Pharmaceutical composition for prevention or treatment of non-alcoholic fatty liver disease, comprising bombyx mori powder or silk fibroin-derived peptide

Abstract

One aspect provides a pharmaceutical composition for the prevention or treatment of non-alcoholic fatty liver disease, comprising a Bombyx mori powder and / or a silk fibroin-derived peptide, wherein 0.005 g / kg to 0.2 g / kg of a Bombyx mori powder is administered, and 10 μg / mL to 200 μg / mL of a silk fibroin-derived peptide is administered. The Bombyx mori powder and / or the silk fibroin-derived peptide, according to one aspect, reduces fat accumulation in the liver, increases fatty acid oxidation, and moreover, significantly reduces liver inflammation caused by non-alcoholic fatty liver. That is, the Bombyx mori powder and / or the silk fibroin-derived peptide exhibits a notably excellent effect in the prevention, alleviation, or treatment of fatty liver and thus may be effectively used as a novel therapeutic agent for non-alcoholic fatty liver disease.

Description

Pharmaceutical composition for the prevention or treatment of non-alcoholic fatty liver disease comprising red silkworm powder or silk fibroin-derived peptides

[0001] The present invention relates to a pharmaceutical composition for the prevention or treatment of non-alcoholic fatty liver disease comprising red silkworm powder or a peptide derived from silk fibroin, wherein the red silkworm powder is administered at a concentration of 0.005 g / kg to 0.2 g / kg.

[0002] Fatty liver refers to a condition in which fat accumulates within liver cells. While fat accounts for about 5% of the total weight of a normal liver, a condition in which more fat is accumulated is called fatty liver. Non-alcoholic fatty liver disease (NAFLD) is the most common chronic disease and refers to a condition in which fat accumulates within liver cells without excessive alcohol consumption.

[0003] The prevalence of non-alcoholic fatty liver disease is rapidly increasing both domestically and abroad, along with the rise in obesity rates, and is closely associated with type 2 diabetes, obesity, and metabolic syndrome. It has been found that some of these patients progress through the stage of non-alcoholic steatohepatitis (NASH) to end-stage liver diseases such as liver fibrosis, cirrhosis, or liver cancer.

[0004] As such, although the number of patients with non-alcoholic fatty liver disease worldwide reaches 60 million, there are currently no non-alcoholic fatty liver disease treatments approved by the U.S. Food and Drug Administration (FDA) or the European Medicines Agency (EMA), and only diabetes, obesity, and hyperlipidemia treatments are being used as non-alcoholic fatty liver disease treatments.

[0005] Therefore, research is needed on treatments that can prevent or treat non-alcoholic fatty liver disease, particularly early-stage non-alcoholic fatty liver disease, without side effects.

[0006] One aspect provides a pharmaceutical composition for the prevention or treatment of non-alcoholic fatty liver disease comprising red silkworm powder, wherein the red silkworm powder is administered at a concentration of 0.005 g / kg to 0.2 g / kg.

[0007] Another aspect provides a health functional food for the prevention or improvement of non-alcoholic fatty liver disease containing red silkworm powder, wherein the red silkworm powder is consumed in an amount of 0.005 g / kg to 0.2 g / kg.

[0008] Another aspect is a step of obtaining cooked silkworms by cooking silkworms at 70 to 130 ℃ for 100 to 160 minutes; and

[0009] The present invention provides a method for manufacturing red silkworm powder comprising the step of drying the above-mentioned cooked silkworm.

[0010] Another aspect provides a method for preventing or treating non-alcoholic fatty liver disease comprising the step of administering red silkworm powder to individuals requiring it at a dose of 0.005 g / kg to 0.2 g / kg.

[0011] Another aspect provides a use of red silkworm powder for the manufacture of a drug for the prevention or treatment of non-alcoholic fatty liver disease, wherein the red silkworm powder is administered at a concentration of 0.005 g / kg to 0.2 g / kg.

[0012] Another aspect provides a pharmaceutical composition for the prevention or treatment of non-alcoholic fatty liver disease comprising a silk fibroin-derived peptide, wherein the silk fibroin-derived peptide is administered at a concentration of 10 μg / mL to 200 μg / mL.

[0013] Another aspect is to provide a health functional food for the prevention or improvement of non-alcoholic fatty liver disease comprising a silk fibroin-derived peptide, wherein the silk fibroin-derived peptide is consumed in an amount of 10 μg / mL to 200 μg / mL.

[0014] Another aspect provides a method for preventing or treating non-alcoholic fatty liver disease comprising the step of administering a silk fibroin-derived peptide to an individual in need of it at a concentration of 10 μg / mL to 200 μg / mL.

[0015] Another aspect provides a use of a silk fibroin-derived peptide for the manufacture of a drug for the prevention or treatment of non-alcoholic fatty liver disease, wherein the silk fibroin-derived peptide is administered at a concentration of 10 μg / mL to 200 μg / mL.

[0016] One aspect provides a pharmaceutical composition for the prevention or treatment of non-alcoholic fatty liver disease comprising red silkworm powder, wherein the red silkworm powder is administered at a concentration of 0.005 g / kg to 0.2 g / kg.

[0017] In one embodiment, the red silkworm powder may be administered in an amount of 0.005 g / kg to 0.2 g / kg, and more specifically, in an amount of 0.005 g / kg to 0.2 g / kg, 0.005 g / kg to 0.1 g / kg, 0.005 g / kg to 0.05 g / kg, 0.005 g / kg to 0.01 g / kg, 0.01 g / kg to 0.2 g / kg, 0.01 g / kg to 0.1 g / kg, 0.01 g / kg to 0.05 g / kg, 0.05 g / kg to 0.2 g / kg, 0.05 g / kg to 0.1 g / kg, or 0.1 g / kg to 0.2 g / kg, and more specifically, in an amount of 0.01 g / kg to 0.1 g / kg.

[0018] The term "Hongjam" may be used interchangeably with "cooked Sukjam," and Hongjam refers to Sukjam, which is the state just before a silkworm spins a cocoon, that has been cooked by steaming with water vapor and freeze-dried. Specifically, the above-mentioned Sukjam refers to a silkworm at the stage where the silk gland begins to rapidly enlarge and the internal organs gradually degenerate when the silkworm reaches the 3rd day of the 5th instar, and by the 7th or 8th day of the 5th instar, it becomes filled only with silk protein.

[0019] The above red silkworm pupae can be produced from silkworm varieties such as Baekgokjam, Geumgangjam, Daebaekjam, Hansaengjam, Juhwangjam, Geumokjam, Baekhwangjam, Daebakjam, Daehwangjam, and Golden Silk. Specifically, it may be Baekgokjam.

[0020] The term "prevention" may refer to any act of suppressing or delaying the occurrence of non-alcoholic fatty liver disease in an individual by administering a pharmaceutical composition according to one aspect.

[0021] The term "treatment" above may refer to any act in which the symptoms of an individual's non-alcoholic fatty liver disease are improved or beneficially altered by the administration of a pharmaceutical composition according to one aspect.

[0022] Additionally, the above pharmaceutical composition may be provided as a pharmaceutical composition comprising one or more pharmaceutically acceptable carriers, excipients, and / or diluents.

[0023] Specifically, the carrier may be, for example, a colloidal suspension, powder, saline solution, lipid, liposome, microsphere, or nano-spherical particle. These may form a complex with or be associated with a transport means and may be transported in vivo using a transport system known in the art, such as lipids, liposomes, microparticles, gold, nanoparticles, polymers, condensation agents, polysaccharides, polyamino acids, dendrimers, saponins, adsorption-enhancing substances, or fatty acids.

[0024] When the above pharmaceutical composition is formulated, it may be prepared using diluents or excipients such as commonly used lubricants, sweeteners, flavorings, emulsifiers, suspending agents, preservatives, fillers, volume expanders, binders, wetting agents, disintegrants, and surfactants. Solid dosage forms for oral administration may include tablets, pills, powders, granules, capsules, orally dissolving films, and such solid dosage forms may be prepared by mixing at least one excipient with the above composition, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition, lubricants such as magnesium stearate and talc may also be used in addition to simple excipients. Liquid formulations for oral administration include suspensions, oral liquids, emulsions, syrups, etc., and may contain various excipients, such as humectants, sweeteners, flavorings, and preservatives, in addition to commonly used simple diluents like water and liquid paraffin. Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspensions. Witepsol, macrogol, Tween 61, cacao oil, laurin oil, glycerogelatin, etc. may be used as bases for suppositories, and known diluents or excipients may be used when manufactured in the form of ophthalmic preparations.

[0025] The above pharmaceutical composition is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level may be determined based on factors including the type and severity of the patient's disease, drug activity, sensitivity to the drug, time of administration, route of administration and elimination rate, duration of treatment, concomitantly used drugs, and other factors well known in the medical field. The administration may be given once a day or divided into several doses. For example, it may be given every other day or once a week.

[0026] The term "administration" above means introducing a specific substance into an individual by an appropriate method, and "individual" refers to all living organisms, including humans, rats, mice, pigs, horses, cattle, and livestock, that may carry non-alcoholic fatty liver disease. Specific examples may include mammals, including humans.

[0027] The above pharmaceutical composition may be administered orally or parenterally, and when administered parenterally, a method of injection may be selected, such as external application to the skin or intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intra-arterial injection, intramedullary injection, intracardiac injection, intrathecal injection, transdermal injection, nasal injection, enteral injection, local injection, sublingual injection, rectal injection, or thoracic injection.

[0028] In one aspect, the pharmaceutical composition may further include other agents for treating non-alcoholic fatty liver disease in addition to red silkworm powder.

[0029] The other non-alcoholic fatty liver disease treatment mentioned above may be included in the pharmaceutical composition in the minimum amount to obtain maximum effect without side effects, which can be easily determined by a person skilled in the art.

[0030] In addition, in one aspect, the pharmaceutical composition may be administered alone or in combination with other known treatments for non-alcoholic fatty liver disease. That is, the pharmaceutical composition may be administered in conjunction with other treatments for non-alcoholic fatty liver disease, and may be administered simultaneously, separately, or sequentially, and may be administered as a single or multiple doses.

[0031] When the above-mentioned other treatment for non-alcoholic fatty liver disease is administered in combination with the above-mentioned pharmaceutical composition, it may be administered in an amount or ratio that can obtain maximum effect without side effects, and this can be easily determined by a person skilled in the art.

