Pharmaceutical composition for preventing or treating high-fat diet-induced non-alcoholic fatty liver disease, comprising abelmoschus manihot extract as active ingredient

Abstract

A pharmaceutical composition for preventing or treating non-alcoholic fatty liver disease, comprising an Abelmoschus manihot extract as an active ingredient, according to the present invention, uses a natural product, and has no toxicity and has excellent antioxidant activity. In addition, in particular, the composition effectively inhibits lipid accumulation in the liver by exhibiting activities of reducing lipid accumulation, reducing adipocyte size, and inhibiting adipocyte differentiation, and thus can be usefully applied as a therapeutic agent for non-alcoholic fatty liver disease.

Description

Pharmaceutical composition for preventing or treating high-fat diet-induced non-alcoholic fatty liver disease, containing extract of Geumhwagyu as an active ingredient

[0001] The present invention relates to a pharmaceutical composition for preventing or treating high-fat diet-induced non-alcoholic fatty liver disease, comprising an extract of Abelmoschus manihot as an active ingredient.

[0002] This invention claims the benefit of priority from Korean Patent Application No. 10-2024-0092037, filed July 11, 2024, and Korean Patent Application No. 10-2025-0093306, filed July 10, 2025, the entire contents of which are incorporated herein by reference.

[0003] Fatty liver disease (FALD) occurs when normal fat metabolism is disrupted by factors such as excessive intake, increased fat accumulation and synthesis within the liver, and decreased excretion. This leads to excessive lipid accumulation, particularly triglycerides, exceeding 5% of the liver's fat content. With recent shifts in dietary habits toward a Western diet, the prevalence of fatty liver disease and related diseases has been rapidly increasing, both in Western societies and in Korea, along with the rising prevalence of obesity. Furthermore, some patients may progress from steatohepatitis to high-risk liver diseases such as cirrhosis or liver cancer, making prevention and improvement strategies crucial.

[0004] However, to date, diabetes, obesity, and hyperlipidemia medications are used at the discretion of physicians to alleviate or prevent the worsening of symptoms of fatty liver and related diseases. For example, polyenephosphatidylcholine is clinically used as a treatment for fatty liver, and fibrates, such as clofibrate, and statins, which are used to treat hyperlipidemia, are known to be effective against fatty liver. However, while fibrates improve lipid metabolism, such as by increasing high-density lipoprotein cholesterol through fatty liver beta-oxidation enzymes, they are known to cause side effects that cause liver dysfunction. In other words, there is currently a lack of fundamental preventive or therapeutic methods that are effective but free of side effects.

[0005] Meanwhile, Abelmoschus manihot (Golden Flower) is a precious medicinal plant, with its roots, stems, leaves, and flowers all possessing medicinal properties. It is only harvested during the July-August harvest season. Rich in collagen, Abelmoschus manihot is also effective for skin care. It also contains numerous beneficial ingredients, including palmitic acid, gossypetin, oleic acid, betaine, and linolenic acid, making it a popular flower tea.

[0006] Nevertheless, the effect of Geumhwagyu extract on improving fatty liver has not yet been reported.

[0007]

[0008] The purpose of the present invention is to provide a pharmaceutical composition for preventing or treating non-alcoholic fatty liver disease, which contains an extract of Abelmoschus manihot as an active ingredient.

[0009] Another object of the present invention is to provide a food composition for preventing or improving non-alcoholic fatty liver disease, which contains an extract of Abelmoschus manihot as an active ingredient.

[0010] Another object of the present invention is to provide a kit for preventing, improving, or treating non-alcoholic fatty liver disease, comprising an extract of Abelmoschus manihot and instructions.

[0011]

[0012] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by a person having ordinary skill in the technical field to which the present invention belongs from the description below.

[0013]

[0014] The present invention provides a pharmaceutical composition for preventing or treating non-alcoholic fatty liver disease, comprising an extract of Abelmoschus manihot as an active ingredient.

[0015] In one embodiment of the present invention, the non-alcoholic fatty liver disease may be, but is not limited to, high-fat diet-induced non-alcoholic fatty liver disease.

[0016] In one embodiment of the present invention, the gold extract may be extracted from a part including at least one selected from the group consisting of stems, roots, leaves, fruits, and flowers, but is not limited thereto.

[0017] In one embodiment of the present invention, the extract may be extracted with any one selected from the group consisting of water, alcohol having 1 to 6 carbon atoms, acetone, ether, benzene, chloroform, ethyl acetate, methylene chloride, hexane, cyclohexane, petroleum ether, dichloromethane, subcritical fluid, and supercritical fluid, but is not limited thereto.

[0018] In one embodiment of the present invention, the composition may be characterized by at least one selected from the group consisting of, but not limited to:

[0019] (a) Reduction of lipid accumulation in the liver;

[0020] (b) reduction in the size or weight of fat cells within the liver;

[0021] (c) Inhibition of intrahepatic adipocyte differentiation;

[0022] (d) Reduction of oxidative stress occurring during lipolysis through antioxidant activity in the liver;

[0023] (e) Inhibition of lipid metabolism in the liver;

[0024] (f) inhibition of liver cell degeneration or necrosis; and

[0025] (g) Inhibition of weight gain.

[0026] In one embodiment of the present invention, the composition may be characterized by at least one selected from the group consisting of, but not limited to:

[0027] (a) inhibiting the expression or activity of intrahepatic lipogenic genes or proteins; and

[0028] (b) Promoting the expression or activity of intrahepatic fat-decomposing genes or proteins.

[0029] In one embodiment of the present invention, the composition may promote the expression or activity of an antioxidant gene or protein, but is not limited thereto.

[0030] In one embodiment of the present invention, the composition can increase the expression of adiponectin, but is not limited thereto.

[0031] The present invention provides a food composition for preventing or improving non-alcoholic fatty liver disease, comprising an extract of Abelmoschus manihot as an active ingredient.

[0032] In one embodiment of the present invention, the food may be a health functional food, but is not limited thereto.

[0033] The present invention provides a kit for preventing, improving, or treating non-alcoholic fatty liver disease, comprising an extract of Abelmoschus manihot and instructions.

[0034] The present invention provides a method for preventing, improving, or treating non-alcoholic fatty liver disease, comprising administering to a subject in need thereof an extract of Abelmoschus manihot or a composition containing the same as an active ingredient in a pharmaceutically effective amount.

[0035] The present invention provides a use of an extract of Abelmoschus manihot or a composition containing the same as an active ingredient for preventing, improving, or treating non-alcoholic fatty liver disease.

[0036] The present invention provides a use for manufacturing a preparation for preventing, improving, or treating non-alcoholic fatty liver disease using an extract of Abelmoschus manihot or a composition containing the same as an active ingredient.

[0037] The pharmaceutical composition for preventing or treating non-alcoholic fatty liver disease, comprising an extract of Abelmoschus manihot as an active ingredient, is a natural product, non-toxic, and exhibits excellent antioxidant activity. Furthermore, it effectively suppresses lipid accumulation in the liver by exhibiting activities such as reducing lipid accumulation, reducing adipocyte size, and inhibiting adipocyte differentiation, making it a useful therapeutic agent for non-alcoholic fatty liver disease.

[0038]

[0039] Figures 1a and 1b show PCR primers used for gene expression analysis.

[0040] Figures 1c and 1d illustrate antibodies used in examples of the present invention.

[0041] Figures 2a to 2d show the antioxidant activity induced by the extract of the present invention.

[0042] Figures 3a and 3b show the cell survival rate of each cell when treated with the extract of the present invention.

[0043] Figures 3c to 3f show the inhibition of lipid accumulation and adipocyte differentiation of the extract of the present invention through ORO staining. Specifically, Figures 3c and 3d show that lipid accumulation is reduced when the extract of the present invention is treated in FL83B hepatocytes in which lipid accumulation is induced by oleic acid, and Figures 3e and 3f show that fat differentiation is reduced when the extract of the present invention is treated in differentiated 3T3-L1 preadipocytes.

[0044] Figure 3g shows changes in the expression of genes related to lipid metabolism when treated with the extract of the present invention.

[0045] Figure 4a is a schematic diagram showing the process of forming a non-alcoholic fatty liver mouse model.

[0046] Figure 4b shows the histological changes in the liver that occurred when the extract of the present invention was treated, and the decrease in lipid accumulation and the change in the size of fat cells that occurred when the extract of the present invention was treated, as shown by H&E staining.

[0047] Figures 4c and 4d show changes in body weight and fat tissue weight when treated with the extract of the present invention.

[0048] Figures 4e and 4f show abdominal CT images and changes in fat tissue area when treated with the extract of the present invention.

[0049] Figure 4g shows the changes in the concentrations of AST, ALT, and adiponectin when treated with the extract of the present invention.

[0050] Figure 5a shows the results of RT-qPCR to confirm changes in the expression of mRNA of biomarkers related to adipogenesis and lipogenesis when the extract of the present invention was treated in vivo (data are geometric means ± standard deviations, and statistical analysis was performed using Tukey's test after one-way analysis of variance, and P < 0.05 was considered statistically significant).

[0051] Figure 5b shows the results of RT-qPCR confirming changes in the expression of mRNA of biomarkers related to lipolysis and fatty acid oxidation when the extract of the present invention was treated in vivo (statistical analysis was performed using one-way ANOVA followed by Tukey's test, and P < 0.05 was considered statistically significant).

[0052] Figure 5c shows the results of RT-qPCR confirming changes in the expression of mRNA of oxidative stress-related biomarkers when the extract of the present invention was treated in vivo (data are geometric means ± standard deviations, and statistical analysis was performed using Tukey's test after one-way ANOVA, and P < 0.05 was considered statistically significant).

[0053] Figure 6a shows the results of immunohistochemical staining to confirm changes in protein expression of biomarkers related to adipogenesis and lipogenesis when the extract of the present invention was treated in vivo (scale bar is 100 μm, data is geometric mean ± standard deviation, statistical analysis was performed using Tukey's test after one-way ANOVA, and P < 0.05 was considered statistically significant).

[0054] Figure 6b shows the results of Western blot analysis to confirm changes in protein expression of p-ACC / ACC and p-AMPK / AMPK when the extract of the present invention was treated in vivo (relative protein expression data were normalized to the expression level of GAPDH, and data are geometric means ± SD. Statistical analysis was performed using Tukey's test after one-way ANOVA, and P < 0.05 was considered statistically significant).

[0055] Figure 6c shows the results of Western blot analysis to confirm changes in protein expression of SREBP-1c, FAS, PPARγ, and C / EBPα, which are proteins related to adipogenesis and lipogenesis, when the extract of the present invention was treated in vivo (relative protein expression data were normalized to the expression level of GAPDH, and data are geometric means ± standard deviations. Statistical analysis was performed using Tukey's test after one-way ANOVA, and P < 0.05 was considered statistically significant).

