Composition for preventing, alleviating or treating fatty liver, comprising extract of cucumis melo var. makuwa leaves

Abstract

The present invention relates to the preventive, alleviative, or therapeutic effects of an extract of Cucumis melo var. makuwa leaves, particularly an alcoholic extract of Cucumis melo var. makuwa leaves, on fatty liver disease associated with metabolic dysfunction. The extract of Cucumis melo var. makuwa leaves according to the present invention can reduce liver weight and the accumulation of fat in liver tissue, and can significantly inhibit ALT activity in the blood, and thus can provide an effective fatty liver prevention, alleviation, or treatment effect. In addition, the extract of Cucumis melo var. makuwa leaves according to the present invention has a high yield of about 21% and thus may be highly economical as an excellent material for preventing, alleviating or treating fatty liver disease.

Description

Composition for the prevention, improvement, or treatment of fatty liver containing Korean melon leaf extract

[0001] This application claims priority to Korean Patent Application No. 10-2024-0141226 filed on October 16, 2024, and the entire specification is a reference to this application.

[0002]

[0003] The present invention relates to the preventive, improving, or therapeutic effects of a Korean melon leaf extract, particularly an alcoholic extract of Korean melon leaves, on metabolic disorders of fatty liver.

[0004]

[0005] The present invention was completed with the support of the Ministry of Science and ICT of the Republic of Korea by Project No. RS-2023-00274576 (2710003021) and Project No. 2021R1A6A1A03039211 (RS-2021-NR060118, 2340006719).

[0006]

[0007] The Korean melon (*Cucumis melo var. makuwa*) is a plant belonging to the Cucurbitaceae family. Its origin is believed to be India, and depending on the region of its spread, it developed into Asian varieties and Western varieties. Currently, it is mainly cultivated in Korea, China, Japan, and other regions. It is estimated that Korean melons have been cultivated in Korea since before the Silla Dynasty, and various native varieties were grown in different regions. As a fruit and vegetable with a sweet taste and unique aroma, its refreshing flavor suits Korean palates, and it has been widely consumed as a summer fruit since ancient times.

[0008] However, the leaves generated after harvesting Korean melons at farms are difficult to dispose of, and disposal incurs costs. Therefore, research is needed to utilize the leaves produced during the cultivation process as a high-value-added material.

[0009] Metabolic dysfunction-associated steatotic liver disease (MASLD) is a type of disease that exhibits histological findings similar to alcoholic hepatitis despite being unrelated to alcohol consumption. It is a type of metabolic syndrome that encompasses metabolic dysfunction-associated steatotic liver, metabolic dysfunction-associated steatohepatitis (MASH), cirrhosis, and hepatocellular carcinomas. Metabolic dysfunction is on the rise alongside the increase in the obese and diabetic populations, and the annual incidence rate in Korea reaches approximately 16%.

[0010] There is currently no established treatment for metabolic fatty liver disease, as the condition is associated with various factors. Consequently, there is a continuous demand for research on materials derived from natural products—rather than synthetic compounds—that can provide preventive, mitigating, or therapeutic effects for metabolic fatty liver disease through non-invasive methods.

[0011]

[0012] [Prior Art Literature]

[0013] [Patent Literature]

[0014] (Patent Document 1) KR 10-2013-0125282 (2013-10-21)

[0015]

[0016] The inventors of the present invention have made diligent efforts to provide a material among natural materials that can provide excellent effects in preventing, improving, or treating metabolic disorders of fatty liver disease, and have confirmed that an extract of Korean melon leaves can provide excellent effects in preventing, improving, or treating metabolic disorders of fatty liver disease, such as reducing liver weight, liver fat, ALT, and AST levels, and have completed the present invention.

[0017] Therefore, the objective of the present invention is to provide the effect of preventing, improving, or treating metabolic disorders of fatty liver disease using a Korean melon leaf extract, particularly an alcoholic extract of Korean melon leaves.

[0018]

[0019] The present invention provides a pharmaceutical composition for the prevention or treatment of fatty liver disease comprising a leaf extract of Korean melon (Cucumis melo var. makuwa).

[0020] According to a preferred embodiment of the present invention, the Korean melon leaf extract is,

[0021] i) Step of extracting Korean melon leaf powder;

[0022] ii) a step of separating the extract and the residue after the above extraction and re-extracting the residue; and

[0023] iii) A step of volatilizing the solvent after the above re-extraction;

[0024] It was manufactured through.