[0032] The other non-alcoholic fatty liver disease treatment mentioned above may be a conventionally known non-alcoholic fatty liver disease treatment or a newly developed non-alcoholic fatty liver disease treatment.

[0033] In one embodiment, the non-alcoholic fatty liver disease may be one or more selected from the group consisting of non-alcoholic fatty liver, non-alcoholic steatohepatitis, liver fibrosis, liver cirrhosis, and liver cancer, and specifically may be non-alcoholic fatty liver.

[0034] In one embodiment, the red silkworm may contain silk fiber protein.

[0035] The above silk protein includes fibroin, a fibrous protein, and sericin, a rubbery protein surrounding it. In addition, the above silk protein is rich in amino acids such as glycine (Gly), serine (Ser), and alanine (Ala).

[0036] In one embodiment, the red silkworm powder may include aspartic acid (Asp), threonine (Thr), serine (Ser), glutamic acid (Glu), glycine (Gly), alanine (Ala), valine (Val), methionine (Met), isoleucine (Ile), leucine (Leu), tyrosine (Tyr), phenylalanine (Phe), lysine (Lys), histidine (His), and arginine (Arg).

[0037] In one embodiment, the components of the red silkworm powder were analyzed, and it was confirmed that among the 15 detected amino acids, glycine (Gly) was the most abundant, followed by serine (Ser) and alanine (Ala), thus confirming that silkworm protein is the main component of red silkworm powder that affects the treatment of non-alcoholic fatty liver disease (see Example 1).

[0038] In one embodiment, the red silkworm powder may reduce fat accumulated in the liver.

[0039] Reducing the aforementioned fat accumulated in the liver includes reducing it by removing it through decomposition, oxidation, etc., and reducing it by inhibiting fat synthesis.

[0040] In one example, it was confirmed that in red silkworm pupae administration groups 1 and 2, in which red silkworm pupae powder was administered along with a high-fat diet (HFD), body weight and liver weight decreased compared to the HFD administration group, lipid globules in liver tissue decreased, and hepatic steatosis and ORO staining scores also decreased. Accordingly, it was confirmed that red silkworm pupae powder has preventive, aggravating, or therapeutic effects on NAFLD, particularly non-alcoholic fatty liver disease (see Example 2).

[0041] In one embodiment, the red silkworm powder may reduce one or more concentrations selected from the group consisting of blood triglyceride concentration, blood glucose concentration, blood total cholesterol concentration, blood LDL-cholesterol concentration, blood alanine transaminase (ALT) concentration, blood aspartate aminotransferase (AST) concentration, blood gamma-glutamyl transferase (GGT) concentration, and blood bilirubin concentration; specifically, blood triglyceride concentration, blood glucose concentration, blood total cholesterol concentration, blood LDL-cholesterol concentration, blood alanine transaminase (ALT) concentration, blood aspartate aminotransferase (AST) concentration, blood It may reduce the blood concentration of all components selected from the group consisting of GGT (gamma-glutamyl transferase) concentration and blood bilirubin concentration.

[0042] In one embodiment, the red silkworm powder may increase the concentration of HDL-cholesterol in the blood.

[0043] In one embodiment, it was confirmed that in red silkworm pupae administration groups 1 and 2, in which red silkworm pupae powder was administered along with a high-fat diet (HFD), the concentrations of blood triglycerides, glucose, total cholesterol, and LDL-cholesterol—indicators of fatty liver—decreased and the concentration of blood HDL-cholesterol increased compared to the HFD administration group, while the concentrations of blood ALT, AST, GGT, and bilirubin—indicators of liver damage—significantly decreased. Accordingly, it was confirmed that red silkworm pupae powder is effective in treating fatty liver and liver tissue damaged by fatty liver, and can be utilized as a preventive, remedial, or therapeutic agent for NAFLD, particularly non-alcoholic fatty liver disease (see Example 3).

[0044] In one embodiment, the red silkworm powder may increase the expression of one or more proteins selected from the group consisting of p-AMPK (p-adenosine monophosphate-activated protein kinase), SIRT1 (sirtuin 1), p-ACC (p-acetyl-CoA carboxylase), and CPT-1 (carnitine palmitoyltransferase 1), and specifically, may increase the expression of all proteins selected from the group consisting of p-AMPK (p-adenosine monophosphate-activated protein kinase), SIRT1 (sirtuin 1), p-ACC (p-acetyl-CoA carboxylase), and CPT-1 (carnitine palmitoyltransferase 1).

[0045] In one embodiment, the red silkworm powder may reduce the expression of p-STAT3 (p-signal transducer and activator of transcription 3) protein.

[0046] In one embodiment, the red silkworm powder may increase the expression of one or more genes selected from the group consisting of the GPR35 (G protein-coupled receptor 35) gene, the PKA (Protein kinase A) gene, the CREB (cAMP-response element binding protein) gene, the PGC-1α (Peroxisome proliferator-activated receptor gamma coactivator 1-alpha) gene, the G6Pase (glucose 6-phosphatase) gene, and the PCK1 (phosphoenolpyruvate carboxykinase 1) gene; specifically, the GPR35 (G protein-coupled receptor 35) gene, the PKA (Protein kinase A) gene, the CREB (cAMP-response element binding protein) gene, the PGC-1α (Peroxisome proliferator-activated receptor gamma coactivator 1-alpha) gene, the G6Pase (glucose 6-phosphatase) gene, and the PCK1 (phosphoenolpyruvate carboxykinase 1) may increase the expression of all selected genes in a group consisting of genes.

[0047] In one embodiment, the red silkworm powder may reduce the expression of one or more genes selected from the group consisting of the SREBP1c (sterol regulatory element-binding protein 1) gene, FASN (fatty acid synthase) gene, IL-6 (interleukin 6) gene, TNF-α (tumor necrosis factor alpha) gene, IL-1β (interleukin-1 beta) gene, c-fos (cellular fos proto-oncogene) gene, HIF-1α (hypoxia-inducible factor 1-alpha) gene, and c-myc (cellular myelocytomatosis oncogene) gene, and specifically, the SREBP1c (sterol regulatory element-binding protein 1) gene, FASN (fatty acid synthase) gene, IL-6 (interleukin 6) gene, TNF-α (tumor necrosis factor alpha) gene, and IL-1β (interleukin-1 beta) It may reduce the expression of all genes selected from the group consisting of the gene, c-fos (cellular fos proto-oncogene) gene, HIF-1α (hypoxia-inducible factor 1-alpha) gene and c-myc (cellular myelocytomatosis oncogene).

[0048] In one example, it was confirmed that red silkworm powder activates the SIRT1 / AMPK signaling pathway to inhibit triglyceride accumulation and increase fatty acid oxidation, and regulates the GPR35 signaling pathway and gluconeogenesis pathway to maintain hepatic lipid homeostasis (see Examples 4 and 6).

[0049] In addition, in another example, the expression of liver inflammation markers (IL-6, TNF-α, and IL-1β) caused by fatty liver was significantly reduced in the red silkworm pupae administration group compared to the HFD administration group, and the expression of STAT3 and its target genes (c-fos, HIF-1α, and c-myc), which are associated with the occurrence of liver inflammation, was significantly reduced, confirming that red silkworm pupae powder is effective in treating inflammation induced by fatty liver (see Example 5).

[0050] A pharmaceutical composition containing red silkworm powder according to one aspect reduces fat accumulation in the liver, increases fatty acid oxidation, and furthermore, significantly reduces liver inflammation caused by non-alcoholic fatty liver disease. In other words, since red silkworm powder exhibits remarkably superior effects in the prevention or treatment of fatty liver, it can be effectively utilized as a novel treatment for non-alcoholic fatty liver disease.

[0051]

[0052] Another aspect provides a health functional food for the prevention or improvement of non-alcoholic fatty liver disease comprising red silkworm powder, wherein the red silkworm powder is consumed in an amount of 0.005 g / kg to 0.2 g / kg.

[0053] The above "red silkworm," "prevention," "non-alcoholic fatty liver disease," "silk protein," etc. may be within the aforementioned range.

[0054] In one embodiment, the red silkworm powder may be consumed in an amount of 0.005 g / kg to 0.2 g / kg, more specifically, in an amount of 0.005 g / kg to 0.2 g / kg, 0.005 g / kg to 0.1 g / kg, 0.005 g / kg to 0.05 g / kg, 0.005 g / kg to 0.01 g / kg, 0.01 g / kg to 0.2 g / kg, 0.01 g / kg to 0.1 g / kg, 0.01 g / kg to 0.05 g / kg, 0.05 g / kg to 0.2 g / kg, 0.05 g / kg to 0.1 g / kg, or 0.1 g / kg to 0.2 g / kg, and more specifically, in an amount of 0.01 g / kg to 0.1 g / kg.

[0055] The term "improvement" may refer to any action that at least reduces parameters related to the condition being treated, such as the severity of symptoms. In this case, the health functional food may be used for the prevention or improvement of non-alcoholic fatty liver disease, either simultaneously with or separately from medications for treatment, either before or after the onset of the disease.

[0056] The term "functional food" is synonymous with "food for special health use (FosHu)" and includes functional health foods, health foods, and dietary supplements. The aforementioned functional food refers to a food with high medical or health effects that is processed to efficiently exhibit bio-regulatory functions in addition to providing nutrition. Here, "functionality" means obtaining useful effects for health purposes, such as regulating nutrients or physiological actions regarding the structure and function of the human body.

[0057] In addition, "health food" refers to food that has active effects on maintaining or promoting health compared to general food, and "health supplement food" refers to food intended for the purpose of health support.

[0058] In the above-mentioned health functional food, red silkworm powder may be added directly to the food or mixed with other foods or food ingredients, and may be used appropriately according to conventional methods. The amount of red silkworm powder mixed may be appropriately determined according to its purpose of use (for prevention or improvement). Generally, when manufacturing food or beverages, the red silkworm powder may be added in an amount of about 15% by weight or less, specifically about 10% by weight or less, with respect to the raw materials. However, in the case of long-term consumption for the purpose of health and hygiene or health control, the above amount may be less than the above range.

[0059] The above-mentioned health functional food may be formulated into one selected from the group consisting of tablets, pills, powders, granules, powders, capsules, liquids, and oral dissolving film formulations, by further including one or more of a carrier, a diluent, an excipient, and an additive. Foods to which red silkworm powder may be added according to one aspect include various food products, powders, granules, tablets, capsules, syrups, beverages, gum, tea, vitamin complexes, and health functional foods.