[0056] Figure 6d shows the results of Western blot analysis to confirm the change in protein expression of p-HSL / HSL, a protein related to lipolysis, when the extract of the present invention was treated in vivo (relative protein expression data were normalized to the expression level of GAPDH, and the data are geometric means ± standard deviations. Statistical analysis was performed using Tukey's test after one-way ANOVA, and P < 0.05 was considered statistically significant).

[0057] Figure 6e shows the results of Western blot analysis to confirm the change in protein expression of HO-1 and Nrf2, which are proteins related to oxidative stress, when the extract of the present invention was treated in vivo (relative protein expression data were normalized to the protein expression data of GAPDH, and the data are geometric means ± standard deviations. Statistical analysis was performed using Tukey's test after one-way ANOVA, and P < 0.05 was considered statistically significant).

[0058] Figure 7 is a schematic diagram showing the relationship between each factor.

[0059]

[0060] The present invention provides a pharmaceutical composition for preventing or treating non-alcoholic fatty liver disease, comprising an extract of Abelmoschus manihot as an active ingredient.

[0061] In the present invention, “Golden Flower” is an annual flowering plant belonging to the Malvaceae family. In China, it is widely used as a traditional medicine, such as by making an ethanol extract of the flower of Golden Flower into capsules and using it to treat patients with kidney disease. The main components of Golden Flower in the present invention may include, but are not limited to, flavonoids, amino acids, nucleosides, polysaccharides, organic acids, steroids, and volatile oils. In the present invention, “Golden Flower” is an annual plant belonging to the Malvaceae family and the Okra genus, also called “Dakpul.” In one embodiment, Golden Flower may be, but is not limited to, Abelmoschus manihot. In addition, in the present invention, Golden Flower may be, but is not limited to, something that a person skilled in the art can commonly purchase in a traditional market or electronic transaction.

[0062] In one embodiment of the present invention, the non-alcoholic fatty liver disease may be, but is not limited to, high-fat diet-induced non-alcoholic fatty liver disease.

[0063] The term “non-alcoholic fatty liver disease” used in the present invention refers to fatty liver disease caused by a high-calorie diet, and may occur in association with metabolic diseases such as obesity, diabetes, and hyperlipidemia, and if left untreated, may worsen to non-alcoholic steatohepatitis, cirrhosis, or liver cancer.

[0064] In one embodiment of the present invention, it may be confirmed that the gold extract of the present invention has antioxidant, anti-adipogenic, anti-inflammatory, antiviral, angiogenesis-promoting, cardioprotective, immunomodulatory, and hepatoprotective effects, but is not limited thereto.

[0065] The extract of the present invention has been completed by confirming that it can be used as a pharmaceutical composition or food composition, as it exhibits a remarkable effect of inhibiting fat differentiation and reducing lipid accumulation, and in particular, exhibits an effect of inhibiting lipid accumulation in liver tissue.

[0066] In one embodiment of the present invention, the gold extract may be extracted from a part including at least one selected from the group consisting of stems, roots, leaves, fruits, and flowers, but is not limited thereto.

[0067] The gold flower of the present invention can be used as is without damaging its original form, or can be used after performing a pretreatment process in consideration of the process speed and process (manufacturing) efficiency intended by a person skilled in the art, and the pretreatment process can include, for example, conventional steps such as selection, washing, cutting, powdering, and drying. In addition, the gold flower used in the present invention can be used regardless of the source as long as it is in a dried form, and regardless of the source, the gold flower extract can contain the same components as the extract of the present invention, and thus can exhibit the same non-alcoholic fatty liver prevention, improvement, or treatment effect.

[0068] In the present invention, “Golden Flower Extract” includes all forms of extracts that can be formed using the extract itself and the extract, such as commercially available extracts, extracts obtained by extraction treatment of the gold flower, diluted or concentrated extracts, dried products obtained by drying the extracts, adjusted or purified products of the extracts, or mixtures thereof.

[0069] In the present invention, the method for extracting the gold powder is not particularly limited, and extraction can be performed according to a method commonly used in the relevant technical field. Non-limiting examples of the extraction method include hot water extraction, ultrasonic extraction, filtration, and reflux extraction, and these may be performed alone or in combination of two or more methods, but are not limited thereto.

[0070] In one embodiment of the present invention, the gold extract may be extracted by a method including, but not limited to, the following steps:

[0071] (s1) a step of extracting the pulverized gold powder by adding a solvent; and

[0072] (s2) Step of filtering the extract.

[0073] In one embodiment of the present invention, when the solvent is water, the method may be characterized by at least one selected from the group consisting of, but not limited to:

[0074] (a) The step (s1) above is to add to the pulverized gold powder 25 to 35 times the weight, 25 to 34 times the weight, 25 to 33 times the weight, 25 to 32 times the weight, 25 to 31 times the weight, 25 to 30 times the weight, 26 to 35 times the weight, 26 to 34 times the weight, 26 to 33 times the weight, 26 to 32 times the weight, 26 to 31 times the weight, 26 to 30 times the weight, 27 to 35 times the weight, 27 to 34 times the weight, 27 to 33 times the weight, 27 to 32 times the weight, 27 to 31 times the weight, 27 to 30 times the weight, 28 to 35 times the weight, 28 to 34 times the weight, Adding water of 28 to 33 times the weight, 28 to 32 times the weight, 28 to 31 times the weight, 28 to 30 times the weight, 29 to 35 times the weight, 29 to 34 times the weight, 29 to 33 times the weight, 29 to 32 times the weight, 29 to 31 times the weight, 29 to 30 times the weight, 30 to 35 times the weight, 30 to 34 times the weight, 30 to 33 times the weight, 30 to 32 times the weight, or 30 to 31 times the weight, and heating at 70 to 130°C, 70 to 120°C, 70 to 110°C, 70 to 100°C, 80 to 130°C, 80 to 120°C, 80 to 110°C, 80 to A step of extracting at 100°C, 90 to 130°C, 90 to 120°C, 90 to 110°C, 90 to 100°C, 100 to 130°C, 100 to 120°C, 100 to 110°C, or 100°C;

[0075] (b) The step (s2) is to extract the pore size of the extract to be 1 to 20 μm, 1 to 15 μm, 1 to 13 μm, 1 to 11 μm, 1 to 10 μm, 1 to 9 μm, 1 to 8 μm, 5 to 20 μm, 5 to 15 μm, 5 to 13 μm, 5 to 11 μm, 5 to 10 μm, 5 to 9 μm, 5 to 8 μm, 6 to 20 μm, 6 to 15 μm, 6 to 13 μm, 6 to 11 μm, 6 to 10 μm, 6 to 9 μm, 6 to 8 μm, 7 to 20 μm, 7 to 15 μm, 7 to 13 μm, 7 to 11 μm, 7 to A step of filtering with a filter having a size of 10 μm, 7 to 9 μm, 7 to 8 μm, 8 to 20 μm, 8 to 15 μm, 8 to 13 μm, 8 to 11 μm, 8 to 10 μm, 8 to 9 μm, or 8 μm; and

[0076] (c) the method may further comprise a step of concentrating the extract,

[0077] The above-mentioned concentrating step is a step of concentrating the extract at 40 to 80°C, 40 to 75°C, 40 to 70°C, 40 to 65°C, 40 to 60°C, 45 to 80°C, 45 to 75°C, 45 to 70°C, 45 to 65°C, 45 to 60°C, 50 to 80°C, 50 to 75°C, 50 to 70°C, 50 to 65°C, 50 to 60°C, 55 to 80°C, 55 to 75°C, 55 to 70°C, 55 to 65°C, 55 to 60°C, 60 to 80°C, 60 to 75°C, 60 to 70°C, 60 to 65°C, or 60°C;

[0078] (d) The method further comprises a step of freeze-drying in a freeze dryer.

[0079] In one embodiment of the present invention, when the solvent is ethanol, the method may be characterized by at least one selected from the group consisting of, but not limited to:

[0080] (a) The step (s1) above is to add to the pulverized gold powder 25 to 35 times the weight, 25 to 34 times the weight, 25 to 33 times the weight, 25 to 32 times the weight, 25 to 31 times the weight, 25 to 30 times the weight, 26 to 35 times the weight, 26 to 34 times the weight, 26 to 33 times the weight, 26 to 32 times the weight, 26 to 31 times the weight, 26 to 30 times the weight, 27 to 35 times the weight, 27 to 34 times the weight, 27 to 33 times the weight, 27 to 32 times the weight, 27 to 31 times the weight, 27 to 30 times the weight, 28 to 35 times the weight, 28 to 34 times the weight, 28 to 33 times the weight, 28 to 32 times the weight, 28 to 31 times the weight, 28 to 30 times the weight, 29 to 35 times the weight, 29 to 34 times the weight, 29 to 33 times the weight, 29 to 32 times the weight, 29 to 31 times the weight, 29 to 30 times the weight, 30 to 35 times the weight, 30 to 34 times the weight, 30 to 33 times the weight, 30 to 32 times the weight, or 30 to 31 times the weight of ethanol is added and the mixture is heated to 10 to 40°C, 10 to 35°C, 10 to 30°C, 10 to 28°C, 10 to 26°C, 10 to 25°C, 10 to 24°C, 10 to 23°C, 10 to 22℃, 15 to 40℃, 15 to 35℃, 15 to 30℃, 15 to 28℃, 15 to 26℃, 15 to 25℃, 15 to 24℃, 15 to 23℃, 15 to 22℃, 17 to 40℃, 17 to 35℃, 17 to 30℃, 17 to 28℃, 17 to 26℃, 17 to 25℃, 17 to 24℃, 17 to 23℃, 17 to 22℃, 19 to 40℃, 19 to 35℃, 19 to 30℃, 19 to 28℃, 19 to 26℃, 19 to 25℃, 19 to 24℃, 19 to 23℃, 19 to 22℃, 20 to 40℃, 20 to 35℃,A step of extracting at 20 to 30°C, 20 to 28°C, 20 to 26°C, 20 to 25°C, 20 to 24°C, 20 to 23°C, or 20 to 22°C;

[0081] (b) The step (s2) is a method of extracting a pore size of the extract having a pore size of 0.10 to 0.40 μm, 0.10 to 0.35 μm, 0.10 to 0.30 μm, 0.10 to 0.28 μm, 0.10 to 0.26 μm, 0.10 to 0.25 μm, 0.15 to 0.40 μm, 0.15 to 0.35 μm, 0.15 to 0.30 μm, 0.15 to 0.28 μm, 0.15 to 0.26 μm, 0.15 to 0.25 μm, 0.20 to 0.40 μm, 0.20 to 0.35 μm, 0.20 to 0.30 μm, 0.20 to 0.28μm, 0.20 to 0.26μm, 0.20 to 0.25μm, 0.22 to 0.40μm, 0.22 to 0.35μm, 0.22 to 0.30μm, 0.22 to 0.28μm, 0.22 to 0.26μm, 0.22 to 0.25μm, 0.24 to 0.40μm, 0.24 to 0.35μm, 0.24 to 0.30μm, 0.24 to 0.28μm, 0.24 to 0.26μm, 0.24 to 0.25μm, 0.25 to 0.40μm, 0.25 to 0.35μm, 0.25 to 0.30μm, A step of filtering with a filter of 0.25 to 0.28 μm, 0.25 to 0.26 μm, or 0.25 μm;

[0082] (c) the method may further comprise a step of concentrating the extract,

[0083] The above-mentioned concentrating step is a step of concentrating the extract at 40 to 80°C, 40 to 75°C, 40 to 70°C, 40 to 65°C, 40 to 60°C, 45 to 80°C, 45 to 75°C, 45 to 70°C, 45 to 65°C, 45 to 60°C, 50 to 80°C, 50 to 75°C, 50 to 70°C, 50 to 65°C, 50 to 60°C, 55 to 80°C, 55 to 75°C, 55 to 70°C, 55 to 65°C, 55 to 60°C, 60 to 80°C, 60 to 75°C, 60 to 70°C, 60 to 65°C, or 60°C;

[0084] (d) the method further comprises a step of vacuum filtering the extract before the concentrating step; and

[0085] (e) The method further comprises a step of freeze-drying in a freeze dryer.

