryMIX (enzynomics, Daejeon, Korea), TOPsimple TM Polymerase chain reaction (PCR) was performed on inflammation-related genes using DryMIX-nTaq (enzynomics, Daejeon, Korea), and the results were run on a 1.5% agarose gel to compare the mRNA expression levels of the markers under each sample treatment condition. The primer sequences of the genes used in the experiment and the primer sequence of the GAPDH gene as a control are shown in Table 4.

[0158]

[0159] Primer sequence (5'-> 3')SEQ ID NO:IL-1β Forward(5') TTC GAC ACA TGG GAT AAC GA (3')21IL-1β Reverse(5') TCT TTC AAC ACG CAG GAC AG (3')22TNFα Forward(5') AAC ATC CAA CCT TCC CAA ACG (3')23TNFα Reverse(5') GAC CCT AAG CCC CCA ATT CTC (3')24IL-6 Forward(5') AAA GAG GCA CTG CCA GAA AA (3')25IL-6 Reverse(5') ATC TGA GGT GCC CAT GCT AC (3')26GAPDH Forward(5') GAG TCA ACG GAT TTG GTC GT (3')9GAPDH Reverse(5') GAC AAG CTT CCC GTT CTC AG (3')10

[0160] As a result of the experiment, it was confirmed that the expression of inflammation-related genes IL-1β, TNFα, and IL-6 was significantly increased by palmitate treatment in HepG2 hepatocytes, but when the peptide complex was treated, the expression of the increased genes was significantly reduced (Fig. 8).

[0161]

[0162] Experimental Example 8: Hepatocellular protective activity analysis

[0163] To confirm whether the peptide complex manufactured in Manufacturing Example 1 has hepatoprotective activity, hepatocytes were treated with palmitate to induce hepatotoxicity, and then the expression levels of ALT and AST, which are hepatotoxicity-related proteins, were evaluated using ELISA after treatment with the peptide complex.

[0164] HepG2 cells (human hepatocyte cell line) were cultured in DMEM medium containing 1% P / S and 10% FBS. When cell confluency reached approximately 70 to 80%, 2 x 10 5Cells were seeded into 12-well culture plates at a density of 1 ml per well. When the cell confluency reached approximately 70-80% in the 12-well culture plates, the medium was replaced with serum-free DMEM medium. After 4 hours, 25 μM palmitate (Sigma, St. Louis, MO, USA) was added to induce lipid accumulation, and simultaneously, the peptide complex was added at a concentration of 0.2, 2, or 20 μM / ml, followed by incubation for 48 hours. The culture medium was harvested, spun down at 2,000 g for 10 minutes, and the supernatant was collected and analyzed using a Human ALT ELISA kit (Abcam, Cambridge, UK) and a Human AST ELISA kit (Abcam, Cambridge, UK).

[0165] As a result of the experiment, it was confirmed that the expression of ALT and AST, which are hepatotoxicity-related proteins, was significantly increased by palmitate treatment in HepG2 hepatocytes, but when the peptide complex was treated, the expression of the increased proteins was decreased in a concentration-dependent manner of the peptide complex (Fig. 9).

[0166]

[0167] Experimental Example 9: Analysis of the degree of fat accumulation in liver tissue

[0168] To confirm whether the peptide complex manufactured in Manufacturing Example 1 can inhibit fat accumulation in liver tissue, the level of fat accumulation was evaluated through H&E staining.

[0169] An obese mouse model was created by feeding 8-week-old mice a high-fat diet (65 kcal%) for 10 weeks. After daily feeding of the peptide complex for 2 weeks, the mice were sacrificed and liver tissue staining was performed. Liver tissues obtained from the mice were washed with phosphate-buffered saline (PBS) at room temperature and fixed in a 10% formalin solution (Thermo Fisher Scientific, Pittsburgh, PA, USA) for 24 hours. After fixation, the tissues were washed with distilled water (DW) for 1 hour, dehydrated in a gradient ethanol series from 70% to 100%, and then placed in xylene for culture. Paraffin blocks were prepared, sectioned at 5 μm thickness using a microtome (LEICA RM 2255, Leica, Wetzlar, Germany), and liver tissue sample fragments were mounted on glass slides. The attached samples were immersed in xylene twice for 5 minutes each, and then deparaffinized and rehydrated using a gradient ethanol series from 100% to 70%. After washing again in deionized water (DW), the nuclei were stained by immersing them in a hematoxylin solution (Agilent Dako, Glostrup, Denmark) for 5 minutes at room temperature, and washed with deionized water. Afterwards, the cytoplasm was stained by immersing them in an eosin Y solution (Thermo Fisher Scientific, Pittsburgh, PA, USA) for 5 minutes at room temperature, and washed with deionized water. Afterwards, a gradient ethanol series from 70% to 100% was performed, and the slides were dehydrated by immersing them in xylene, and the tissue slides were mounted using a xylene-based mounting medium (Sigma, St. Louis, MO, USA). After drying, the tissue morphology was observed and images were taken using an optical microscope (FLEXACAM C1, Leica, Wetzlar, Germany).

[0170] As a result of the experiment, it was confirmed that the lipid area in the liver tissue of obese mice was significantly increased compared to that of normal mice, but when the peptide complex was treated, the increased liver lipid area was significantly reduced (Fig. 10).

[0171]

[0172] Experimental Example 10: Analysis of Fibrosis Levels in Liver Tissue

[0173] To determine whether the peptide complex prepared in Manufacturing Example 1 can suppress the level of fibrosis in liver tissue, the level of liver fibrosis was evaluated by staining collagen in liver tissue using Sirius red staining.