[0060] Specific examples of the above carrier, excipient, diluent, and additive may be one or more selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, erythritol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium phosphate, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, polyvinylpyrrolidone, methylcellulose, water, sugar syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0061] In addition to containing the above-mentioned red silkworm powder, the above-mentioned health functional food may include other ingredients as essential components without special limitations. For example, it may include various flavoring agents or natural carbohydrates as additional ingredients, such as in ordinary beverages. Examples of the above-mentioned natural carbohydrates may be monosaccharides, e.g., glucose, fructose, etc.; disaccharides, e.g., maltose, sucrose, etc.; polysaccharides, e.g., dextrin, cyclodextrin, etc., and conventional sugars, and sugar alcohols such as xylitol, sorbitol, erythritol, etc. As flavoring agents other than those mentioned above, natural flavoring agents (thaumatin, stevia extract (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 the choice of a person skilled in the art.

[0062] In addition to the above, a health functional food according to one aspect may include various nutritional supplements, vitamins, minerals (electrolytes), flavoring agents such as synthetic 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 colloidal 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 proportion of these additives may also be appropriately selected by a person skilled in the art.

[0063] In one aspect, the above-mentioned health functional food may further include other health functional foods for the prevention or improvement of non-alcoholic fatty liver disease in addition to red silkworm powder.

[0064] The above-mentioned other health functional food for the prevention or improvement of non-alcoholic fatty liver disease may be included in the health functional food in the minimum amount that can obtain maximum effect without side effects, and this can be easily determined by a person skilled in the art.

[0065] In addition, in one aspect, the health functional food may be consumed alone or in combination with the other health functional food for the prevention or improvement of non-alcoholic fatty liver disease. That is, the health functional food may be consumed in conjunction with the other health functional food for the prevention or improvement of non-alcoholic fatty liver disease, and may be consumed simultaneously, separately, or sequentially, and may be consumed as a single or multiple times.

[0066] When consumed in combination with other health functional foods for the prevention or improvement of non-alcoholic fatty liver disease mentioned above, it may be consumed in an amount or ratio that can obtain maximum effect without side effects, and this can be easily determined by a person skilled in the art.

[0067] The above-mentioned other health functional foods for the prevention or improvement of non-alcoholic fatty liver disease may be conventionally known health functional foods for the prevention or improvement of non-alcoholic fatty liver disease or newly developed health functional foods for the prevention or improvement of non-alcoholic fatty liver disease.

[0068] A health functional food containing red silkworm powder according to one aspect reduces fat accumulation in the liver, increases fatty acid oxidation, and furthermore, significantly reduces liver inflammation caused by non-alcoholic fatty liver disease. In other words, since red silkworm powder exhibits remarkably superior effects in preventing or improving fatty liver, it can be effectively utilized for the prevention or improvement of non-alcoholic fatty liver disease.

[0069]

[0070] Another aspect is a step of obtaining cooked silkworms by cooking silkworms at 70 to 130 ℃ for 100 to 160 minutes; and

[0071] A method for manufacturing red silkworm powder is provided, comprising the step of drying the cooked silkworms.

[0072] The above "red silkworm," "silk fiber protein," etc. may be within the aforementioned range.

[0073] In one embodiment, the step of cooking the silkworm at 70 to 130 ℃ for 100 minutes to 160 minutes may be included. Specifically, the step of cooking at 80 to 120 ℃ for 110 minutes to 150 minutes, more specifically, at 90 to 110 ℃ for 120 minutes to 140 minutes may be included.

[0074] When the above silkworm is cooked at 100°C for 130 minutes and then vacuum freeze-dried for 24 hours, the silk protein of the silkworm becomes soft enough to be edible and / or processable, and since the amino acid components of the silk protein present inside the silkworm are not broken down, it can exhibit a significantly superior therapeutic effect for non-alcoholic fatty liver disease.

[0075] The above "cooked silkworm" can be obtained by methods such as cooking the silkworm by placing it in water or cooking the silkworm using the heat of steam.

[0076] The above "drying" may include all methods such as freeze-drying, hot-air drying, natural drying, and vacuum drying, and specifically, it may be a freeze-drying method.

[0077] In one embodiment, the silkworm may contain silk fiber protein.

[0078] In one embodiment, the manufacturing method may further include a step of crushing the dried silkworm after the step of drying the cooked silkworm.

[0079] The above "grinding" may include all methods such as household mixers, disc mills, hammer mills, cutter mills, etc., and specifically, may be a disc mill method.

[0080] Red silkworm powder produced according to a manufacturing method based on a specific aspect exhibits significantly superior preventive, aggravating, or therapeutic effects for non-alcoholic fatty liver disease, as the silk protein is softened to a level suitable for consumption and / or processing, and the major amino acid components of the silkworm containing the silk protein are not degraded during the manufacturing process.

[0081]

[0082] Another aspect provides a method for preventing or treating non-alcoholic fatty liver disease comprising the step of administering red silkworm powder to individuals requiring it at a dose of 0.005 g / kg to 0.2 g / kg.

[0083] The above "red silkworm," "prevention," "treatment," "administration," "non-alcoholic fatty liver disease," "silk protein," etc. may be within the aforementioned range.

[0084] In one embodiment, the method for preventing or treating non-alcoholic fatty liver disease may include the step of administering red silkworm powder to an individual requiring it at a concentration of 0.005 g / kg to 0.2 g / kg, specifically, the red silkworm powder may be administered at a concentration of 0.005 g / kg to 0.2 g / kg, 0.005 g / kg to 0.1 g / kg, 0.005 g / kg to 0.05 g / kg, 0.005 g / kg to 0.01 g / kg, 0.01 g / kg to 0.2 g / kg, 0.01 g / kg to 0.1 g / kg, 0.01 g / kg to 0.05 g / kg, 0.05 g / kg to 0.2 g / kg, 0.05 g / kg to 0.1 g / kg, or 0.1 g / kg to 0.2 g / kg, and more specifically, It can be administered at a dose of 0.01 g / kg to 0.1 g / kg.

[0085] A pharmaceutical composition containing red silkworm pupae powder according to one aspect reduces fat accumulation in the liver, increases fatty acid oxidation, and furthermore, significantly reduces liver inflammation caused by non-alcoholic fatty liver disease. In other words, since red silkworm pupae powder exhibits remarkably superior effects in the prevention, improvement, or treatment of fatty liver, it can be effectively utilized as a novel treatment for non-alcoholic fatty liver disease.

[0086]

[0087] Another aspect provides a use of red silkworm powder for the manufacture of a drug for the prevention or treatment of non-alcoholic fatty liver disease, wherein the red silkworm powder is administered at a concentration of 0.005 g / kg to 0.2 g / kg.

[0088] The above "red silkworm," "prevention," "treatment," "non-alcoholic fatty liver disease," "silk protein," etc. may be within the aforementioned range.

[0089] Specifically, in one embodiment, the red silkworm powder may be administered at a concentration of 0.005 g / kg to 0.2 g / kg, and more specifically, at a concentration of 0.005 g / kg to 0.2 g / kg, 0.005 g / kg to 0.1 g / kg, 0.005 g / kg to 0.05 g / kg, 0.005 g / kg to 0.01 g / kg, 0.01 g / kg to 0.2 g / kg, 0.01 g / kg to 0.1 g / kg, 0.01 g / kg to 0.05 g / kg, 0.05 g / kg to 0.2 g / kg, 0.05 g / kg to 0.1 g / kg, or 0.1 g / kg to 0.2 g / kg, and more specifically, at a concentration of 0.01 g / kg to 0.1 g / kg. there is.

[0090] According to one mechanism, red silkworm powder reduces fat accumulation in the liver, increases fatty acid oxidation, and furthermore, significantly reduces liver inflammation caused by non-alcoholic fatty liver disease. In other words, since red silkworm powder exhibits remarkably superior effects in the prevention, improvement, or treatment of fatty liver, it can be effectively utilized as a novel treatment for non-alcoholic fatty liver disease.

[0091]

[0092] One aspect provides a pharmaceutical composition for the prevention or treatment of non-alcoholic fatty liver disease comprising a silk fibroin-derived peptide, wherein the silk fibroin-derived peptide is administered at a concentration of 10 μg / mL to 200 μg / mL.

[0093] The above "prevention," "treatment," "non-alcoholic fatty liver disease," "pharmaceutical composition," etc. may be within the scope described above.

[0094] In one embodiment, the silk fibroin-derived peptide may be administered at a concentration of 10 μg / mL to 200 μg / mL, and more specifically, at a concentration of 1 μg / mL to 1000 μg / mL, 1 μg / mL to 500 μg / mL, 1 μg / mL to 300 μg / mL, 1 μg / mL to 200 μg / mL, 1 μg / mL to 100 μg / mL, 5 μg / mL to 1000 μg / mL, 5 μg / mL to 500 μg / mL, 5 μg / mL to 300 μg / mL, 5 μg / mL to 200 μg / mL, 5 μg / mL to 100 μg / mL, 10 μg / mL to 1000 μg / mL, 10 μg / mL to 500 μg / mL, 10 It may be administered in amounts of μg / mL to 300 μg / mL, 10 μg / mL to 200 μg / mL, 10 μg / mL to 100 μg / mL, 30 μg / mL to 1000 μg / mL, 30 μg / mL to 500 μg / mL, 30 μg / mL to 300 μg / mL, 30 μg / mL to 200 μg / mL, 30 μg / mL to 100 μg / mL, 50 μg / mL to 1000 μg / mL, 50 μg / mL to 500 μg / mL, 50 μg / mL to 300 μg / mL, 50 μg / mL to 200 μg / mL, or 50 μg / mL to 100 μg / mL, and more specifically, as 50 μg / mL to 100 μg / mL. It can be administered.

[0095] The term "silk" refers to the fiber obtained from the cocoon made by the silkworm, and the term "silk fibroin" refers to the natural protein obtainable from the silkworm cocoon and is the main component that makes up silk. Specifically, silkworm cocoon threads are composed of fibroin and sericin; fibroin is located in the center of the thread and is responsible for structural strength and luster, while sericin wraps around the fibroin strands and acts to bind them together.