[0086]

[0087] In one embodiment of the present invention, the extract may be extracted with any one selected from the group consisting of water, alcohol having 1 to 6 carbon atoms, acetone, ether, benzene, chloroform, ethyl acetate, methylene chloride, hexane, cyclohexane, petroleum ether, dichloromethane, subcritical fluid, and supercritical fluid, but is not limited thereto.

[0088] In one embodiment of the present invention, the gold flower extract can be prepared by a hot water extraction method by soaking in distilled water and is selected from the group consisting of 80°C to 120°C, 85°C to 120°C, 90°C to 120°C, 95°C to 120°C, 98°C to 120°C, 99°C to 120°C, 80°C to 110°C, 85°C to 110°C, 90°C to 110°C, 95°C to 110°C, 98°C to 110°C, 99°C to 110°C, 80°C to 105°C, 85°C to 105°C, 90°C to 105°C, 95°C to 105°C, 98°C to 105°C, 99°C to 105°C, 80°C to 100°C, 85°C to It may be a hot water extraction method at a temperature condition of 100°C, 90°C to 100°C, 95°C to 100°C, 98°C to 100°C, or 99°C to 100°C, and in one embodiment of the present invention, it may be a hot water extraction method at 100°C, but is not limited thereto.

[0089] In one embodiment of the present invention, the gold flower extract can be prepared by soaking in alcohol having 1 to 6 carbon atoms, and in one embodiment of the present invention, it can be prepared by soaking in ethanol, and the ethanol is 30 to 100 v / v%, 40 to 100 v / v%, 50 to 100 v / v%, 60 to 100 v / v%, 30 to 90 v / v%, 40 to 90 v / v%, 50 to 90 v / v%, 60 to 90 v / v%, 30 to 80 v / v%, 40 to 80 v / v%, 50 to 80 v / v%, 60 to 80 v / v%, 30 to 70 v / v%, 40 to 70 v / v%, 50 to 70 v / v%, 60 to It may be ethanol at a concentration of 70 v / v%, 30 to 60 v / v%, 40 to 60 v / v%, 50 to 60 v / v%, 55 to 60 v / v%, or 60 to 65 v / v%, and in one embodiment of the present invention, it may be 60 v / v% ethanol, but is not limited thereto.

[0090] In one embodiment of the present invention, the composition may be characterized by at least one selected from the group consisting of, but not limited to:

[0091] (a) Reduction of lipid accumulation in the liver;

[0092] (b) reduction in the size or weight of fat cells within the liver;

[0093] (c) Inhibition of intrahepatic adipocyte differentiation;

[0094] (d) Reduction of oxidative stress occurring during lipolysis through antioxidant activity in the liver;

[0095] (e) Inhibition of lipid metabolism in the liver;

[0096] (f) inhibition of liver cell degeneration or necrosis; and

[0097] (g) Inhibition of weight gain.

[0098] The term “lipid” used in the present invention is a broad concept that collectively refers to biomolecules that dissolve in nonpolar solvents, and includes fat.

[0099] The term "differentiation" used in the present invention refers to a phenomenon in which structures or functions become specialized during the growth of cells through division and proliferation, that is, it can refer to, but is not limited to, a change in form or function in order for cells, tissues, etc. of a living organism to perform their respective tasks. In general, it refers to a phenomenon in which a relatively simple system is divided into two or more qualitatively different subsystems. For example, during ontogeny, a distinction is made between parts of an egg that were initially homogeneous, such as the head or torso, or a distinction is made among cells, such as muscle cells or nerve cells, etc., such that qualitative differences arise between parts of a certain biological system that were initially almost homogeneous, or a state in which they are divided into qualitatively distinguishable subregions or subsystems as a result is called differentiation.

[0100] The term "antioxidant" used in the present invention refers to the action of inhibiting oxidation. The human body has a balance of prooxidants and antioxidants, but if this balance becomes unbalanced due to various factors and tilts toward oxidation promotion, oxidative stress is induced, causing potential cell damage and pathological diseases. Reactive oxygen species (ROS), which are the direct cause of this oxidative stress, are unstable and highly reactive, easily reacting with various biological substances and attacking macromolecules in the body, causing irreversible damage to cells and tissues or leading to mutations, cytotoxicity, and carcinogenesis.

[0101] In one embodiment of the present invention, the reduction of oxidative stress occurring during fat decomposition through antioxidant activity in the liver is evaluated as an indicator of, but not limited to, an increase in radical scavenging activity of at least one radical selected from the group consisting of DPPH radical, ABTS radical, and hydroxyl radical; or an increase in metal chelating activity.

[0102] In the present invention, “body weight” means the weight or mass of the entire object, and may include, but is not limited to, the weight or mass of body components including organs, tissues, body fluids, fat, muscles, etc. In one embodiment of the present invention, weight gain may be, but is not limited to, an increase in the weight of one or more tissues selected from the group consisting of liver, epididymal adipose tissue, subcutaneous adipose tissue, visceral adipose tissue, and abdominal adipose tissue.

[0103] In one embodiment of the present invention, inhibition of lipid metabolism in the liver may be, but is not limited to, a decrease in the expression or activity of a lipogenic gene or protein in the liver (inhibition of activity); or an increase in the expression or activity of a lipolytic gene or protein in the liver (promotion of activity).

[0104] In one embodiment of the present invention, the composition may be characterized by at least one selected from the group consisting of, but not limited to:

[0105] (a) inhibiting the expression or activity of intrahepatic lipogenic genes or proteins; and

[0106] (b) Promoting the expression or activity of intrahepatic fat-decomposing genes or proteins.

[0107] In one embodiment of the present invention, the composition can promote an increase in the expression ratio of any one or more genes or proteins selected from the group consisting of p-ACC / ACC, p-AMPK / AMPK, and p-HSL / HSL, but is not limited thereto.

[0108] In the present invention, a fat-producing gene or protein may mean a gene or protein that promotes fat differentiation, fat formation, fat cell differentiation, fat cell production, or fat synthesis.

[0109] In one embodiment of the present invention, the lipogenic gene or protein may be at least one selected from the group consisting of fatty acid synthase (FAS), peroxisome proliferator-activated receptor gamma (PPARγ), CCAAT-enhancer-binding-protein alpha (C / EBPα), sterol regulatory element binding protein-1c (SREBP-1c), acetyl-CoA carboxylase 1 (ACC1), stearoyl-CoA desaturase 1 (SCD1), and AMP-activated protein kinase (AMPK), but is not limited thereto.

[0110] In the present invention, a fat decomposition gene or protein may mean a gene or protein that inhibits or suppresses lipid oxidation; or fat differentiation, fat formation, adipocyte differentiation, adipocyte production, or fat synthesis.

[0111] In one embodiment of the present invention, the lipolytic gene or protein may be at least one selected from the group consisting of peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC1α), hormone-sensitive lipase (HSL), adipose triglyceride lipase (ATGL), carnitine palmitoyltransferase-1 (CPT-1), and acyl-CoA oxidase (ACOX), but is not limited thereto.

[0112] In one embodiment of the present invention, the composition may promote the expression or activity of an antioxidant gene or protein, but is not limited thereto.

[0113] In one embodiment of the present invention, the antioxidant gene or protein may be at least one selected from the group consisting of nuclear factor erythroid 2-related factor (Nrf2), heme oxygenase-1 (HO-1), glutathione peroxidase (GPx), and catalase (CAT), but is not limited thereto.

[0114] In one embodiment of the present invention, the composition can increase the expression of adiponectin, but is not limited thereto.

[0115] In one embodiment of the present invention, the subject or patient of the present invention may be characterized by one or more of the following, but is not limited thereto:

[0116] (a) increased expression or activity of any one or more lipogenic genes or proteins selected from the group consisting of fatty acid synthase (FAS), peroxisome proliferator-activated receptor gamma (PPARγ), CCAAT-enhancer-binding-protein alpha (C / EBPα), sterol regulatory element binding protein-1c (SREBP-1c), acetyl-CoA carboxylase 1 (ACC1), stearoyl-CoA desaturase 1 (SCD1), and AMP-activated protein kinase (AMPK);

[0117] (b) decreased expression or activity of any one or more lipolytic genes or proteins selected from the group consisting of peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC1α), hormone-sensitive lipase (HSL), adipose triglyceride lipase (ATGL), carnitine palmitoyltransferase-1 (CPT-1), and acyl-CoA oxidase (ACOX); and

[0118] (c) decreased expression or activity of any one or more antioxidant genes or proteins selected from the group consisting of nuclear factor erythroid 2-related factor (Nrf2), heme oxygenase-1 (HO-1), glutathione peroxidase (GPx), and catalase (CAT).

[0119] In the present invention, the composition of the present invention may be used for one or more patients selected from the group consisting of patients with non-alcoholic fatty liver disease accompanied by obesity, patients with non-alcoholic fatty liver disease accompanied by abdominal obesity, patients with non-alcoholic fatty liver disease accompanied by type 2 diabetes, patients with non-alcoholic fatty liver disease accompanied by insulin resistance, patients with non-alcoholic fatty liver disease with reduced fatty acid oxidation, and patients with non-alcoholic fatty liver disease with reduced antioxidant defense mechanisms, but is not limited thereto.