[0025] According to a preferred embodiment of the present invention, the extract is an alcohol extract of Korean melon leaves.

[0026] According to a preferred embodiment of the present invention, the alcohol is 1 to 100% ethanol.

[0027] According to a preferred embodiment of the present invention, the fatty liver disease is a metabolic dysfunction-associated steatotic liver disease.

[0028] In addition, the present invention provides a health functional food composition for preventing or improving fatty liver disease comprising a leaf extract of Korean melon (Cucumis melo var. makuwa).

[0029] According to a preferred embodiment of the present invention, the fatty liver disease is a metabolic dysfunction-associated steatotic liver disease.

[0030] In addition, the present invention provides a composition for improving liver function comprising a leaf extract of Korean melon (Cucumis melo var. makuwa).

[0031] In addition, the present invention comprises: i) a step of extracting Korean melon (Cucumis melo var. makuwa) leaf powder;

[0032] ii) a step of separating the extract and the residue after the above extraction and re-extracting the residue; and

[0033] iii) A step of volatilizing the solvent after the above re-extraction;

[0034] A method for preparing a composition for preventing, improving, or treating fatty liver is provided, comprising a Korean melon (Cucumis melo var. makuwa) leaf extract.

[0035] In addition, the present invention provides a method for treating fatty liver comprising the step of administering a Korean melon (Cucumis melo var. makuwa) leaf extract to an individual in need.

[0036] According to a preferred embodiment of the present invention, the extract is an alcohol extract of Korean melon leaves.

[0037] According to a preferred embodiment of the present invention, the alcohol is 1 to 100% ethanol.

[0038] According to a preferred embodiment of the present invention, the fatty liver disease is a metabolic dysfunction-associated steatotic liver disease.

[0039]

[0040] The present invention will be described in more detail below.

[0041]

[0042] The term "prevention" in this invention refers to any act that causes fatty liver or fatty liver-related diseases to be suppressed or their onset to be delayed due to the Korean melon leaf ethanol extract of this invention.

[0043] The term "improvement" or "treatment" of the present invention refers to any act that causes parameters related to fatty liver or fatty liver-related diseases, such as the severity of symptoms, to improve or become beneficial due to the Korean melon leaf ethanol extract of the present invention.

[0044]

[0045] The present invention relates to the preventive, improving, or therapeutic effects of an ethanol extract of Korean melon leaves on metabolic disorders of fatty liver, or to the liver health-promoting effects. The inventors investigated the effects of the ethanol extract of Korean melon leaves on fatty liver and liver health-related indicators in C57BL / 6J mice in which obesity was induced by a high-fat diet.

[0046] The Korean melon leaf ethanol extract of the present invention significantly reduced liver weight in the Korean melon leaf ethanol extract group (0.8%) compared to the high-fat diet group and also reduced fat accumulation in liver tissue. In addition, it was confirmed that there is a liver function recovery effect as the activity of blood ALT and AST was significantly reduced in the Korean melon leaf ethanol extract group compared to the high-fat diet group.

[0047]

[0048] Accordingly, the present invention can provide a pharmaceutical composition for the prevention or treatment of fatty liver disease comprising a leaf extract of Korean melon (Cucumis melo var. makuwa).

[0049] According to a preferred embodiment of the present invention, the Korean melon leaf extract is,

[0050] i) Step of extracting Korean melon leaf powder;

[0051] ii) a step of separating the extract and the residue after the above extraction and re-extracting the residue; and

[0052] iii) A step of volatilizing the solvent after the above re-extraction;

[0053] It may be manufactured through.

[0054] In step i) above, the weight ratio of Korean melon leaf powder to extraction solvent may be 1:10 to 1:20, and preferably, the weight ratio of Korean melon leaf powder to extraction solvent may be 1:12 to 1:17.

[0055] In step ii) above, the weight ratio of the residue to the extraction solvent may be 1:5 to 1:15, and preferably, the weight ratio of the residue to the extraction solvent may be 1:7 to 1:12.

[0056] According to a preferred embodiment of the present invention, the extract may be an alcohol extract of Korean melon leaves.

[0057] According to a preferred embodiment of the present invention, the alcohol is 1 to 100% ethanol. Preferably, the alcohol may be 50 to 80% ethanol.

[0058] According to a preferred embodiment of the present invention, the fatty liver disease may be a metabolic dysfunction-associated steatotic liver disease.