[0096] The term “silk fibroin-derived peptide” refers to a peptide processed by hydrolyzing silk fibroin protein to a size suitable for absorption into the body, or synthesized from a compound. Specifically, silk fibroin generally possesses an amino acid sequence characterized by the repeated alternation of glycine and alanine, or alanine alone; since repetitions of specific amino acid sequences such as 'Gly-Ala-Gly-Ala-Gly-Ser' appear, it can be divided into repeat sequence units. The repeat sequence units of the silk fibroin may include, for example, the following amino acid sequences: (GAGAGS) 1-15 (Sequence No. 31); (GX) 5-15 (X=V, I, A) (Sequence No. 32); GAAS (Sequence No. 33); (S 1-2 A 11-13 ) (Sequence No. 34); GX 1-4 GGX (X=A, S, Y, R, DV, W, R, D) (Sequence No. 35); GGGX (X=A, S, Y, R, DV, W, R, D) (Sequence No. 36); (S 1-2 A 1-4 ) 1-2 (Sequence No. 37); GXGGXG (X=L, I, V, P) (Sequence No. 38); GPX (X=L, Y, I) (Sequence No. 39); (GP(GGX) 1-4 Y) (X=Y, V, S, A) (Sequence No. 40); GRGGA (Sequence No. 41); GGX (X=A, T, V, S) (Sequence No. 42); GAG(A) 6-7 GGA (sequence number 43); and GGXGXGXX (X=Q, Y, L, A, S, R) (sequence number 44).

[0097] In one embodiment, the silk fibroin-derived peptide may be composed of any one amino acid sequence selected from the group consisting of the amino acid sequence of SEQ ID NO. 31, the amino acid sequence of SEQ ID NO. 32, the amino acid sequence of SEQ ID NO. 33, the amino acid sequence of SEQ ID NO. 34, the amino acid sequence of SEQ ID NO. 35, the amino acid sequence of SEQ ID NO. 36, the amino acid sequence of SEQ ID NO. 37, the amino acid sequence of SEQ ID NO. 38, the amino acid sequence of SEQ ID NO. 39, the amino acid sequence of SEQ ID NO. 40, the amino acid sequence of SEQ ID NO. 41, the amino acid sequence of SEQ ID NO. 42, the amino acid sequence of SEQ ID NO. 43, and the amino acid sequence of SEQ ID NO. 44, or a repeat sequence thereof. Specifically, the silk fibroin-derived peptide may be composed of the amino acid sequence of SEQ ID NO. 31 or a repeat sequence thereof. The above repeat sequence may be 1 to 1000 repeats, 1 to 500 repeats, 1 to 300 repeats, 1 to 100 repeats, 1 to 50 repeats, 1 to 30 repeats, 1 to 10 repeats, 1 to 5 repeats, or 1 to 3 repeats. More specifically, the silk fibroin-derived peptide may be composed of the amino acid sequence of SEQ ID NO. 45.

[0098] In one embodiment, the peptide composed of the amino acid sequence of SEQ ID NO. 45, which is a silk fibroin-derived peptide, was treated to a liver cancer HepG2 cell line in which fat accumulation was induced with palmitic acid. As a result, it was confirmed that fat accumulation was reduced, SIRT1 and AMPK activities regulating fatty acid synthesis and oxidation were increased, ACC activity regulating fatty acid oxidation was increased, SREBP1 protein expression induced by fat accumulation was decreased, and GPR35, PKA, and CREB protein expression regulating gluconeogenesis were increased. This confirmed that the silk fibroin-derived peptide has the effect of improving fatty liver by activating the SIRT1 / AMPK pathway to reduce fat synthesis and promote fatty acid oxidation, and restores the gluconeogenesis pathway in liver tissue by activating the GPR / PKA pathway (see Example 7).

[0099] In one embodiment, the silk fibroin-derived peptide may be obtained from silkworms, insects, spiders, bees, synthetic silk peptides, and biotechnological variants thereof. In one embodiment, the silkworm may be selected from the group consisting of Antheraea mylitta, Antheraea pernyi, Antheraea yamamai, Galleria mellonella, Bombyx mori, and Bombyx mandarina, the insect may be Galleria mellonella, and the spider may be Nephila clavipes, Nephila senegalensis, Gasteracantha mammosa, Argiope aurantia, Araneus diadematus, Latrodectus geometricus, Araneus It may be selected from the group consisting of Araneus bicentenarius, Tetragnatha versicolor, Araneus ventricosus, Dolomedes tenebrosus, Euagrus chisoseus, Plectreurys tristis, Argiope trifasciata, and Nephila madagascariensis. In one embodiment, the silkworm may be a silkworm cocoon, a mature silkworm, or a red silkworm. More specifically, the silk fibroin-derived peptide may be obtained from a red silkworm.

[0100] In one aspect, the pharmaceutical composition may further include other therapeutic agents for non-alcoholic fatty liver disease in addition to silk fibroin-derived peptides.

[0101] The other non-alcoholic fatty liver disease treatment mentioned above may be included in the pharmaceutical composition in the minimum amount to obtain maximum effect without side effects, which can be easily determined by a person skilled in the art.

[0102] In addition, in one aspect, the pharmaceutical composition may be administered alone or in combination with other known treatments for non-alcoholic fatty liver disease. That is, the pharmaceutical composition may be administered in conjunction with other treatments for non-alcoholic fatty liver disease, and may be administered simultaneously, separately, or sequentially, and may be administered as a single or multiple doses.

[0103] When the above-mentioned other treatment for non-alcoholic fatty liver disease is administered in combination with the above-mentioned pharmaceutical composition, it may be administered in an amount or ratio that can obtain maximum effect without side effects, and this can be easily determined by a person skilled in the art.

[0104] The other non-alcoholic fatty liver disease treatment mentioned above may be a conventionally known non-alcoholic fatty liver disease treatment or a newly developed non-alcoholic fatty liver disease treatment.

[0105] In one embodiment, the non-alcoholic fatty liver disease may be one or more selected from the group consisting of non-alcoholic fatty liver, non-alcoholic steatohepatitis, liver fibrosis, liver cirrhosis, and liver cancer, and specifically may be non-alcoholic fatty liver.

[0106] In one embodiment, the silk fibroin-derived peptide may reduce fat accumulated in the liver.

[0107] Reducing the aforementioned fat accumulated in the liver includes reducing it by removing it through decomposition, oxidation, etc., and reducing it by inhibiting fat synthesis.

[0108] In one embodiment, the silk fibroin-derived peptide may increase the expression of one or more proteins selected from the group consisting of p-AMPK, SIRT1, and p-ACC, and specifically, may increase the expression of all proteins selected from the group consisting of p-AMPK, SIRT1, and p-ACC.

[0109] In one embodiment, the silk fibroin-derived peptide may increase the expression of one or more genes selected from the group consisting of the GPR35 gene, the PKA gene, and the CREB gene, and specifically, may increase the expression of all genes selected from the group consisting of the GPR35 gene, the PKA gene, and the CREB gene.

[0110] In one embodiment, the silk fibroin-derived peptide may reduce SREBP1 protein expression.

[0111] A pharmaceutical composition containing a silk fibroin-derived peptide according to one aspect reduces fat accumulation in the liver, increases fatty acid oxidation, and restores the gluconeogenesis pathway. That is, since the silk fibroin-derived peptide exhibits significantly superior effects in the prevention or treatment of fatty liver, it can be effectively utilized as a new treatment for non-alcoholic fatty liver disease.

[0112]

[0113] Another aspect provides a health functional food for the prevention or improvement of non-alcoholic fatty liver disease comprising a silk fibroin-derived peptide, wherein the silk fibroin-derived peptide is consumed in an amount of 10 μg / mL to 200 μg / mL.

[0114] The above "silk fibroin-derived peptide," "prevention," "improvement," "non-alcoholic fatty liver disease," "health functional food," etc. may be within the aforementioned scope.

[0115] In one embodiment, the silk fibroin-derived peptide may be consumed in an amount of 10 μg / mL to 200 μg / mL, and more specifically, 1 μg / mL to 1000 μg / mL, 1 μg / mL to 500 μg / mL, 1 μg / mL to 300 μg / mL, 1 μg / mL to 200 μg / mL, 1 μg / mL to 100 μg / mL, 5 μg / mL to 1000 μg / mL, 5 μg / mL to 500 μg / mL, 5 μg / mL to 300 μg / mL, 5 μg / mL to 200 μg / mL, 5 μg / mL to 100 μg / mL, 10 μg / mL to 1000 μg / mL, 10 μg / mL to 500 μg / mL, 10 It may be consumed in amounts of μg / mL to 300 μg / mL, 10 μg / mL to 200 μg / mL, 10 μg / mL to 100 μg / mL, 30 μg / mL to 1000 μg / mL, 30 μg / mL to 500 μg / mL, 30 μg / mL to 300 μg / mL, 30 μg / mL to 200 μg / mL, 30 μg / mL to 100 μg / mL, 50 μg / mL to 1000 μg / mL, 50 μg / mL to 500 μg / mL, 50 μg / mL to 300 μg / mL, 50 μg / mL to 200 μg / mL, or 50 μg / mL to 100 μg / mL, and more specifically, as 50 μg / mL to 100 μg / mL. It can be consumed.

[0116] In one aspect, the above-mentioned health functional food may further include other health functional foods for the prevention or improvement of non-alcoholic fatty liver disease in addition to silk fibroin-derived peptides.

[0117] The above-mentioned other health functional food for the prevention or improvement of non-alcoholic fatty liver disease may be included in the health functional food in the minimum amount that can obtain maximum effect without side effects, and this can be easily determined by a person skilled in the art.

[0118] In addition, in one aspect, the health functional food may be consumed alone or in combination with the other health functional food for the prevention or improvement of non-alcoholic fatty liver disease. That is, the health functional food may be consumed in conjunction with the other health functional food for the prevention or improvement of non-alcoholic fatty liver disease, and may be consumed simultaneously, separately, or sequentially, and may be consumed as a single or multiple times.

[0119] When consumed in combination with other health functional foods for the prevention or improvement of non-alcoholic fatty liver disease mentioned above, it may be consumed in an amount or ratio that can obtain maximum effect without side effects, and this can be easily determined by a person skilled in the art.

[0120] The above-mentioned other health functional foods for the prevention or improvement of non-alcoholic fatty liver disease may be conventionally known health functional foods for the prevention or improvement of non-alcoholic fatty liver disease or newly developed health functional foods for the prevention or improvement of non-alcoholic fatty liver disease.