[0120] The pharmaceutical composition according to the present invention may further comprise suitable carriers, excipients, and diluents commonly used in the manufacture of pharmaceutical compositions. The excipients may be, for example, at least one selected from the group consisting of diluents, binders, disintegrants, lubricants, adsorbents, moisturizers, film-coating materials, and controlled-release additives.

[0121] The content of the gold leaf extract in the composition of the present invention can be appropriately adjusted depending on the symptoms of the disease, the degree of progression of the symptoms, the condition of the patient, etc., and may be, for example, 0.0001 to 99.9 wt% or 0.001 to 50 wt% based on the total weight of the composition, but is not limited thereto. The content ratio is a value based on the dry amount after removing the solvent.

[0122] The pharmaceutical composition according to the present invention may be formulated and used in the form of external preparations such as powders, granules, sustained-release granules, enteric-coated granules, liquids, eye drops, ellipsoids, emulsions, suspensions, alcohols, troches, aromatic waters, limonades, tablets, sustained-release tablets, enteric-coated tablets, sublingual tablets, hard capsules, soft capsules, sustained-release capsules, enteric capsules, pills, tinctures, soft extracts, dry extracts, fluid extracts, injections, capsules, irrigation solutions, ointments, lotions, pastes, sprays, inhalants, patches, sterile injection solutions, or aerosols, according to a conventional method, and the external preparations may have formulations such as creams, gels, patches, sprays, ointments, ointments, lotions, liniments, pastes, or cataplasmas.

[0123] Carriers, excipients and diluents that may be included in the pharmaceutical composition according to the present invention include lactose, dextrose, sucrose, oligosaccharides, 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, and mineral oil.

[0124] When formulating, it is usually prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants.

[0125] The additives of the tablets, powders, granules, capsules, pills, and troches according to the present invention include excipients such as corn starch, potato starch, wheat starch, lactose, sucrose, glucose, fructose, D-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, calcium hydrogen phosphate, calcium sulfate, sodium chloride, sodium bicarbonate, purified lanolin, microcrystalline cellulose, dextrin, sodium alginate, methylcellulose, sodium carboxymethylcellulose, kaolin, urea, colloidal silica gel, hydroxypropyl starch, hydroxypropyl methylcellulose (HPMC), HPMC 1928, HPMC 2208, HPMC 2906, HPMC 2910, propylene glycol, casein, calcium lactate, and Primogel; Gelatin, gum arabic, ethanol, agar powder, cellulose acetate phthalate, carboxymethylcellulose, calcium carboxymethylcellulose, glucose, purified water, sodium caseinate, glycerin, stearic acid, sodium carboxymethylcellulose, sodium methylcellulose, methylcellulose, microcrystalline cellulose, dextrin, hydroxycellulose, hydroxypropyl starch, hydroxymethylcellulose, refined shellac, starch starch, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl alcohol, polyvinyl pyrrolidone, and binders such as hydroxypropyl methylcellulose, corn starch, agar powder, methylcellulose, bentonite, hydroxypropyl starch, sodium carboxymethylcellulose, sodium alginate, Disintegrants such as carboxymethylcellulose calcium, calcium citrate, sodium lauryl sulfate, anhydrous silicic acid, 1-hydroxypropyl cellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde-treated casein and gelatin, alginic acid, amylose, guar gum, baking soda, polyvinylpyrrolidone, calcium phosphate, gelled starch, gum arabic, amylopectin, pectin, sodium polyphosphate, ethylcellulose, sucrose, magnesium aluminum silicate, di-sorbitol solution, and light anhydrous silicic acid;Lubricants such as calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, lycopodium dentata, kaolin, petrolatum, sodium stearate, cacao butter, sodium salicylate, magnesium salicylate, polyethylene glycol (PEG) 4000, PEG 6000, liquid paraffin, hydrogenated soybean oil (Lubri wax), aluminum stearate, zinc stearate, sodium lauryl sulfate, magnesium oxide, macrogol, synthetic aluminum silicate, anhydrous silicic acid, higher fatty acids, higher alcohols, silicone oil, paraffin oil, polyethylene glycol fatty acid ether, starch, sodium chloride, sodium acetate, sodium oleate, dl-leucine, and light anhydrous silicic acid can be used.

[0126] As additives of the liquid formulation according to the present invention, water, dilute hydrochloric acid, dilute sulfuric acid, sodium citrate, monostearate sucrose, polyoxyethylene sorbitol fatty acid esters (twin esters), polyoxyethylene monoalkyl ethers, lanolin ethers, lanolin esters, acetic acid, hydrochloric acid, ammonia water, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone, ethylcellulose, sodium carboxymethylcellulose, etc. can be used.

[0127] The syrup according to the present invention may include a solution of white sugar, other sugars, or sweeteners, and may also include a fragrance, a coloring agent, a preservative, a stabilizer, a suspending agent, an emulsifier, a viscosity modifier, and the like, as needed.

[0128] Purified water may be used in the emulsion according to the present invention, and emulsifiers, preservatives, stabilizers, fragrances, etc. may be used as needed.

[0129] The suspension according to the present invention may include suspending agents such as acacia, tragacanth, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropylmethylcellulose (HPMC), HPMC 1828, HPMC 2906, HPMC 2910, and the like. Surfactants, preservatives, stabilizers, colorants, and fragrances may also be used as needed.

[0130] The injection according to the present invention includes a solvent such as distilled water for injection, 0.9% sodium chloride injection, Ringer's injection, dextrose injection, dextrose + sodium chloride injection, PEG, lactated Ringer's injection, ethanol, propylene glycol, non-volatile oils - sesame oil, cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristate, benzene benzoate; a solubilizing agent such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide, butazolidine, propylene glycol, tween, nitrile acid amide, hexamine, dimethylacetamide; a buffer such as weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, albumins, peptones, gums; It may include isotonic agents such as sodium chloride; stabilizers such as sodium bisulfite (NaHSO3), carbon dioxide gas, sodium metabisulfite (Na2S2O5), sodium sulfite (Na2SO3), nitrogen gas (N2), and ethylenediaminetetraacetic acid; oxidizing agents such as sodium bisulfide 0.1%, sodium formaldehyde sulfoxylate, thiourea, disodium ethylenediaminetetraacetic acid, and acetone sodium bisulfite; analgesics such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, and calcium gluconate; and suspending agents such as sodium cis-methylenediamine, sodium alginate, Tween 80, and aluminum monostearate.

[0131] The suppository according to the present invention comprises cocoa butter, lanolin, withepsol, polyethylene glycol, glycerogelatin, methylcellulose, carboxymethylcellulose, a mixture of stearic acid and oleic acid, Subanal, cottonseed oil, peanut oil, palm oil, cocoa butter + cholesterol, lecithin, ranet wax, glycerol monostearate, Tween or Span, Imhausen, monolene (propylene glycol monostearate), glycerin, Adeps solidus, Buytyrum Tego-G, Cebes Pharma 16, hexalide base 95, Cotomar, Hydroxycote SP, S-70-XXA, S-70-XX75 (S-70-XX95), Hydroxycote Mechanisms such as (Hydrokote) 25, Hydrokote 711, Idropostal, Massa estrarium (A, AS, B, C, D, E, I, T), Massa-MF, Masupol, Masupol-15, Neosupostal-N, Paramound-B, Suposiro (OSI, OSIX, A, B, C, D, H, L), Suppository base type IV (AB, B, A, BC, BBG, E, BGF, C, D, 299), Supostal (N, Es), Wecovi (W, R, S, M, Fs), Tezester triglyceride base (TG-95, MA, 57) can be used.

[0132] Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid dosage forms are prepared by mixing the extract with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.

[0133] Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups. In addition to commonly used simple diluents such as water and liquid paraffin, they may contain various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

[0134] The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment. The effective dosage level can be determined based on factors including the type and severity of the patient's disease, drug activity, drug sensitivity, administration time, administration route and excretion rate, treatment period, concurrently used drugs, and other factors well known in the medical field.

[0135] The pharmaceutical composition according to the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents. It can be administered sequentially or simultaneously with conventional therapeutic agents, or in single or multiple doses. Taking all of the above factors into account, it is important to administer an amount that achieves maximum efficacy with minimal side effects. This amount can be readily determined by those skilled in the art to which the present invention pertains.

[0136] The pharmaceutical composition of the present invention can be administered to a subject via various routes. All modes of administration are conceivable, including oral ingestion, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, intrathecal injection, sublingual administration, buccal mucosa administration, rectal insertion, vaginal insertion, ocular administration, otic administration, nasal administration, inhalation, oral or nasal spraying, dermal administration, and transdermal administration.

[0137] The pharmaceutical composition of the present invention is determined according to the type of drug as an active ingredient along with various related factors such as the disease to be treated, route of administration, age, sex, weight of the patient, and severity of the disease.

[0138] In the present invention, the term "subject" refers to a subject requiring treatment for a disease, and more specifically, refers to mammals such as human or non-human primates, mice, rats, dogs, cats, horses, and cows. In the present invention, the term "subject" may be used interchangeably with a patient, but is not limited thereto.

[0139] In the present invention, “administration” means providing a predetermined composition of the present invention to an individual by any appropriate method.

[0140] In the present invention, “prevention” means any action that suppresses or delays the onset of a target disease, “treatment” means any action that improves or beneficially changes a target disease and its metabolic abnormality symptoms by administering a pharmaceutical composition according to the present invention, and “improvement” means any action that reduces a parameter related to a target disease, for example, the severity of a symptom, by administering a composition according to the present invention.

[0141] In addition, the present invention provides a method for preventing, improving, or treating non-alcoholic fatty liver disease, comprising administering to a subject in need thereof an extract of Abelmoschus manihot or a composition containing the same as an active ingredient in a pharmaceutically effective amount.

[0142] In addition, the present invention provides a method for preventing, improving, or treating non-alcoholic fatty liver disease, comprising administering to a subject in need thereof a pharmaceutically effective amount of an extract of Abelmoschus manihot or a composition containing the same as an active ingredient.

[0143] In addition, the present invention provides a use of an extract of Abelmoschus manihot or a composition containing the same as an active ingredient for preventing, improving, or treating non-alcoholic fatty liver disease.

[0144] In addition, the present invention provides a use for manufacturing a preparation for preventing, improving, or treating non-alcoholic fatty liver disease using an extract of Abelmoschus manihot or a composition containing the same as an active ingredient.

[0145]

[0146] The present invention provides a food composition for preventing or improving non-alcoholic fatty liver disease, comprising an extract of Abelmoschus manihot as an active ingredient.