[0059] The pharmaceutical composition of the present invention may be in various oral or parenteral formulations. When formulating the composition, it may be prepared using one or more buffers (e.g., saline solution or PBS), antioxidants, bacteriostatic agents, chelating agents (e.g., EDTA or glutathione), fillers, extenders, binders, adjuvants (e.g., aluminum hydroxide), suspending agents, thickening agents, wetting agents, disintegrants or surfactants, diluents or excipients.

[0060] Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid dosage forms are prepared by mixing at least one excipient with one or more compounds, for example, starch (including corn starch, wheat starch, rice starch, potato starch, etc.), calcium carbonate, sucrose, lactose, dextrose, sorbitol, mannitol, xylitol, erythritol, maltitol, cellulose, methyl cellulose, sodium carboxymethylcellulose and hydroxypropylmethylcellulose, or gelatin, etc. For example, tablets or sugar-coated tablets can be obtained by combining an active ingredient with a solid excipient, grinding the mixture, adding a suitable auxiliary agent, and processing it into a granular mixture.

[0061] In addition, lubricants such as magnesium stearate and talc are also used in addition to simple excipients. Liquid formulations for oral administration include suspensions, liquid formulations, emulsions, or syrups, and may contain various excipients, such as humectants, sweeteners, flavorings, or preservatives, in addition to commonly used simple diluents like water and liquid paraffin. Additionally, cross-linked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate may be added as disintegrants, and may additionally include anticoagulants, flavorings, emulsifiers, solubilizers, dispersants, flavorings, antioxidants, packaging agents, pigments, and preservatives.

[0062] Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, or suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspensions. Witepsol, macrogol, Tween 61, cocoa paste, laurin paste, glycerol, gelatin, etc. may be used as bases for suppositories.

[0063] The composition of the present invention may be administered orally or parenterally, and when administered parenterally, it may be formulated into the abdominal cavity, rectum, vein, muscle, or subcutaneous according to methods known in the art.

[0064] The above-mentioned injectable must be sterilized and protected from contamination by microorganisms such as bacteria and fungi. Suitable carriers for the injectable may include, but are not limited to, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), mixtures thereof, and / or vegetable oils, as solvents or dispersion media. More preferably, suitable carriers may include Hanks' solution, Ringer's solution, PBS (phosphate buffered saline) containing triethanolamine, sterile water for injection, isotonic solutions such as 10% ethanol, 40% propylene glycol, and 5% dextrose. To protect the injectable from microbial contamination, various antimicrobial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, and thimerosal may be additionally included. Additionally, the injectable may, in most cases, further include isotonic agents such as sugars or sodium chloride.

[0065] The composition of the present invention may be administered in a pharmaceutically effective amount. A pharmaceutically effective amount refers to an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level may be determined based on factors including the type and severity of the patient's disease, drug activity, sensitivity to the drug, time of administration, route of administration and elimination rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered as a single or multiple doses. That is, the total effective amount of the composition of the present invention may be administered to the patient as a single dose, or administered via a fractionated treatment protocol involving long-term administration of multiple doses. It is important to administer an amount that obtains maximum effect with a minimum amount without side effects by considering all the aforementioned factors, and this can be easily determined by a person skilled in the art.

[0066] The dosage of the pharmaceutical composition of the present invention may vary depending on the patient's body weight, age, gender, health condition, diet, time of administration, method of administration, excretion rate, and severity of the disease.

[0067] The composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers.

[0068]

[0069] In addition, the present invention may provide a health functional food composition for preventing or improving fatty liver disease comprising a leaf extract of Korean melon (Cucumis melo var. makuwa).

[0070] According to a preferred embodiment of the present invention, the Korean melon leaf extract is,

[0071] i) Step of extracting Korean melon leaf powder;

[0072] ii) a step of separating the extract and the residue after the above extraction and re-extracting the residue; and

[0073] iii) A step of volatilizing the solvent after the above re-extraction;

[0074] It may be manufactured through

[0075] In step i) above, the weight ratio of Korean melon leaf powder to extraction solvent may be 1:10 to 1:20, and preferably, the weight ratio of Korean melon leaf powder to extraction solvent may be 1:12 to 1:17.

[0076] In step ii) above, the weight ratio of the residue to the extraction solvent may be 1:5 to 1:15, and preferably, the weight ratio of the residue to the extraction solvent may be 1:7 to 1:12.

[0077] According to a preferred embodiment of the present invention, the extract may be an alcohol extract of Korean melon leaves.