[0121] A health functional food containing silk fibroin-derived peptides according to one aspect reduces fat accumulation in the liver, increases the oxidation of fatty acids, and furthermore, significantly reduces liver inflammation caused by non-alcoholic fatty liver disease. In other words, since silk fibroin-derived peptides show remarkably superior effects in preventing or improving fatty liver, they can be effectively utilized for the prevention or improvement of non-alcoholic fatty liver disease.

[0122]

[0123] Another aspect provides a method for preventing or treating non-alcoholic fatty liver disease comprising the step of administering a silk fibroin-derived peptide to an individual in need at a concentration of 10 μg / mL to 200 μg / mL.

[0124] The above "silk fibroin-derived peptide," "prevention," "treatment," "administration," "non-alcoholic fatty liver disease," etc. may be within the aforementioned range.

[0125] In one embodiment, the method for preventing or treating non-alcoholic fatty liver disease may include the step of administering a silk fibroin-derived peptide to an individual requiring it at a concentration of 10 μg / mL to 200 μg / mL, and specifically, the silk fibroin-derived peptide may be 1 μg / mL to 1000 μg / mL, 1 μg / mL to 500 μg / mL, 1 μg / mL to 300 μg / mL, 1 μg / mL to 200 μg / mL, 1 μg / mL to 100 μg / mL, 5 μg / mL to 1000 μg / mL, 5 μg / mL to 500 μg / mL, 5 μg / mL to 300 μg / mL, 5 μg / mL to 200 μg / mL, 5 μg / mL to 100 μg / mL, 10 μg / mL to 1000 μg / mL, 10 μg / mL to 500 μg / mL, 10 μg / mL to 300 μg / mL, 10 μg / mL to 200 μg / mL, 10 μg / mL to 100 μg / mL, 30 μg / mL to 1000 μg / mL, 30 μg / mL to 500 μg / mL, 30 μg / mL to 300 μg / mL, 30 μg / mL to 200 μg / mL, 30 μg / mL to 100 μg / mL, 50 μg / mL to 1000 μg / mL, 50 μg / mL to 500 μg / mL, 50 μg / mL to 300 μg / mL, 50 μg / mL to 200 μg / mL, or 50 μg / mL to 100 It can be administered at μg / mL, and more specifically at 50 μg / mL to 100 μg / mL.

[0126] Silk fibroin-derived peptides, depending on the pattern, reduce fat accumulation in the liver, increase fatty acid oxidation, and furthermore, significantly reduce liver inflammation caused by non-alcoholic fatty liver disease. In other words, since silk fibroin-derived peptides show remarkably superior effects in preventing or improving fatty liver, they can be effectively utilized for the prevention or improvement of non-alcoholic fatty liver disease.

[0127]

[0128] Another aspect provides a use of a silk fibroin-derived peptide for the manufacture of a drug for the prevention or treatment of non-alcoholic fatty liver disease, wherein the silk fibroin-derived peptide is administered at a concentration of 10 μg / mL to 200 μg / mL.

[0129] The above "silk fibroin-derived peptide," "prevention," "treatment," "non-alcoholic fatty liver disease," etc. may be within the aforementioned range.

[0130] Specifically, in one embodiment, the silk fibroin-derived peptide may be administered at a concentration of 10 μg / mL to 200 μg / mL, and more specifically, at a concentration of 1 μg / mL to 1000 μg / mL, 1 μg / mL to 500 μg / mL, 1 μg / mL to 300 μg / mL, 1 μg / mL to 200 μg / mL, 1 μg / mL to 100 μg / mL, 5 μg / mL to 1000 μg / mL, 5 μg / mL to 500 μg / mL, 5 μg / mL to 300 μg / mL, 5 μg / mL to 200 μg / mL, 5 μg / mL to 100 μg / mL, 10 μg / mL to 1000 μg / mL, 10 μg / mL to 500 μg / mL, 10 It may be administered in amounts of μg / mL to 300 μg / mL, 10 μg / mL to 200 μg / mL, 10 μg / mL to 100 μg / mL, 30 μg / mL to 1000 μg / mL, 30 μg / mL to 500 μg / mL, 30 μg / mL to 300 μg / mL, 30 μg / mL to 200 μg / mL, 30 μg / mL to 100 μg / mL, 50 μg / mL to 1000 μg / mL, 50 μg / mL to 500 μg / mL, 50 μg / mL to 300 μg / mL, 50 μg / mL to 200 μg / mL, or 50 μg / mL to 100 μg / mL, and more specifically, as 50 μg / mL to 100 μg / mL. It can be administered.

[0131] Silk fibroin-derived peptides, depending on the pattern, reduce fat accumulation in the liver, increase fatty acid oxidation, and furthermore, significantly reduce liver inflammation caused by non-alcoholic fatty liver disease. In other words, since silk fibroin-derived peptides show remarkably superior effects in preventing or improving fatty liver, they can be effectively utilized for the prevention or improvement of non-alcoholic fatty liver disease.

[0132] According to one aspect, red silkworm powder and / or silk fibroin-derived peptides reduce fat accumulation in the liver, increase fatty acid oxidation, and furthermore, significantly reduce liver inflammation caused by non-alcoholic fatty liver disease. In other words, red silkworm powder and / or silk fibroin-derived peptides show remarkably superior effects in preventing, improving, or treating fatty liver disease, so they can be effectively utilized as a new treatment for non-alcoholic fatty liver disease.

[0133] Figure 1 is an HPLC graph analyzing the components of red silkworm powder.

[0134] Figure 2 is a graph showing the change in body weight according to the diet of mice (normal feed, high-fat feed, high-fat feed + red silkworm pupae 0.01 kg / g, high-fat feed + red silkworm pupae 0.1 kg / g, and high-fat feed + silymarin 0.1 kg / g).

[0135] Figure 3 is a graph showing the weight gain of mice according to their diet (normal feed, high-fat feed, high-fat feed + red silkworm pupae 0.01 kg / g, high-fat feed + red silkworm pupae 0.1 kg / g, and high-fat feed + silymarin 0.1 kg / g).

[0136] Figure 4 is a graph showing liver weight according to the diet of mice (normal feed, high-fat feed, high-fat feed + red silkworm pupae 0.01 kg / g, high-fat feed + red silkworm pupae 0.1 kg / g, and high-fat feed + silymarin 0.1 kg / g).

[0137] Figure 5 is a graph showing the liver weight / body weight ratio according to the diet of mice (normal feed, high-fat feed, high-fat feed + red silkworm pupae 0.01 kg / g, high-fat feed + red silkworm pupae 0.1 kg / g, and high-fat feed + silymarin 0.1 kg / g).

[0138] Figure 6 shows liver photographs, H&E and ORO stained micrographs according to the diet of mice (normal feed, high-fat feed, high-fat feed + red silkworm pupae 0.01 kg / g, high-fat feed + red silkworm pupae 0.1 kg / g and high-fat feed + silymarin 0.1 kg / g).

[0139] Figure 7 is a graph showing hepatic steatosis and ORO scores according to the diet of mice (normal feed, high-fat feed, high-fat feed + red silkworm pupae 0.01 kg / g, high-fat feed + red silkworm pupae 0.1 kg / g, and high-fat feed + silymarin 0.1 kg / g).

[0140] Figure 8 is a graph showing the concentrations of liver triglycerides (A), blood triglycerides (B), glucose (C), total cholesterol (D), HDL-cholesterol (E), and LDL-cholesterol (F) according to the diet of mice (normal feed, high-fat feed, high-fat feed + red silkworm pupae 0.01 kg / g, high-fat feed + red silkworm pupae 0.1 kg / g, and high-fat feed + silymarin 0.1 kg / g).

[0141] Figure 9 is a graph showing the concentrations of ALT(G), AST(H), GGT(I), and bilirubin(J) according to the diet of mice (normal feed, high-fat feed, high-fat feed + red silkworm 0.01 kg / g, high-fat feed + red silkworm 0.1 kg / g, and high-fat feed + silymarin 0.1 kg / g).

[0142] Figure 10 shows the Western blotting results of p-AMPK, SIRT1, p-ACC, and CPT-1 according to the diets of mice (normal feed, high-fat feed, high-fat feed + red silkworm pupae 0.01 kg / g, high-fat feed + red silkworm pupae 0.1 kg / g, and high-fat feed + silymarin 0.1 kg / g).

[0143] Figure 11 is a graph quantifying the Western blotting results of p-AMPK, SIRT1, p-ACC, and CPT-1 according to the diets of mice (normal feed, high-fat feed, high-fat feed + red silkworm pupae 0.01 kg / g, high-fat feed + red silkworm pupae 0.1 kg / g, and high-fat feed + silymarin 0.1 kg / g).

[0144] Figure 12 is a graph comparing the expression levels of SREBP1c, FASN, and CPT-1 mRNA according to the diet of mice (normal feed, high-fat feed, high-fat feed + red silkworm pupae 0.01 kg / g, high-fat feed + red silkworm pupae 0.1 kg / g, and high-fat feed + silymarin 0.1 kg / g).

[0145] Figure 13 is a graph showing the results of measuring TNF-α(A) and IL-1β(B) concentrations according to the diet of mice (normal feed, high-fat feed, high-fat feed + red silkworm pupae 0.01 kg / g, high-fat feed + red silkworm pupae 0.1 kg / g and high-fat feed + silymarin 0.1 kg / g).

[0146] Figure 14 is a graph showing the Western blotting results of p-STAT3 according to mouse diets (normal feed, high-fat feed, high-fat feed + red silkworm pupae 0.01 kg / g, high-fat feed + red silkworm pupae 0.1 kg / g, and high-fat feed + silymarin 0.1 kg / g) and the quantification thereof.

[0147] Figure 15 is a graph comparing the expression levels of IL-6, TNF-α, IL-1β, c-fos, HIF-1α, and c-myc mRNA according to the diet of mice (normal feed, high-fat feed, high-fat feed + red silkworm pupae 0.01 kg / g, high-fat feed + red silkworm pupae 0.1 kg / g, and high-fat feed + silymarin 0.1 kg / g).

[0148] Figure 16 shows the Western blotting results of GPR35, PKA, CREB, and PGC-1α according to mouse diets (normal feed, high-fat feed, high-fat feed + red silkworm pupae 0.01 kg / g, high-fat feed + red silkworm pupae 0.1 kg / g, and high-fat feed + silymarin 0.1 kg / g).