[0147] In one embodiment of the present invention, the pharmaceutical composition for preventing or treating non-alcoholic fatty liver disease of the present invention can be equally applied to the food composition of the present invention, but is not limited thereto.

[0148] In one embodiment of the present invention, the food may be a health functional food, but is not limited thereto.

[0149] In the present invention, “food” means a natural product or processed product containing one or more nutrients, preferably a product that has gone through a certain degree of processing to become directly edible, and in its usual sense, includes all health functional foods, beverages, food additives, and beverage additives.

[0150] In the present invention, the term “health functional food” is the same as food for special health use (FoSHU), and refers to a food with high medical or healthcare effects that is processed to efficiently exhibit a bioregulatory function in addition to providing nutrition, and can be manufactured in the form of tablets, capsules, pills, granules, powders, liquids, flakes, pastes, syrups, gels, jellies, bars, or films. Here, “functionality” means regulating nutrients for the structure and functions of the human body or obtaining a useful effect for health purposes such as physiological actions.

[0151] In the present invention, there is no particular limitation on the type of the health functional food. Specifically, examples of foods to which the composition of the present invention can be added include dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and particularly, all foods designed to sufficiently exert the body's regulatory functions, such as regulating the biological defense rhythm, disease prevention, and recovery, of the food group or food composition that has added value so that the function of the food in the conventional sense can be performed and expressed for a specific purpose.

[0152] In the present invention, “food additive” refers to a substance that is added to, mixed with, infiltrated into, or used in any other way in the manufacture, processing, or preservation of food, and must be harmless to the human body when consumed over a long period of time, such as a health functional food.

[0153] When the composition of the present invention is used as a food additive, the food additive may be added as is or used together with other foods or food ingredients, and may be appropriately used according to a conventional method.

[0154] The amount of active ingredients mixed can be appropriately determined depending on the intended use (prevention, health, or therapeutic treatment). Generally, when manufacturing a food or beverage, the composition of the present invention can be added in an amount of 15% by weight or less, or 10% by weight or less, based on the raw material. However, for long-term intake for health and hygiene purposes or health control purposes, the amount may be less than the above range, and since there are no safety issues, the active ingredients can also be used in amounts greater than the above range.

[0155] In the present invention, the composition may include various food additives that are food-related and acceptable, and may further include suitable carriers, excipients, and diluents commonly used in the manufacture of foods.

[0156] In addition to the above, the composition of the present invention may contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, 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. In addition, the composition of the present invention may contain fruit pulp for the production of natural fruit juice, fruit juice drinks, and vegetable drinks. These ingredients may be used independently or in combination. The ratio of these additives is not particularly important, but is generally selected in the range of 0.01-0.20 parts by weight per 100 parts by weight of the composition of the present invention, but is not limited thereto, and may be an optimal or arbitrary amount depending on the type and function of the product utilized.

[0157] In the present invention, there is no particular limitation on the type of the food. Examples of foods to which the substance can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and all health functional foods in the conventional sense can be included, but are not limited thereto.

[0158] In addition, the composition according to the present invention can be added to health drinks, and, like conventional beverages, can contain various flavoring agents or natural carbohydrates as additional ingredients. The natural carbohydrates mentioned above are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As sweeteners, natural sweeteners such as thaumatin and stevia extract, or synthetic sweeteners such as saccharin and aspartame can be used. The proportion of the above natural carbohydrates may be generally about 0.01-0.20 g, or about 0.04-0.10 g per 100 mL of the composition of the present invention, but is not limited thereto, and may be a general amount added in the art, or may include the maximum range to enhance the efficacy of the composition of the present invention, and may include an optimal, arbitrary amount considering the synergistic effect with other substances added together.

[0159]

[0160] The present invention provides a kit for preventing, improving, or treating non-alcoholic fatty liver disease, comprising an extract of Abelmoschus manihot and a description. Furthermore, the description may describe, but is not limited to, a method for preventing, improving, or treating non-alcoholic fatty liver disease.

[0161] In the present invention, a "kit" refers to a tool that can prevent or treat non-alcoholic fatty liver disease, including the composition of the present invention. In addition to the above-described substances, the kit of the present invention may also include other components, compositions, solutions, devices, etc. typically required for their storage and processing. Specifically, each component may be applied at least once without limitation, there is no restriction on the order in which each substance is applied, and the application of each substance may be performed simultaneously or in small increments.

[0162] In the present invention, the kit may include a container; instructions; and the like. The container may serve to package the substance, and may also serve to store and fix the substance. The material of the container may take the form of, for example, a bottle, a tub, a sachet, an envelope, a tube, an ampoule, and the like, and these may be formed partially or wholly from plastic, glass, paper, foil, wax, and the like. The container may be initially equipped with a completely or partially detachable stopper, which may be part of the container or may be attached to the container by mechanical, adhesive, or other means, and may also be equipped with a stopper for allowing access to the contents by means of a syringe needle. The kit may include an outer package, and the outer package may include instructions for the use of the components.

[0163] In the present invention, when the term "comprising" is used, it does not exclude other components unless specifically stated otherwise, but rather means that other components can be included. As used throughout the present invention, the terms "step of ~" or "step of ~" do not mean "step for ~."

[0164]

[0165] The above-described matters may be applied without limitation to the compositions, kits, and methods of the present invention, where applicable.

[0166]

[0167] Hereinafter, preferred examples are presented to aid in understanding the present invention. However, the following examples are provided solely to facilitate a better understanding of the present invention, and the scope of the present invention is not limited by the following examples.

[0168]

[0169] [Example]

[0170]

[0171] Example 1. Method for producing a gold extract

[0172] Dried goldenrod flowers were harvested in 2022, purchased from a local Korean market, and stored at room temperature before extraction.

[0173]

[0174] Example 1-1. Method for producing distilled extract of goldenrod flower

[0175] Dried Abelmoschus manihotaquatic flowers were finely chopped, accurately weighed (10 g), and added to 300 ml of distilled water. The mixture was boiled at 100°C for 2 hours to produce a distilled water extract. The distilled water extract was then cooled and concentrated at 60°C using a rotary evaporator. The extract was then filtered using a filter with a pore size of 8 μm (Whatman, No. 2) and freeze-dried in a freeze-dryer for 1 week to produce a distilled Abelmoschus manihotaquatic flower extract (FAA).

[0176]

[0177] Example 1-2. Method for producing ethanol extract of goldenrod flower

[0178] Dried Abelmoschus manioc flowers were finely chopped, 10 g was precisely weighed, and an ethanol extract was prepared using 300 ml of 60 v / v% ethanol at room temperature for 2 hours. The extract was then vacuum filtered three times. The extract was then cooled and evaporated and concentrated at 60°C. The extract was then filtered through a 0.5 mm pore size, then filtered once more through a 0.25 μm pore size, and freeze-dried in a freeze dryer for 7 days to prepare an ethanol extract of Abelmoschus manioc flowers (Flower of Abelmoschus manioc, EtOH extract, FAE).

[0179]

[0180] Example 2. Confirmation of antioxidant activity of gold leaf extract

[0181]

[0182] Fatty acid oxidation that occurs during fat breakdown causes protein damage, lipid peroxidation, and DNA damage, and induces intracellular oxidative stress, which induces inflammation in the body. Therefore, it is necessary to neutralize this through antioxidant effects.

[0183] Accordingly, it was confirmed through the following examples whether the extract of Geumhwagyu has an antioxidant effect.

[0184]

[0185] Example 2-1. Confirmation of DPPH radical scavenging activity of gold leaf extract

[0186] To confirm the antioxidant activity of the extracts prepared in Example 1, DPPH radical scavenging activity was measured according to the method disclosed in Blois, MS (1958). Antioxidant determinations by the use of a stable free radical. (Nature, 181(4617), 1199-1200.).

[0187] Specifically, DPPH (2,2-diphenyl-1-picrylhydrazyl) solution (1.5 x10 -4M, 100 μL) of each extract prepared in Example 1 was mixed or left unmixed and aged at room temperature for 30 minutes. After standing, the absorbance at a wavelength of 540 nm was recorded using an enzyme measuring device, and the scavenging activity was calculated as a percentage using the following formula.

[0188] [Official 1]

[0189]

[0190] Here A Control is the absorbance of the unmixed extract, and A Sample is the absorbance of the mixed extract.

[0191] As a result, as shown in Fig. 2a, it was confirmed that the DPPH radical scavenging activity increased in a concentration-dependent manner in each extract.

[0192]

[0193] Example 2-2. Confirmation of ABTS radical scavenging activity of gold leaf extract

[0194] To confirm the antioxidant activity of the extracts prepared in Example 1, ABTS radical scavenging activity was measured according to the method disclosed in Arnao, MB, Cano, A., & Acosta, M. (2001). The hydrophilic and lipophilic contribution to total antioxidant activity. (Food chemistry, 73(2), 239-244.).

[0195] The stock solution containing ABTS (2,2'-aziono-bis(3-ethylbenzthiazoline-6-sulphonic acid))-+ solution and potassium persulphate solution was diluted with pure ABTS-+ solution, and each extract prepared in Example 1 was mixed or left unmixed, and then aged for 2 hours, and the absorbance of each solution was recorded at a wavelength of 735 nm. The scavenging activity was calculated as a percentage using Equation 1 of Example 2-1.

[0196] As a result, as shown in Fig. 2b, it was confirmed that ABTS radical scavenging activity increased in a concentration-dependent manner in each extract.

[0197]

[0198] Example 2-3. Confirmation of the hydroxyl radical scavenging activity of the extract of Geumhwagyu

[0199] To confirm the antioxidant activity of the extracts prepared in Example 1, the hydroxyl radical scavenging activity was measured according to the method disclosed in Chung ShinKyo, CS, Osawa, T., & Kawakishi, S. (1997). Hydroxyl radical-scavenging effects of spices and scavengers from brown mustard (Brassica nigra).

[0200] Specifically, hydroxyl radicals were generated by the Fenton reaction in the presence of FeSO4. A reaction mixture containing 0.1 mL of FeSO4, 10 mM EDTA, and 10 mM 2-deoxyribose was mixed with 0.1 mL of each extract prepared in Example 1, and then 0.1 mM phosphate buffer (pH 7.4) was added to make a total volume of 0.9 mL. 0.1 mL of 10 mM H2O2 was then added to the mixture, and the mixture was aged at 37°C for 4 h. After aging, 0.5 mL of 2.8% trichloroacetic acid (TCA) and 1.0% thiobarbituric acid (TBA) were added to each mixture, and then each mixture was placed in a boiling water bath for 10 min. The absorbance was then measured at a wavelength of 532 nm. Hydroxyl radical scavenging activity was calculated as a percentage using formula 1.

[0201] As a result, as shown in Fig. 2c, it was confirmed that the hydroxyl radical scavenging activity increased in a concentration-dependent manner.