[0174] An obese mouse model was created by feeding 8-week-old mice a high-fat diet (65 kcal%) for 10 weeks. After daily feeding of the peptide complex for 2 weeks, the mice were sacrificed and liver tissue staining was performed. Liver tissues obtained from the mice were washed with phosphate-buffered saline (PBS) at room temperature and fixed in a 10% formalin solution (Thermo Fisher Scientific, Pittsburgh, PA, USA) for 24 hours. After fixation, the tissues were washed with deionized water (DW) for 1 hour, dehydrated in a gradient ethanol series from 70% to 100%, and placed in xylene for incubation. Paraffin blocks were then prepared, sectioned at 5 μm thickness using a microtome (LEICA RM 2255, Leica, Wetzlar, Germany), and liver tissue sample fragments were mounted on slide glass. The attached samples were immersed in xylene twice for 5 minutes each, and then deparaffinized and rehydrated using a gradient ethanol series from 100% to 70%. After washing the samples with distilled water, the fibrotic areas (collagen) were stained with Picro Sirius Red solution (Abcam, Cambridge, MA, USA) at room temperature for 1 hour. After staining, the samples were washed twice with acetic acid solution and 100% ethanol, and the tissue slides were dehydrated by immersing them in xylene. Afterwards, the tissue slides were mounted using a xylene-based mounting medium (Sigma, St. Louis, MO, USA). After drying, the tissue morphology was observed and images were taken using an optical microscope (FLEXACAM C1, Leica, Wetzlar, Germany).

[0175] As a result of the experiment, it was confirmed that the collagen area in the liver tissue of obese mice was significantly increased compared to that of normal mice, but when the peptide complex was treated, the increased collagen area was significantly reduced, thereby alleviating the level of fibrosis (Fig. 11).

[0176]

[0177] Through the above experiments, it was confirmed that the peptide complex of the present application can suppress the expression of genes and proteins related to fat synthesis in hepatocytes and significantly suppress the degree of fat accumulation. In addition, it was confirmed that the peptide complex of the present application can increase the degree of lipolysis in hepatocytes and also increase the expression of genes and proteins related to fatty acid oxidation. Furthermore, it was confirmed that the peptide complex of the present application can suppress the expression of inflammation-related genes in hepatocytes, suppress hepatotoxicity, and have hepatocellular protective activity, and can suppress the degree of fat accumulation and liver fibrosis in liver tissue. Therefore, the peptide complex of the present application is expected to be effectively utilized for the prevention or treatment of fatty liver disease.

[0178]

[0179] Although representative embodiments of the present application have been described above as examples, the scope of the present application is not limited to the specific embodiments described above, and a person with ordinary knowledge in the relevant field will be able to make appropriate changes within the scope described in the claims of the present application.

Claims

1. A pharmaceutical composition for preventing or treating fatty liver disease, comprising a peptide complex comprising (i) a peptide comprising the amino acid sequence of sequence number 1; and (ii) a peptide comprising the amino acid sequence of sequence number 2; as an active ingredient.

2. In claim 1, A pharmaceutical composition for preventing or treating fatty liver disease, wherein the fatty liver disease is non-alcoholic fatty liver disease.

3. In claim 1, A pharmaceutical composition for preventing or treating fatty liver disease, wherein the peptide complex inhibits the expression of one or more genes selected from the group consisting of SREBP1, FAS, and ELOVL6 in hepatocytes.

4. In claim 1, A pharmaceutical composition for preventing or treating fatty liver disease, wherein the peptide complex inhibits the expression of one or more proteins selected from the group consisting of SREBP1 and FAS in hepatocytes.

5. In claim 1, A pharmaceutical composition for preventing or treating fatty liver disease, wherein the peptide complex increases the expression of pACCα protein in hepatocytes.

6. In claim 1, A pharmaceutical composition for preventing or treating fatty liver disease, wherein the peptide complex inhibits the expression of one or more genes selected from the group consisting of PGC1α, CPT1, Acox, PPARα, and ACADL in hepatocytes.

7. In claim 1, A pharmaceutical composition for preventing or treating fatty liver disease, wherein the peptide complex inhibits the expression of one or more proteins selected from the group consisting of pAMPKα, PGC1α, CPT1, and GLUT4 in hepatocytes.

8. In claim 1, A pharmaceutical composition for preventing or treating fatty liver disease, wherein the above peptide complex inhibits fat accumulation in hepatocytes.

9. In claim 1, A pharmaceutical composition for preventing or treating fatty liver disease, wherein the peptide complex promotes fat decomposition in hepatocytes.

10. In claim 1, A pharmaceutical composition for preventing or treating fatty liver disease, wherein the peptide complex inhibits the expression of one or more genes selected from the group consisting of IL-1β, TNFα, and IL-6 in hepatocytes.

11. In claim 1, A pharmaceutical composition for preventing or treating fatty liver disease, wherein the peptide complex inhibits the expression of one or more proteins selected from the group consisting of ALT and AST in hepatocytes.

12. In claim 1, A pharmaceutical composition for preventing or treating fatty liver disease, wherein the peptide complex has hepatocellular protective activity.

13. In claim 1, A pharmaceutical composition for preventing or treating fatty liver disease, wherein the peptide complex inhibits liver fibrosis in liver tissue.

14. A food composition for preventing or improving fatty liver disease, comprising a peptide complex comprising (i) a peptide comprising the amino acid sequence of sequence number 1; and (ii) a peptide comprising the amino acid sequence of sequence number 2; as an active ingredient.

Images