[0149] Figure 17 is a graph quantifying the Western blotting results of GPR35, PKA, CREB, and PGC-1α according to mouse diets (normal feed, high-fat feed, high-fat feed + red silkworm pupae 0.01 kg / g, high-fat feed + red silkworm pupae 0.1 kg / g, and high-fat feed + silymarin 0.1 kg / g).

[0150] Figure 18 is a graph comparing the mRNA expression levels of G6Pase, PCK1, GPR35, PKA, CREB, and PGC-1α according to the diet of mice (normal feed, high-fat feed, high-fat feed + red silkworm pupae 0.01 kg / g, high-fat feed + red silkworm pupae 0.1 kg / g, and high-fat feed + silymarin 0.1 kg / g).

[0151] Figure 19 shows the chemical structure of a silk fibroin-derived peptide (sequence number 45: Gly-Ala-Gly-Ala-Gly-Ser).

[0152] Figure 20 shows the results of treating a liver cancer HepG2 cell line, in which fat accumulation was induced with palmitic acid (PA), with a silk fibroin-derived peptide. Figure 20a is a micrograph showing ORO staining, and Figure 20b is a graph comparing the amount of fat accumulation.

[0153] Figure 21 shows the results of treating liver cancer HepG2 cell lines, in which fat accumulation was induced with palmitic acid (PA), with silk fibroin-derived peptides. Figure 21a shows the Western blotting results of SIRT1, AMPK, ACC, and SREBP1, and Figure 21b shows the SRTI1 / β-actin graph and p-ACC / ACC graph calculated by quantifying the Western blotting results.

[0154] Figure 22 shows the results of treating liver cancer HepG2 cell lines, in which fat accumulation was induced with palmitic acid (PA), with silk fibroin-derived peptides. Figure 22a shows the Western blotting results of GPR35, PKA, and CREB1, and Figure 22b shows the GPR35 / β-actin graph, PKA / β-actin graph, and CREB1 / β-actin graph calculated by quantifying the Western blotting results.

[0155] The present invention will be explained in more detail below through examples. However, these examples are intended to illustrate the invention and the scope of the invention is not limited to these examples.

[0156]

[0157] <Preparation Example> Preparation of Red Silkworm Powder

[0158] To prepare red silkworm powder, 7-day-old 5th instar raw silkworms (Bombyx mori; variety: white-jade) containing silk fiber protein were steamed at 100°C for 130 minutes using an electric non-pressure cooker (KumSeong Ltd.). Then, they were freeze-dried for 24 hours using a freeze dryer (FDT-8612, Operon Ltd.) and ground using a disc mill (HM001, Korean Pulverizing Machinery Co. Ltd.) to a particle size of less than 0.1 mm. The prepared red silkworm powder was stored at -50°C for the experiment.

[0159]

[0160] <Reference Example 1> Preparation of Animals

[0161] All animal experiment procedures were performed in accordance with the guidelines and approvals of the Institutional Animal Care and Use Committees (IACUC) of CHA University (Reference No.: 220115). Five-week-old male C57BL / 6N mice (Orient Bio) were used as experimental animals, and the mice were housed in a pathogen-free sterile room at room temperature with a 12-hour day / night cycle. The mice were given a one-week acclimatization period during which they were fed commercially available feed (Haran 2018s) and allowed to drink tap water freely. Subsequently, the mice were divided into five groups (n=8 / group). Specifically, the mice were divided into groups fed a normal diet (hereinafter, control group), a high-fat diet (hereinafter, HFD group), and experimental groups administered a high-fat diet along with 0.01 g / kg of red silkworm pupae (hereinafter, red silkworm pupae group 1), 0.1 g / kg of red silkworm pupae (hereinafter, red silkworm pupae group 2), or 0.1 g / kg of silymarin (hereinafter, silymarin group). The experimental diet was provided for 12 weeks, during which time the mice were allowed to consume food and water freely. Body weight and food intake were measured weekly throughout the experiment. After the experiment, the mice were anesthetized, blood was collected from the abdominal aorta and placed in a heparin-coated tube, and plasma was obtained by centrifuging the blood at 5,000 rpm for 30 minutes at 4°C. The livers were resected, rinsed with PBS, and weighed; for further analysis, a portion of each liver was fixed in 10% formalin. Plasma and liver samples were stored at -80°C until analysis.

[0162]

[0163] <Reference Example 2> Serum Analysis

[0164] Serum triglyceride, glucose, total cholesterol, LDL-cholesterol (low density lipoprotein-cholesterol), HDL-cholesterol (high density lipoprotein-cholesterol), ALT (alanine aminotransferase), AST (aspartate aminotransferase), GGT (gamma glutamyl transpeptidase), and bilirubin levels were analyzed using a Hitachi Automated Analyzer 7600-210 (Hitachi High-Technologies Corporation), and the concentrations of TNF-α (tumor necrosis factor-alpha) and IL-1β (interleukin-1 beta) were measured using a commercial mouse ELISA kit (R&D System) according to the manufacturer's protocol.

[0165]

[0166] <Reference Example 3> Analysis of triglycerides in liver tissue

[0167] Triglyceride levels in liver tissue were measured using a triglyceride analysis kit (ab65336, Abcam) according to the manufacturer's protocol.

[0168]

[0169] <Reference Example 4> Histological Analysis

[0170] Liver samples were fixed in paraffin, sliced ​​to 5 μm, sectioned, and stained with hematoxylin and eosin (H&E). The levels of fat infiltration and fatty degeneration were evaluated as follows: 0 points for no effect on hepatocytes, 0.5 points for slight effect on hepatocytes (fat infiltration and fatty degeneration at the 0-5% level), 1 point for mild effect (fat infiltration and fatty degeneration at the 5-20% level), 2 points for moderate effect (fat infiltration and fatty degeneration at the 20-50% level), and 3 points for severe effect (fat infiltration and fatty degeneration at the greater than 50% level).

[0171]

[0172] <Reference Example 5> Oil-red O (ORO) staining

[0173] Tissue sections were immersed in 60% isopropyl alcohol for 20–30 seconds, then stained with ORO staining solution (O1391, Sigma-Aldrich) at room temperature for 15 minutes, and the sections were washed again with 60% isopropanol and distilled water to remove non-specifically bound stained regions. The degree of ORO staining was evaluated as follows: no staining = 0; less than 30% staining = 1; less than 60% staining = 2; greater than 90% staining = 3.

[0174]

[0175] <Reference Example 6> RNA Isolation and Gene Analysis (qRT-PCR)

[0176] Total mRNA was isolated from mouse livers using Trizol reagent (Invitrogen), and cDNA was synthesized using the SuperScript® II Reverse Transcriptase Kit (Invitrogen) according to the manufacturer's protocol. mRNA levels were analyzed by qRT-PCR (quantitative real-time polymerase chain reaction). The primers used for qRT-PCR are shown in Table 1 below.

[0177] Sequence Number Primer Direction Sequence 1 SREBP-1c Forward 5'-CTG GTG AGT GGA GGG ACC AT-3'2 Reverse 5'-GGT GGA TGG GCA GTT TGT CT-3'3 FASN Forward 5'-GGA GGT GGT GAT AGC CGG TAT-3'4 Reverse 5'-TGG GTA ATC CAT AGA GCC CAG-3'5 CPT-1 Forward 5'-GGC ATA AAC GCA GAG CAT TCC TG-3'6 Reverse 5'-CAG TGT CCA TCC TCT GAG TAG C-3'7 c-fos Forward 5'-GGG AAT GGT GAA GAC CGT GTC A-3'8 Reverse 5'-GCA GCC ATC TTA TTC CGT TCC C-3'9 HIF-1α Forward 5'-CCT GCA CTG AAT CAA GAG GTT GC-3'10 Reverse 5'- CCA TCA GAA GGA CTT GCT GGC T-3'11c-myc Forward 5'-TGC GAC GAG GAA GAG AAT TT-3'12 Reverse 5'-AAC CGC TCC ACA TAC AGT CC-3'13TNF-α Forward 5'-ACT GAA CTT CGG GGT GAT CG-3'14 Reverse 5'-GCT TGG TGG TTT GCT ACG AC-3'15IL-1β Forward 5'-AAA TCT CGC AGC AGC ACA TCA A-3'16 Reverse 5'-CCA CGG GAA AGA CAC AGG TAG C-3'17IL-6 Forward 5'-TAC CAC TTC ACA AGT CGG AGG C-3'18 Reverse 5'-CTG CAA GTG CAT CAT CGT TGT TC-3'19GPR35 Forward 5'-CTC TGC TCC TTG CCA TTT GTG C-3'20 Reverse 5'-AGC AAT GGC AGT GAC CAG GCT T-3'21PKA Forward 5'- CAG ACT TCG GTT TTG CCA AGC G-3'22 Reverse 5'- GCC ATC TCG TAG ATG AGG ACT C-3'23CREB Forward 5'-CAC AGA CCA CTG GAC AGC A-3'24 Reverse 5'- AGG ACG CGA TAA CAA CTC CAGG-3'25PGC-1αForward 5'- GAA TCA AGC CAC TAC AGA CAC CG-3'26Reverse 5'-CAT CCC TCT TGA GCC TTT CGT G-3'27G6PaseForward 5'-AGG TCG TGG CTG GAG TCT TGT C-3'28Reverse 5'-GTA GCA GGT AGA ATC CAA GCG C-3'29PCK1Forward 5'-GGC GAT GAC ATT GCC TGG ATG A-3'30Reverse 5'-TGT CTT CAC TGA GGT GCC AGG A-3'

[0178]

[0179] <Reference Example 7> Western blotting

[0180] Liver tissue was homogenized with cold cell lysis buffer containing a protease inhibitor (Roche Applied Science), incubated on ice with vortexing for 5 minutes, and then centrifuged at 13,000 rpm for 15 minutes. The protein concentration of each supernatant was quantified according to the manufacturer's protocol using the Pierce™ BCA Protein Analysis Kit (Thermo Fisher Scientific). Proteins were loaded onto 10% SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and transferred to a polyvinylidene fluoride membrane (Millipore). Subsequently, the membrane was blocked with 3% bovine serum albumin (BSA) in Tris-buffered saline (TBS-T) containing 0.05% tween 20 and hybridized with the designated primary antibody (diluted 1:1000) at 4°C for one day. The membrane was washed and incubated with an appropriate secondary antibody in TBS-T for 1 hour, and the blot was verified using an enhanced chemiluminescence system (Thermo Fisher Scientific). Antibodies against p-AMPK, AMPK, SIRTI, CPT-1, PKA, CREB, PGC-1α, and β-actin were purchased from Santa Cruz Biotechnology, antibodies against p-ACC, ACC, p-STAT3, and STAT3 were purchased from Cell Signaling Technology, and the antibody against GPR35 was purchased from Protein Technology.