[0202]

[0203] Example 2-4. Confirmation of the metal chelating activity of the extract of Geumhwagyu

[0204] Fe by the extract prepared in Example 1 2+ The chelation reaction was measured according to the method disclosed in Carter, P. (1971). Spectrophotometric determination of serum iron at the submicrogram level with a new reagent (ferrozine). (Analytical biochemistry, 40(2), 450-458.).

[0205] Specifically, each extract prepared in Example 1 was mixed with a reaction mixture consisting of 5 μL of FeCl3, 2 mM 4H2O, and 130 μL of distilled water (DW), and then aged in a dark room for 5 minutes. Thereafter, 5 mM ferrozine was added, and the mixture was aged in a dark room for 10 minutes to induce a reaction. The absorbance was measured at 562 nm. The sample to which ethylenediaminetetraacetic acid (EDTA) was added was used as a control, and among these, the sample to which EDTA was added was used as a positive control, and the sample without EDTA was used as a negative control. The metal chelating activity was calculated as a percentage using Equation 1.

[0206] As a result, as shown in Fig. 2d, it was confirmed that activity increased in a concentration-dependent manner.

[0207]

[0208] In summary, the extract of the present invention suggests that it can prevent cell damage caused by oxidative stress by relieving oxidative stress that occurs during fat decomposition.

[0209]

[0210] Example 3. Confirmation of the effect of gold leaf extract on improving fatty liver in a cell model.

[0211]

[0212] Example 3-1. Cell culture method

[0213] To confirm the effect of the extract prepared in Example 1 on improving fatty liver, FL83B hepatocytes and 3T3-L1 preadipocytes were cultured as follows.

[0214] First, FL83B hepatocytes (American Type Culture Collection, USA) were cultured in F12K medium (Sigma-Aldrich, St. Louis, USA) containing 10% fetal bovine serum, 1% penicillin, and 1% streptomycin (Sigma-Aldrich). Depending on the experimental purpose, they were seeded in 100 nm dishes, 12-well plates, and 6-well plates, and cultured in an incubator maintained at 37°C in a humidified 5% CO2 atmosphere.

[0215] Then, to induce lipid accumulation, oleic acid (OA, Sigma-Aldrich, St. Louis, USA) dissolved in 100% ethanol and 2% (w / v) bovine serum albumin were diluted in the F12K medium and cultured for 48 hours. Then, to confirm the effect of the extracts prepared in Example 1, the extracts prepared in Example 1 were dissolved in distilled water, filtered through a 2 μm pinhole filter, and then treated in the F12K medium at various concentrations (25, 50, 100, and 200 μg / mL).

[0216] 3T3-L1 preadipocytes obtained from the Korean Cell Line Bank (Seoul, Korea) were cultured in Dulbecco's Modified Eagle Medium (DMEM) medium containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (P / S) at 37°C. The cells were plated in 100 μm dishes, 12-well plates, and 6-well plates depending on the experimental purpose and cultured for 2 days. To induce differentiation into adipocytes, the medium was changed to DMEM medium supplemented with 10% fetal bovine serum (FBS), 0.5 mM isobutylmethylxanthine, 0.5 mM dexamethasone, and 5 μg / mL insulin, and differentiation was induced by treating with various concentrations (0, 25, 50, 100, and 200 μg / mL) of the extract prepared in Example 1. Forty-eight hours after differentiation induction, the medium was replaced with DMEM medium supplemented with 10% FBS and 5 g / mL insulin, and the extract prepared in Example 1 was treated at various concentrations (0, 25, 50, 100, and 200 μg / mL). Thereafter, the medium was replaced with DMEM medium containing 10% FBS every other day until day 8, when the preadipocytes acquired intracellular lipid droplets.

[0217] In addition, to confirm the effect of the extract prepared in Example 1 on differentiation into adipocytes, it was treated at various concentrations (0, 25, 50, 100, and 200 μg / mL) at every medium replacement time (every other day).

[0218]

[0219] Example 3-2. Confirmation of cytotoxicity measurement results of gold leaf extract

[0220] To confirm the cytotoxicity of the extract prepared in Example 1, the cytotoxicity of the extract was measured using the MTT assay (Sigma-Aldrich, USA).

[0221] Specifically, on the first day of the experiment, the cultured cells were seeded into 96-well plates at a density of 1,000 cells per well. On the second day, the cells were treated with different concentrations (0-2,000 μg / mL) of the extract (FAA or FAE) and cultured for 24 hours. Afterwards, 5 mg / mL MTT solution was additionally treated and cultured at 37°C for another 4 hours. Afterwards, insoluble methylation products were dissolved using DMSO (Dimethyl Sulfoxide), and the absorbance was measured at a wavelength of 540 nm using a microplate reader to analyze cell viability for cytotoxicity induced by the extract.

[0222] As a result, as shown in Figs. 3a and 3b, it was confirmed that the cell viability was maintained at approximately 80% or higher even when the concentration of FAA or FAE increased in FL83B hepatocytes (Fig. 3a) and 3T3-L1 preadipocytes (Fig. 3b).

[0223] Through this, it was confirmed that the extract of the present invention does not exhibit significant toxicity in cell survival.

[0224]

[0225] Example 3-3. Confirmation of the lipid accumulation and fat differentiation inhibition effects of the extract of Geumhwagyu

[0226] The effect of the extract prepared in Example 1 on lipid accumulation and adipocyte differentiation was confirmed through ORO (Oil Red O) staining.

[0227] Specifically, FL83B hepatocytes and 3T3-L1 preadipocytes were seeded in 12-well plates, and lipid accumulation and differentiation into adipocytes were induced according to the method disclosed in Example 3-1. Thereafter, the induced cells were washed with PBS, fixed with 10% buffered formalin, and stained with Oil Red O solution (0.7 g in 200 ml isopropanol) for 30 minutes at room temperature. After the lipid droplets were stained red, the Oil Red O solution was removed, washed with distilled water, and then dried.

[0228] Images of intracellular stained lipid droplets were obtained using a binocular microscope (DMi1; Leica Microsystems, Germany). The remaining dye was then eluted with isopropanol and quantified by measuring the absorbance at a wavelength of 520 nm using a microplate reader.

[0229]

[0230] Figures 3c and 3d show the effect of the extract (FAA, FAE) of the present invention on FL38B hepatocytes in which lipids are accumulated as oleic acid (OA), as confirmed through ORO staining, and it was confirmed that lipid accumulation was reduced in a concentration-dependent manner.

[0231] In addition, FIGS. 3e and 3f show that the effect of the extract of the present invention on adipocyte differentiation of 3T3-L1 preadipocytes was confirmed through ORO staining, and it was confirmed that adipocyte differentiation decreased in a concentration-dependent manner.

[0232] Through this, it was confirmed that the extract of the present invention can exert significant lipid accumulation inhibition and adipocyte differentiation inhibition effects.

[0233]

[0234] Example 3-4. Confirmation of the lipid metabolism inhibitory effect of the extract of Geumhwagyu

[0235] It was confirmed whether the extract (FAE) prepared in Example 1 regulates the expression of genes related to lipid metabolism in 3T3-L1 preadipocytes.

[0236] Specifically, total cellular RNA was isolated using TRIzol according to the manufacturer's protocol, followed by reverse transcription-polymerase chain reaction (RT-PCR). The first cDNA (complementary DNA) was synthesized using Superscript II reverse transcriptase (Invitrogen).

[0237] Quantitative real-time PCR was performed using a Thermal Cycler Dice TP850 (Takarabio Inc., Japan) according to the manufacturer's protocol. PCR conditions included mixing 2 μL of cDNA (100 ng), 1 μL of sense and antisense primer solutions (0.4 μM), 12.5 μL of SYBR Premix Ex Taq (Takarabio Inc.), and 9.5 μL of distilled water in each reaction tube to obtain a final reaction mixture of 25 μL. The PCR primers used for gene expression analysis are listed in Table 1. Amplification was performed under the following conditions: 95°C for 10 s; 40 cycles of 95°C for 5 s and 60°C for 30 s; and 40 cycles of 95°C for 15 s, 60°C for 30 s, and 95°C for 15 s. The mRNA levels of target genes compared to GAPDH were normalized as follows: relative mRNA expression = 2-(ΔCt of target gene - ΔCt of GAPDH), where Ct is the threshold cycle value. The expression levels of the genes analyzed in each sample were normalized to the expression levels of GAPDH and expressed as relative mRNA levels.

[0238] As shown in Fig. 3g, it was confirmed that in differentiated 3T3-L1 preadipocytes, adipogenic genes (FAS, PPARγ, C / EBPα, SREBP-1c, ACC1) increased and adipogenic genes (PGC1α) decreased. When treated with the extract of the present invention, it was confirmed that the expression of adipogenic genes decreased in a concentration-dependent manner and the expression of adipogenic genes increased.

[0239]

[0240] Through this, it was confirmed that the extract of the present invention inhibits lipid metabolism by affecting the expression of genes related to lipid metabolism.

[0241] Gene nameSequenceSequence numberFASForward5-CTT GGG TGC TGA CTA CAA CC-3Sequence number 1Reverse5-GCC CTC CCG TAC ACT CAC TC-3Sequence number 2SREBP-1cForward5-CTT CTG GAG ACA TCG CAA AC-3Sequence number 3Reverse5-GGT AGA CAA CAG CCG CAT C-3Sequence number 4C / EBPαForward5-CGT CTA AGA TGA GGG AGT C-3Sequence number 5Reverse5-GGC ACA AGG TTA CTT CCT-3Sequence number 6PPARγForward5-GAA AGA CAA CGG ACA AAT CAC-3Sequence number 7Reverse5-GAA ACT GGC ACC CTT GAA-3Sequence number 8HSLForward5-GAA AGA CAA CGG ACA AAT CAC-3Sequence number 9Reverse5-GAA ACT GGC ACC CTT GAA-3SEQ ID NO: 10ACC1Forward5-GGG CTA CCT CTA ATG GTC TT-3SEQ ID NO: 11Reverse5-CTA CCT GAT GGT AAA TGG GA-3SEQ ID NO: 12PGC-1αForward5-ATT CGG GAG CTG GAT GGC TT-3SEQ ID NO: 13Reverse5-CCG ATT GGT CGC TAC ACC AC-3SEQ ID NO: 14CPT-1Forward5-TGT GTG AGG ATG CTG CTT CC-3SEQ ID NO: 15Reverse5-CTC GGA GAG CTA AGC TTG TC -3SEQ ID NO: 16GAPDHForward5-GCA CAG TCA AGG CCG AGA AT-3SEQ ID NO: 17Reverse5-GCC TTC TCC ATG GTG GTG AA-3SEQ ID NO: 18

[0242] Gene nameSequenceSequence numberSCD1Forward5-CGG AAA TGA ACG AGA GAA GG-3Sequence number 19Reverse5-CCG AAG AGG CAG GTG TAG AG-3Sequence number 20ATGLForward5-GAC CTG ATG ACC ACC CTT TCC-3Sequence number 21Reverse5-TGC TAC CCG TCT GCT CTT TCA-3Sequence number 22ACOXForward5-GCA CCA TTG CCA TTC GAT ACA-3Sequence number 23Reverse5-CCA CTG CTG TGA GAA TAG CCG-3Sequence number 24Nrf2Forward5-AGC ACA TCC AGA CAG ACA CCA GT-3Sequence number 25Reverse5-TTC AGC GTG GCT GGG GAT AT-3Sequence number 26HO-1Forward5-ACA GGG TGA CAG AAG AGG CTA AGA C-3 SEQ ID NO: 27 Reverse 5-ATT TTC CTC GGG GCG TCT CT-3 SEQ ID NO: 28 GPxForward 5-ACA TTC CCA GTC ATT CTA CC-3 SEQ ID NO: 29 Reverse 5-TTC AAG CAG GCA GAT ACG-3 SEQ ID NO: 30 CAT Forward 5-GAA CGA GGA GGA GAG GAA AC-3 SEQ ID NO: 31 Reverse 5-TGA AAT TCT TGA CCG CTT TC-3 SEQ ID NO: 32

[0243] Example 4. Confirmation of the effect of Geumhwagyu extract on improving fatty liver in an animal model.