[0181]

[0182] <Example 1> Component Analysis of Red Silkworm Powder

[0183] HPLC analysis was performed to identify the major components of red silkworm powder. Specifically, amino acids and 0.05% mercaptoethanol were added to hydrochloric acid containing red silkworm powder, and the hydrochloric acid was removed by repeated drying at 40°C. Subsequently, sodium citrate buffer or hydrochloric acid solution was added to be used as the test solution. Precipitates were removed using a membrane filter.

[0184] As shown in Figure 1, HPLC analysis results revealed that aspartic acid (Asp), threonine (Thr), serine (Ser), glutamic acid (Glu), glycine (Gly), alanine (Ala), valine (Val), methionine (Met), isoleucine (Ile), leucine (Leu), tyrosine (Tyr), phenylalanine (Phe), lysine (Lys), histidine (His), and arginine (Arg) were the major components of red silkworm pupae. Among them, glycine (Gly) was the most abundant, followed by serine (Ser) and alanine (Ala) in terms of abundance (Figure 1).

[0185]

[0186] <Example 2> Confirmation of the effect of red silkworm powder on fat accumulation in liver tissue

[0187] To evaluate the effect of red silkworm powder on improving symptoms of nonalcoholic fatty liver disease (NAFLD) in mice administered a High Fat Diet (HFD), body weight, liver weight, and the level of fat accumulation in the liver were evaluated.

[0188] The mice used in the experiment were prepared according to Reference Example 1, and histological analysis of fat accumulation in the liver was performed according to Reference Examples 4 and 5.

[0189] As a result, as shown in Figure 2, in the red silkworm pupae administration groups 1 (red silkworm pupae 0.01 g / kg) and 2 (red silkworm pupae 0.1 g / kg) administered red silkworm pupae powder along with HFD for 12 weeks, body weight was significantly reduced compared to the HFD administration group administered only HFD, and as shown in Figure 3, the red silkworm pupae administration groups 1 and 2 administered red silkworm pupae powder showed a significantly reduced increase in body weight compared to the HFD administration group administered only HFD.

[0190] In addition, liver weight (Fig. 4) and liver weight relative to body weight (Fig. 5) in red silkworm pupae administration groups 1 and 2, which were administered red silkworm pupae powder, were significantly reduced compared to the HFD administration group. That is, it was confirmed that liver weight was significantly reduced when red silkworm pupae were administered compared to when only a high-fat diet was administered (HFD administration group).

[0191] As shown in Fig. 6, histological analysis of liver tissue revealed that the liver size of red silkworm pupae administration groups 1 and 2 was significantly reduced to the level of the control group fed normal feed compared to the HFD administration group. Additionally, histological analysis using H&E and ORO staining confirmed that lipid globules generated by the high-fat diet were significantly reduced in red silkworm pupae administration groups 1 and 2 compared to the HFD administration group (Fig. 6).

[0192] Specifically, as shown in Figure 7, the liver tissue H&E (steatosis score) and ORO staining results (ORO staining score) of the control group administered a normal diet, the HFD group, the red silkworm group 1, the red silkworm group 2, and the silymarin group were evaluated. As a result, both the steatosis score and the ORO score were significantly lower in the red silkworm group 1 and 2 compared to the HFD group. In particular, regarding the steatosis score, the red silkworm group 1 and 2 were lower than the silymarin group, confirming that the red silkworm group is more effective in improving steatosis.

[0193] Based on the above results, it was confirmed that administering red silkworm powder restores fatty liver induced by a high-fat diet to a level similar to that of a normal liver, thereby demonstrating that red silkworm powder is effective in preventing, improving, or treating non-alcoholic fatty liver disease (NAFLD).

[0194]

[0195] <Example 3> Confirmation of the effects of red silkworm powder on fatty liver indicators and liver damage indicators

[0196] To evaluate the effects of red silkworm powder on fatty liver and liver damage indicators in mice with non-alcoholic fatty liver disease (NAFLD) induced by a high-fat diet, the levels of triglycerides, glucose, total cholesterol, LDL-cholesterol, HDL-cholesterol, ALT, AST, GGT, and bilirubin were analyzed in experimental groups administered a normal diet (hereinafter, control group), a high-fat diet (hereinafter, HFD administration group), and 0.01 g / kg of red silkworm pupae (hereinafter, red silkworm pupae administration group 1), 0.1 g / kg of red silkworm pupae (hereinafter, red silkworm pupae administration group 2), or 0.1 g / kg of silymarin (hereinafter, silymarin administration group), respectively, along with the high-fat diet.

[0197] The experiment was conducted using mice prepared according to Reference Example 1, and serum analysis was performed according to Reference Example 2.

[0198] As shown in Figure 8, in the red silkworm pupae administration groups 1 (red silkworm pupae 0.01 g / kg) and 2 (red silkworm pupae 0.1 g / kg) administered red silkworm pupae powder along with HFD for 12 weeks, the levels of liver triglycerides, plasma triglycerides, glucose, total cholesterol, and LDL-cholesterol, which were increased by hepatic fat accumulation due to the high-fat diet, were significantly reduced compared to the HFD administration group administered only HFD, and the levels of HDL-cholesterol, which were decreased by the high-fat diet, were significantly increased.

[0199] In addition, as shown in Figure 9, when comparing the levels of ALT, AST, GGT, and bilirubin, which are indicators of liver damage, it was confirmed that the levels of ALT, AST, GGT, and bilirubin, which were increased by HFD administration, were significantly reduced in red silkworm administration groups 1 and 2.

[0200] Accordingly, it was confirmed that red silkworm powder can be effectively used as a preventive, remedial, or therapeutic agent for non-alcoholic fatty liver disease by reducing blood triglyceride and cholesterol levels, which are increased due to non-alcoholic fatty liver disease, and by reducing levels of liver damage indicators (ALT, AST, GGT, and bilirubin).

[0201]

[0202] <Example 4> Confirmation of the Effect of Red Silkworm Powder on Fatty Acid Oxidation and Lipid Synthesis Mechanisms

[0203] To confirm the effects of red silkworm powder on fatty acid oxidation and lipid synthesis mechanisms, we examined its effects on SIRT1 / AMPK, which is associated with fatty acid oxidation and triglyceride synthesis in liver tissue and is reported to be inhibited in NAFLD patients.

[0204] The mice used in the experiment were prepared according to Reference Example 1, and mRNA levels and protein levels were measured according to Reference Examples 6 and 7, respectively.

[0205] As shown in Figures 10 and 11, it was confirmed that the levels of p-AMPK, SIRT1, p-ACC, and CPT-1 proteins, which were reduced by HFD, were significantly increased in red silkworm administration groups 1 and 2.

[0206] In addition, as shown in Figure 12, SREBP1c and FASN mRNA expression levels, which were increased by HFD in red silkworm administration groups 1 and 2, were significantly reduced, and CPT-1 mRNA expression levels, known as a fatty acid oxidation regulator that was reduced by HFD, were increased again in red silkworm administration groups 1 and 2. In particular, when examining the results of CPT-1 mRNA and protein expression levels related to fatty acid oxidation, it was confirmed that red silkworm powder showed significantly increased expression levels compared to silymarin, a control drug known to be effective in treating NAFLD.

[0207] Accordingly, red silkworm powder can prevent, improve, or treat NAFLD by activating the SIRT1 / AMPK signaling pathway in the livers of mice fed a high-fat diet, thereby inhibiting triglyceride accumulation and increasing fatty acid oxidation. In particular, it was confirmed that red silkworm powder is promising as a preventive, improving, or therapeutic agent for NAFLD by demonstrating significantly superior efficacy compared to silymarin in relation to fatty acid oxidation metabolism.

[0208]

[0209] <Example 5> Confirmation of the therapeutic effect of red silkworm powder on liver inflammation caused by non-alcoholic fatty liver disease

[0210] To determine the effect of red silkworm powder on liver inflammation caused by non-alcoholic fatty liver disease, changes in liver inflammation markers (STAT3, IL-6, TNF-α, IL-1β, c-fos, HIF-1α, and c-myc) were measured.

[0211] The mice used in the experiment were prepared according to Reference Example 1, and changes in the levels of liver inflammation indicator factors were measured according to Reference Examples 6 and 7.

[0212] As shown in Figure 13, it was confirmed that the blood concentrations of TNF-α and IL-1β were significantly reduced in red silkworm pupae administration groups 1 and 2, which were administered red silkworm pupae powder, compared to the HFD administration group.

[0213] Specifically, Western blotting was performed to determine whether red silkworm powder affects STAT3, which is known to be associated with the development of liver inflammation. As a result, as shown in Figure 14, it was confirmed that while the phosphorylation of STAT3 increased in the HFD administration group, the phosphorylation of STAT3 decreased in a dose-dependent manner when red silkworm powder was administered. In particular, red silkworm administration group 2 showed reduced STAT3 phosphorylation compared to salimarin.

[0214] In addition, mRNA expression of inflammatory cytokines (IL-6, TNF-α, and IL-1β) and mRNA expression levels of STAT3 target genes were confirmed through qRT-PCR.

[0215] Measurement results showed that the expression of IL-6, TNF-α, and IL-1β mRNA, known as inflammatory cytokines, increased in the HFD administration group, and when red silkworm powder was administered, the expression levels decreased in a dose-dependent manner to the level of the control group administered normal feed (Fig. 15).

[0216] In the case of STAT3 target genes c-fos, HIF-1α, and c-myc, as shown in Figure 15, it was confirmed that when red silkworm powder was administered (red silkworm administration groups 1 and 2), the mRNA expression levels of c-fos, HIF-1α, and c-myc were significantly reduced compared to the HFD administration group.

[0217] Accordingly, it was confirmed that red silkworm powder significantly reduces liver inflammation caused by non-alcoholic fatty liver disease; specifically, it was confirmed that it reduces the inflammatory response associated with non-alcoholic fatty liver disease by inhibiting the signaling pathway through STAT3 phosphorylation.

[0218] Therefore, it was confirmed that red silkworm powder can be utilized for the prevention, improvement, or treatment of inflammation caused by NAFLD.