[0244]

[0245] Example 4-1. Method for producing a non-alcoholic fatty liver disease animal model

[0246] All animal experiments were approved by the Animal Care and Use Committee of Dong-A University (Approval Number: DIACUC-23-59). Four-week-old male C57BL / 6 mice were purchased from the Nara Bio Animal Center (Nara Biotech, Republic of Korea) and housed under specific pathogen-free conditions. They were housed in groups of four in transparent plastic cages covered with poplar wood chips and provided standard mouse chow and tap water when not being tested. The environment in the cage was controlled with a 12-h light cycle, a temperature of 20–21°C, and a relative humidity of 40–45%.

[0247] After a 7-day acclimation period in the experimental facility, the mice were randomly divided into two groups under the following conditions to conduct an experiment to determine the effect of the extract (FAE) of the present invention on fatty liver: a control group (CON) of 8 mice fed a normal diet containing 10% kcal; a high-fat diet (HFD) group of 32 mice fed a high-fat diet containing 60% kcal. Food intake was recorded every two days, and body weight was measured weekly.

[0248] After 8 weeks, mice that gained more than 20% of their body weight compared to the control group were selected and divided into the following four groups (n=8): high-fat diet group (HFD); low-dose FAE group (FAE_L) fed a high-fat diet with 50 mg / kg FAE; high-dose FAE group (FAE_H) fed a high-fat diet with 100 mg / kg FAE; and EGCG group fed a high-fat diet with 30 mg / kg epigallocatechin-gallate (EGCG). The administration was performed for 12 weeks, and EGCG was used as a positive control (Fig. 4a).

[0249]

[0250] Example 4-2. Confirmation of the pathological improvement effect of the extract of Geumhwagyu

[0251] After 12 weeks of administration, mice were fasted overnight and sacrificed to obtain adipose tissue (epididymis and liver). The tissues were then fixed in 10% formalin at room temperature for 48 hours and embedded in paraffin.

[0252] Liver tissue was rinsed in running water for 24 hours, then immersed in alcohol of concentrations of 70%, 80%, 90%, 95%, and 100% (twice) and dehydrated for 30 minutes. Then, the liver tissue was immersed in xylene I and xylene II solutions for 30 minutes each to make it transparent. After that, the liver tissue was immersed in paraffin I, paraffin II, and paraffin III for 2 hours each at 60℃ to form paraffin blocks.

[0253] After paraffin embedding, paraffin blocks were cut into 4-μm-thick sections, deparaffinized, and stained with hematoxylin and eosin (H&E). Random sections were then examined under a light microscope at 200× magnification to examine hepatocyte degeneration (edema, steatosis) and necrosis. Epididymal tissue was assessed for adipocyte size macroscopically using a microscope.

[0254] As shown in the upper part of Fig. 4b, histological changes were observed in the liver of mice fed a high-fat diet, and when treated with the extract of the present invention, it was confirmed that the liver showed a similar morphology to the normal control group.

[0255] In addition, as shown in Fig. 4b, H&E staining confirmed that lipid accumulation was reduced in a concentration-dependent manner in the liver tissue of the FAE administration group.

[0256] In addition, as shown in the lower part of Figure 4b, it was confirmed that the size of fat cells in the FAE-administered group was reduced compared to the control group in the epididymal fat tissue.

[0257]

[0258] Through this, it was confirmed that the extract of the present invention inhibits degeneration and necrosis of hepatocytes, significantly inhibits lipid accumulation in the liver, and inhibits an increase in the size of fat cells, suggesting that it can improve liver lesions induced by a high-fat diet.

[0259]

[0260] Example 4-3. Confirmation of the weight loss effect of the extract of Geumhwagyu

[0261] Body weight changes in a non-alcoholic fatty liver disease animal model were observed over a 12-week administration period.

[0262] As shown in Figure 4c, it was confirmed that from the 8th week onwards, the body weight decreased significantly in the FAE administration group, and the weight gain also decreased significantly.

[0263] In addition, in order to confirm whether the extract of the present invention suppresses weight gain in organ tissues, after 12 weeks of administration, mice were fasted overnight and sacrificed to obtain liver, epididymal adipose tissue (EAT), subcutaneous adipose tissue (SAT), and visceral adipose tissue (VAT), and their weights were measured.

[0264] As shown in Fig. 4d, it was confirmed that the weight of fat tissue in each organ increased in the HFD group, and it was confirmed that the weight significantly decreased in the FAE administration group.

[0265]

[0266] Through this, it was found that the extract of the present invention can suppress weight gain and significantly suppress fat accumulation in the body.

[0267]

[0268] Example 4-4. Confirmation of the inhibitory effect of Geumhwagyu extract on abdominal fat accumulation.

[0269] To confirm whether the extract of the present invention inhibits fat accumulation induced by a high-fat diet, abdominal CT scans were performed on mice.

[0270] Specifically, after anesthetizing the mouse model at 4 weeks of administration, a CT scan of the abdominal cross-section was performed using SKYSCAN 1076 in vivo micro-CT (BRUKER, Belgium), through which superficial subcutaneous fat, deep subcutaneous fat, and visceral fat tissue were identified.

[0271] As shown in Fig. 4e, it can be confirmed that the superficial subcutaneous fat (green area), deep subcutaneous fat (red area), and visceral fat tissue (yellow area) increased in the HFD group compared to the CON group, and it can be confirmed that the above fat tissues significantly decreased in the FAE administration group.

[0272] In addition, the areas of superficial subcutaneous fat (SSAT), deep subcutaneous fat (DSAT), and visceral adipose tissue (VAT) were shown in a diagram in Figure 4f, and the areas of these tissues (mm) in the FAE administration group 2 ) was confirmed to have significantly decreased.

[0273]

[0274] Through this, it can be confirmed that the extract of the present invention significantly inhibits abdominal fat accumulation, including liver fat accumulation.

[0275]

[0276] Example 4-5. Analysis of serum metabolic indicators through biochemical analysis

[0277] Blood samples were collected by cardiac puncture, and serum was centrifuged (3,000 rpm, 20 min) and stored at -80°C. Serum metabolic markers were analyzed using the following assay kits and measured using a microplate reader. The markers were as follows: Alanine aminotransferase (ALT) (Abcam, No. ab282882); Aspartate aminotransferase (AST) (Abcam, No. ab263882).

[0278] Additionally, the following indicators were analyzed using an ELISA kit and measured with a microplate reader: adiponectin (adiponectin, Abcam, No: ab108785).

[0279] Here, AST and ALT are enzymes present in liver cells. When liver cells are destroyed, they leak into the blood, causing fluctuations in blood levels. Therefore, they are representative indicators of liver disease. (Med Sci Sports Exerc. 2023 Apr 1;55(4):670-679.)

[0280] Adiponectin is a regulatory peptide secreted from adipose tissue. It is known to promote mitochondrial β-oxidation and affect appetite control. Its concentration is also known to decrease in obesity.

[0281] As shown in Figure 4g, in the HFD administration group, the concentration of adiponectin decreased, while the concentrations of AST and ALT increased. On the other hand, in the FAE administration group, the concentration of adiponectin increased in a dose-dependent manner, while the concentrations of AST and ALT decreased.

[0282]

[0283] Through this, it can be confirmed that the extract of the present invention has a positive effect on improving non-alcoholic fatty liver disease.

[0284]

[0285] Example 5. Confirmation of the effect of the extract of Geumhwagyu on altering gene expression in an animal model.

[0286]

[0287] Example 5-1. Confirmation of the effect of gold leaf extract on altering the expression of genes related to lipid metabolism.

[0288] Changes in gene expression of lipid metabolism-related biomarkers in a non-alcoholic fatty liver disease animal model were identified using the quantitative real-time PCR method described in Examples 3-4. The PCR primers used for gene expression analysis are listed in Tables 1 and 2 above.

[0289] In the HFD group, the gene expression of SREBP-1c, ACC1, FAS, PPARγ, SCD1, and C / EBPα, which are biomarkers related to adipogenesis or lipogenesis, was confirmed to be upregulated (Fig. 5a). In contrast, the gene expression of biomarkers related to lipolysis (HSL, ATGL, and PGC-1α) and biomarkers related to lipid oxidation (CPT-1 and ACOX) was confirmed to be downregulated (Fig. 5b).

[0290] When FAE was administered, it was confirmed that the gene expression of biomarkers related to adipocyte production and fat synthesis (biomarkers related to adipogenesis) was down-regulated, and the gene expression of biomarkers related to lipolysis and lipid oxidation (biomarkers related to lipolysis) was up-regulated.

[0291]

[0292] Through this, it was confirmed that the extract of the present invention affects the gene expression of biomarkers related to lipid metabolism in vivo, thereby inducing an effect of inhibiting fat accumulation.

[0293]

[0294] Example 5-2. Confirmation of the effect of the extract of Geumhwagyu on altering the gene expression of oxidative stress-related biomarkers.

[0295] Changes in gene expression of lipid metabolism-related biomarkers in a non-alcoholic fatty liver disease animal model were identified using the quantitative real-time PCR method described in Examples 3-4. The PCR primers used for gene expression analysis are listed in Table 2 above.

[0296] In the HFD group, it was confirmed that the gene expression of oxidative stress-related biomarkers Nrf2, HO-1, GPx, and CAT was downregulated (Fig. 5c).

[0297] When FAE was administered, it was confirmed that the gene expression of oxidative stress-related biomarkers was upregulated.

[0298]

[0299] Through this, it was confirmed that the extract of the present invention upregulates the gene expression of oxidative stress-related biomarkers in vivo, thereby relieving oxidative stress generated during fat decomposition and preventing cell damage caused by oxidative stress.

[0300]

[0301] Example 6. Confirmation of the effect of the extract of Geumhwagyu on changing the protein expression of biomarkers in an animal model.

[0302]

[0303] Example 6-1. Immunohistochemical staining method

[0304] Tissues excised from animals were washed with chilled saline, and then a portion of each tissue was fixed in a 10% formalin solution. The fixed tissues were embedded in paraffin and then sectioned at 3 to 4 μm thickness.

[0305] The sectioned tissues were immersed in 0.01 M citrate buffer (pH 6.0) for antigen retrieval, microwaved for 10 minutes, and then left at room temperature for 10 minutes. They were then washed with distilled water. The washed sections were treated with 3% hydrogen peroxide (H₂O₂) for 5 minutes, and then treated with 10% normal goat serum to block nonspecific binding between antigen and antibody.

[0306] Afterwards, primary antibodies specific to the proteins associated with each section (diluted 1:300) were dispensed and incubated overnight at 4°C. After incubation, each section was washed three times with PBS for 5 minutes each. Subsequently, fluorescently labeled secondary antibodies (diluted 1:1,000) were dispensed and incubated at room temperature for 1 hour. The antibodies used are listed in Tables 3 and 4 below.

[0307] Afterwards, the tissues were stained with hematoxylin and sealed, and the expression patterns of related proteins in the tissues were observed using a Leica DMi1 microscope (Leica, Wetzlar, Germany).

[0308]

[0309] Example 6-2. Confirmation of the effect of the extract of Geumhwagyu on changing the protein expression of biomarkers related to lipid metabolism.

[0310] Changes in protein expression of biomarkers related to lipid metabolism in a non-alcoholic fatty liver animal model were confirmed according to the immunohistochemical staining method described in Example 6-1.

[0311] In mice fed a high-fat diet, it was confirmed that the levels of fatty acid synthase (FAS) and sterol regulatory factor binding protein-1c (SREBP-1c) liver protein expression increased, but in the FAE administration group, it was confirmed that the levels of fatty acid synthase (FAS) and sterol regulatory factor binding protein-1c (SREBP-1c) liver protein expression decreased in a dose-dependent manner (Fig. 6a).

[0312]

[0313] Through this, it was confirmed that the extract of the present invention affects the protein expression of biomarkers related to lipid metabolism in vivo, thereby inducing an effect of inhibiting fat production.

[0314]

[0315] Example 6-3. Western blot analysis method

[0316] Liver tissue was lysed in RIPA buffer containing a protein inhibitor cocktail (Sigma). Protein concentration of the lysed tissue was measured using a BCA assay. The measured proteins were loaded onto an SDS polyacrylamide gel and electrophoresis was performed. The separated proteins were then transferred to membranes using a Mini Trans-Blot® Cell (Bio-Rad).

[0317] The transferred membrane was blocked for 1 hour with a 5% skim milk solution, then treated with primary antibodies and incubated overnight at 4°C. After incubation, the membrane was washed three times for 5 minutes with TBST (TBS + 0.1% Tween-20). Subsequently, the membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 hour at room temperature, and then washed three times for 5 minutes with TBST. The antibodies used are listed in Tables 3 and 4 below.

[0318] Afterwards, color development was performed using HRP substrate (Advansta Inc., San Jose, CA, USA), and the expressed band was detected using Azure c300 imaging system (Azure Biosystems).

[0319] Protein band intensities were analyzed using ImageJ (NIH ver. 1.48, USA) software. Band areas were determined for each sample and quantified by normalization based on the area of ​​the housekeeping gene, GAPDH.

[0320] Anti bodyCompanyCat. #, (Secondary Antibody)FASSanta Cruzsc-55580, (Mouse)SREBP-1cSanta Cruzsc-13551, (Mouse)p-ACCCell Signaling11818S, (Rabbit)ACCCell Signaling3676S, (Rabbit)p-AMPKCell Signaling2535S, (Rabbit)AMPKSanta Cruzsc-25792, (Rabbit)GAPDHSanta Cruzsc-365062, (Mouse)PPAR-γCell Signaling2435S, (Rabbit)C / EBPαCell Signaling8178S, (Rabbit)p-HSLCell Signaling4137S, (Rabbit)HSLCell Signaling4107S, (Rabbit)HO-1Santa Cruzsc-136960, (Mouse)Nrf-2Santa Cruzsc-365949, (Mouse)

[0321] Anti bodyCompanyCat. #Rabbit lgG, HRPBio ActsRSA1221Mouse lgG, HRPBio ActsRSA1122

[0322] Example 6-4. Confirmation of the effect of the extract of Geumhwagyu on altering the expression of proteins related to lipid metabolism.

[0323] Changes in the expression of proteins related to lipid metabolism in a non-alcoholic fatty liver disease animal model were confirmed according to the Western blot analysis method described in Example 6-3 above.

[0324] In mice fed a high-fat diet, it was confirmed that the levels of p-ACC / ACC and p-AMPK / AMPK protein expression decreased, but in the FAE administration group, it was confirmed that the levels of p-ACC / ACC and p-AMPK / AMPK protein expression increased in a concentration-dependent manner (Fig. 6b).

[0325]

[0326] In addition, we confirmed that the expression of SREBP-1c, FAS, PPARγ, and C / EBPα, which are proteins related to adipogenesis or lipogenesis, increased in the HFD group. When FAE was administered, we confirmed that the expression of proteins related to adipogenesis and lipogenesis (biomarkers related to lipogenesis) was downregulated (Fig. 6c). On the other hand, we confirmed that the expression of HSL, a protein related to lipolysis, was decreased in the HFD group. When FAE was administered, we confirmed that the expression of proteins related to lipolysis was upregulated (Fig. 6d).

[0327]

[0328] Through this, it was confirmed that the extract of the present invention affects the protein expression of lipid metabolism-related biomarkers in vivo, thereby inducing a fat accumulation inhibitory effect.

[0329]

[0330] Example 6-5. Confirmation of the effect of the extract of Geumhwagyu on changing the protein expression of oxidative stress-related biomarkers.

[0331] Changes in protein expression of biomarkers related to oxidative stress in a non-alcoholic fatty liver disease animal model were confirmed according to the Western blot analysis method described in Example 6-3 above.

[0332] In the HFD group, we observed a decrease in the protein expression of Nrf2 and HO-1, which are biomarkers related to oxidative stress. When FAE was administered, the protein expression of biomarkers related to oxidative stress was confirmed to be upregulated (Fig. 6e).

[0333]

[0334] Through this, it was confirmed that the extract of the present invention upregulates the protein expression of oxidative stress-related biomarkers in vivo, thereby relieving oxidative stress occurring during fat decomposition and preventing cell damage caused by oxidative stress.

[0335]

[0336] The statistical evaluation of the data presented in the above examples is expressed as the mean standard deviation, and the data were analyzed by one-way ANOVA with Tukey's test using the GraphPad Prism program (Ver 8.4; GraphPad Software, Inc., USA), and p value<0.05 was considered significant.

[0337]

[0338] The foregoing description of the present invention is provided for illustrative purposes only. Those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

[0339]

[0340] The pharmaceutical composition for preventing or treating non-alcoholic fatty liver disease, comprising an extract of Abelmoschus manihot as an active ingredient, is a natural product, non-toxic, and has excellent antioxidant activity. Furthermore, it effectively suppresses lipid accumulation in the liver by exhibiting lipid accumulation reduction, fat cell size reduction, and fat cell differentiation inhibition activities, making it a useful therapeutic agent for non-alcoholic fatty liver disease, and thus possesses industrial applicability.

Claims

1. A pharmaceutical composition for preventing or treating non-alcoholic fatty liver disease, comprising an extract of Abelmoschus manihot as an active ingredient.

2. In paragraph 1, A pharmaceutical composition, wherein the above non-alcoholic fatty liver disease is high-fat diet-induced non-alcoholic fatty liver disease.

3. In paragraph 1, A pharmaceutical composition wherein the above-mentioned extract of Geumhwagyu is extracted from a part including at least one selected from the group consisting of stems, roots, leaves, fruits, and flowers.

4. In paragraph 1, A pharmaceutical composition, wherein the extract is extracted with any one selected from the group consisting of water, alcohol having 1 to 6 carbon atoms, acetone, ether, benzene, chloroform, ethyl acetate, methylene chloride, hexane, cyclohexane, petroleum ether, dichloromethane, subcritical fluid, and supercritical fluid.

5. In paragraph 1, The above composition is a pharmaceutical composition characterized by at least one selected from the group consisting of: (a) Reduction of lipid accumulation in the liver; (b) reduction in the size or weight of fat cells within the liver; (c) Inhibition of intrahepatic adipocyte differentiation; (d) Reduction of oxidative stress occurring during lipolysis through antioxidant activity in the liver; (e) Inhibition of lipid metabolism in the liver; (f) inhibition of liver cell degeneration or necrosis; and (g) Inhibition of weight gain.

6. In paragraph 1, The above composition is a pharmaceutical composition characterized by at least one selected from the group consisting of: (a) inhibiting the expression or activity of intrahepatic lipogenic genes or proteins; and (b) Promoting the expression or activity of intrahepatic fat-decomposing genes or proteins.

7. In paragraph 1, A pharmaceutical composition characterized in that the composition promotes the expression or activity of an antioxidant gene or protein.

8. In paragraph 1, A pharmaceutical composition characterized in that the composition increases the expression of adiponectin.

9. A food composition for preventing or improving non-alcoholic fatty liver disease, comprising an extract of Abelmoschus manihot as an active ingredient.

10. In paragraph 9, A food composition wherein the above food is a health functional food.

11. A kit for preventing, improving, or treating non-alcoholic fatty liver disease, comprising an extract of Abelmoschus manihot and an instruction manual.

12. A method for improving or treating non-alcoholic fatty liver disease, comprising administering to a subject in need thereof a pharmaceutically effective amount of an extract of Abelmoschus manihot or a composition containing the extract as an active ingredient.

13. Use of Abelmoschus manihot extract or a composition containing it as an active ingredient for preventing, improving, or treating non-alcoholic fatty liver disease.

14. Use of an extract of Abelmoschus manihot or a composition containing the same as an active ingredient to manufacture a preparation for preventing, improving, or treating non-alcoholic fatty liver disease.

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