[0219]

[0220] <Example 6> Analysis of the Effects of Red Silkworm Powder on the Mechanism of Glucogenesis

[0221] To confirm the therapeutic mechanism of red silkworm pupae for NAFLD, the effects of red silkworm pupae powder on lipid metabolism and glucogenesis mechanisms related to the maintenance of lipid homeostasis were examined.

[0222] The mice used in the experiment were prepared according to Reference Example 1, and mRNA levels and protein levels were measured according to Reference Examples 6 and 7, respectively.

[0223] As shown in Figures 16 and 17, the expression of the GPR35 protein, known to inhibit lipid accumulation in liver cells and regulate cholesterol metabolism, was significantly increased in red silkworm pupae administration groups 1 and 2. Additionally, when the expression levels of PKA protein, which is a downstream regulator of GPR35 and known to inhibit lipid accumulation, and CREB and PGC-1α proteins related to gluconeogenesis were measured, it was confirmed that the expression levels of PKA, CREB, and PGC-1α proteins were significantly increased in red silkworm pupae administration groups 1 and 2.

[0224] In addition, the mRNA expression levels of genes related to gluconeogenesis, which affect the GPR35 signaling pathway and lipid homeostasis, were measured using qRT-PCR.

[0225] As shown in Figure 18, it was confirmed that the mRNA expression levels of GPR35 and PKA, which are GPR35 signaling pathway genes significantly reduced by the high-fat diet, and CREB, PGC-1α, G6Pase, and PCK1, which are gluconeogenesis-related genes, recovered to the level of the control group administered a normal diet in red silkworm pupae administration groups 1 and 2. In particular, while the administration of silymarin did not effectively regulate the GPR35 / PKA pathway, the administration of red silkworm pupae powder significantly increased the mRNA expression of genes related to the GPR35 / PKA pathway, thereby increasing gluconeogenesis that had decreased in the HFD administration group.

[0226] Accordingly, it was confirmed that red silkworm powder significantly increases gluconeogenesis through the GPR35 / PKA pathway, thereby maintaining hepatic lipid homeostasis, and as a result, can prevent, improve, or treat NAFLD.

[0227]

[0228] <Example 7> Confirmation of the fatty liver improvement effect of silk fibroin-derived peptide

[0229] In Example 1 above, the analysis of the constituent amino acids of red silkworm pupae confirmed that the content of Gly, Ala, and Ser was high, which matched the representative sequence (Gly-Ala-Gly-Ala-Gly-Ser) of silk fibroin-derived peptides (Fig. 19). Therefore, in this example, we intended to further confirm at the molecular level whether silk fibroin-derived peptides, which are the main components of red silkworm pupae, also exhibit improvement and therapeutic effects on non-alcoholic fatty liver disease (NAFLD). To this end, a liver cancer HepG2 cell line in which fat accumulation was induced with palmitic acid was used. The degree of fat accumulation was confirmed through Oil Red O (ORO) staining according to Reference Example 5, and the expression levels of related genes and proteins were analyzed by performing Western blotting and qRT-PCR according to Reference Examples 6 and 7.

[0230] After treating HepG2 cells with palmitic acid-induced fatty liver with a silk fibroin-derived peptide, the degree of intracellular lipid accumulation was observed through ORO staining. As a result, intracellular lipid globules were significantly reduced in the peptide-treated group, confirming that the silk fibroin-derived peptide has a direct inhibitory effect on lipid accumulation in liver cells (Fig. 20).

[0231] Next, lipid metabolism regulatory pathways were analyzed to confirm the molecular mechanism of fatty liver improvement. Western blotting analysis revealed that treatment with silk fibroin-derived peptides significantly increased the expression of SIRT1 and phosphorylated AMPK (p-AMPK), key regulators of fatty acid synthesis and oxidation, leading to increased activity of phosphorylated ACC (p-ACC), which regulates fatty acid oxidation (Fig. 21). This indicates that silk fibroin-derived peptides activate the SIRT1 / AMPK signaling pathway to promote fatty acid oxidation and inhibit triglyceride accumulation in the liver.

[0232] In addition to the effect of promoting fatty acid oxidation through the activation of the SIRT1 / AMPK pathway, a mechanism that inhibits lipid production itself was also confirmed. As a result of measuring changes in protein expression regulating lipid synthesis by Western blot, the protein expression of SREBP1, a key transcription factor that promotes lipid production, was significantly reduced by treatment with silk fibroin-derived peptides (Fig. 21). This demonstrates that silk fibroin-derived peptides improve fatty liver by complexly regulating two pathways: the promotion of fatty acid oxidation and the inhibition of lipid production.

[0233] Furthermore, the effect of silk fibroin-derived peptides on the mechanism of gluconeogenesis was also analyzed. Upon treatment with silk fibroin-derived peptides, the protein expression of GPR35, which inhibits lipid accumulation in hepatocytes and regulates cholesterol metabolism, and its downstream signaling factors, PKA and CREB, were all significantly increased (Fig. 22). This suggests that silk fibroin-derived peptides contribute to maintaining lipid homeostasis in the liver by activating the GPR35 / PKA signaling pathway and promoting gluconeogenesis.

Claims

1. A pharmaceutical composition for the prevention or treatment of non-alcoholic fatty liver disease comprising red silkworm powder, wherein the red silkworm powder is administered at a concentration of 0.005 g / kg to 0.2 g / kg.

2. A pharmaceutical composition according to claim 1, wherein the non-alcoholic fatty liver disease is one or more selected from the group consisting of non-alcoholic fatty liver, non-alcoholic steatohepatitis, liver fibrosis, liver cirrhosis, and liver cancer.

3. The pharmaceutical composition of Claim 1, wherein the red silkworm comprises silk fiber protein.

4. A pharmaceutical composition according to claim 1, wherein the red silkworm powder comprises aspartic acid (Asp), threonine (Thr), serine (Ser), glutamic acid (Glu), glycine (Gly), alanine (Ala), valine (Val), methionine (Met), isoleucine (Ile), leucine (Leu), tyrosine (Tyr), phenylalanine (Phe), lysine (Lys), histidine (His), and arginine (Arg).

5. A pharmaceutical composition according to claim 1, wherein the red silkworm powder reduces fat accumulated in the liver.

6. A pharmaceutical composition according to claim 1, wherein the red silkworm powder reduces one or more concentrations selected from the group consisting of blood triglyceride concentration, blood glucose concentration, blood total cholesterol concentration, blood LDL-cholesterol concentration, blood alanine transaminase (ALT) concentration, blood aspartate aminotransferase (AST) concentration, blood gamma-glutamyl transferase (GGT) concentration, and blood bilirubin concentration.

7. A pharmaceutical composition according to claim 1, wherein the red silkworm powder increases the concentration of HDL-cholesterol in the blood.

8. A pharmaceutical composition according to claim 1, wherein the red silkworm powder increases the expression of one or more proteins selected from the group consisting of p-AMPK (p-adenosine monophosphate-activated protein kinase), SIRT1 (sirtuin 1), p-ACC (p-acetyl-CoA carboxylase), and CPT-1 (carnitine palmitoyltransferase 1).

9. A pharmaceutical composition according to claim 1, wherein the red silkworm powder reduces the expression of p-STAT3 (p-signal transducer and activator of transcription 3) protein.

10. A pharmaceutical composition according to claim 1, wherein the red silkworm powder increases the expression of one or more genes selected from the group consisting of the GPR35 (G protein-coupled receptor 35) gene, the PKA (Protein kinase A) gene, the CREB (cAMP-response element binding protein) gene, the PGC-1α (Peroxisome proliferator-activated receptor gamma coactivator 1-alpha) gene, the G6Pase (glucose 6-phosphatase) gene, and the PCK1 (phosphoenolpyruvate carboxykinase 1) gene.

11. A pharmaceutical composition according to claim 1, wherein the red silkworm powder reduces the expression of one or more genes selected from the group consisting of the SREBP1c (sterol regulatory element-binding protein 1) gene, FASN (fatty acid synthase) gene, IL-6 (interleukin 6) gene, TNF-α (tumor necrosis factor alpha) gene, IL-1β (interleukin-1 beta) gene, c-fos (cellular fos proto-oncogene) gene, HIF-1α (hypoxia-inducible factor 1-alpha) gene and c-myc (cellular myelocytomatosis oncogene) gene.

12. A health functional food for the prevention or improvement of non-alcoholic fatty liver disease comprising red silkworm powder, wherein the red silkworm powder is consumed at a concentration of 0.005 g / kg to 0.2 g / kg.

13. A step of obtaining cooked silkworms by cooking silkworms at 70 to 130 ℃ for 100 to 160 minutes; and A method for manufacturing red silkworm powder comprising the step of drying the above-mentioned cooked silkworm.

14. A method for manufacturing red silkworm powder according to claim 13, wherein the manufacturing method further comprises the step of grinding the dried silkworm after the step of drying the cooked silkworm.

15. A pharmaceutical composition for the prevention or treatment of non-alcoholic fatty liver disease comprising a silk fibroin-derived peptide, wherein the silk fibroin-derived peptide is administered at a concentration of 10 μg / mL to 200 μg / mL.

16. A pharmaceutical composition according to claim 15, wherein the silk fibroin-derived peptide is composed of the amino acid sequence of SEQ ID NO.

31.

17. A pharmaceutical composition according to claim 15, wherein the non-alcoholic fatty liver disease is one or more selected from the group consisting of non-alcoholic fatty liver, non-alcoholic steatohepatitis, liver fibrosis, liver cirrhosis, and liver cancer.

18. A pharmaceutical composition according to claim 15, wherein the silk fibroin-derived peptide reduces fat accumulated in the liver.

19. A pharmaceutical composition according to claim 15, wherein the silk fibroin-derived peptide increases the expression of one or more proteins selected from the group consisting of p-AMPK, SIRT1, and p-ACC.

20. A pharmaceutical composition according to claim 15, wherein the silk fibroin-derived peptide increases the expression of one or more genes selected from the group consisting of the GPR35 gene, the PKA gene, and the CREB gene.

21. A pharmaceutical composition according to claim 15, wherein the silk fibroin-derived peptide reduces SREBP1 protein expression.

22. A health functional food for the prevention or improvement of non-alcoholic fatty liver disease comprising a silk fibroin-derived peptide, wherein the silk fibroin-derived peptide is consumed in an amount of 10 μg / mL to 200 μg / mL.

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