[0078] According to a preferred embodiment of the present invention, the alcohol may be 1 to 100% ethanol. Preferably, the alcohol may be 50 to 80% ethanol.

[0079] According to a preferred embodiment of the present invention, the fatty liver disease may be a metabolic dysfunction-associated steatotic liver disease.

[0080] The food composition according to the present invention can be prepared in various forms according to conventional methods known in the art. General foods may be prepared by adding the Korean melon leaf alcohol extract of the present invention to beverages (including alcoholic beverages), fruits and their processed foods (e.g., canned fruit, bottled fruit, jam, marmalade, etc.), fish, meat and its processed foods (e.g., ham, sausage, corned beef, etc.), breads and noodles (e.g., udon, buckwheat noodles, ramen, spaghetti, macaroni, etc.), fruit juices, various drinks, cookies, malt syrup, dairy products (e.g., butter, cheese, etc.), edible vegetable oils, margarine, vegetable proteins, retort foods, frozen foods, and various seasonings (e.g., soybean paste, soy sauce, sauces, etc.), although not limited thereto. Additionally, nutritional supplements may be prepared by adding the Korean melon leaf alcohol extract of the present invention to capsules, tablets, pills, etc. In addition, as a health functional food, it is not limited to this, but for example, the Korean melon leaf alcohol extract of the present invention itself can be consumed by liquefying, granulating, encapsulating, and powdering it so that it can be prepared in the form of tea, juice, and drink for consumption (health beverage). In addition, to use the Korean melon leaf alcohol extract of the present invention as a food additive, it can be prepared and used in the form of a powder or concentrate. Furthermore, the Korean melon leaf alcohol extract of the present invention can be prepared in the form of a composition by mixing it with a known active ingredient known to have an effect of preventing or improving fatty liver.

[0081] When the Korean melon leaf ethanol extract of the present invention is used as a health drink, the health drink composition may contain various flavoring agents or natural carbohydrates as additional ingredients, as in conventional beverages. The aforementioned natural carbohydrates may be monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; polysaccharides such as dextrin and cyclodextrin; or 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 may be used. The proportion of the natural carbohydrates is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 mL of the composition of the present invention.

[0082] In addition, the Korean melon leaf ethanol extract of the present invention may be included as an active ingredient in a health functional food composition for the prevention or improvement of fatty liver. The amount is not specifically limited to an amount effective for achieving the prevention or improvement of fatty liver, but it is preferable to be 0.01 to 100 weight% with respect to the total weight of the composition. The health functional food composition of the present invention may be prepared by mixing the Korean melon leaf ethanol extract with other active ingredients known to be effective for fatty liver.

[0083] In addition to the above, the health functional food of the present invention may contain various nutritional supplements, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid, salts of pectic acid, alginic acid, salts of alginic acid, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, or carbonating agents. Furthermore, the health functional food of the present invention may contain fruit pulp for the production of natural fruit juice, fruit juice beverages, or vegetable beverages. These ingredients may be used independently or in combination.

[0084]

[0085] In addition, the present invention can provide a composition for improving liver function comprising a leaf extract of Korean melon (Cucumis melo var. makuwa).

[0086] According to a preferred embodiment of the present invention, the Korean melon leaf extract is,

[0087] i) Step of extracting Korean melon leaf powder;

[0088] ii) a step of separating the extract and the residue after the above extraction and re-extracting the residue; and

[0089] iii) A step of volatilizing the solvent after the above re-extraction;

[0090] It may be manufactured through.

[0091] In step i) above, the weight ratio of Korean melon leaf powder to extraction solvent may be 1:10 to 1:20, and preferably, the weight ratio of Korean melon leaf powder to extraction solvent may be 1:12 to 1:17.

[0092] In step ii) above, the weight ratio of the residue to the extraction solvent may be 1:5 to 1:15, and preferably, the weight ratio of the residue to the extraction solvent may be 1:7 to 1:12.

[0093] According to a preferred embodiment of the present invention, the extract may be an alcohol extract of Korean melon leaves.

[0094] According to a preferred embodiment of the present invention, the alcohol may be 1 to 100% ethanol. Preferably, the alcohol may be 50 to 80% ethanol.

[0095]

[0096] In addition, the present invention comprises: i) a step of extracting Korean melon (Cucumis melo var. makuwa) leaf powder;

[0097] ii) a step of separating the extract and the residue after the above extraction and re-extracting the residue; and

[0098] iii) A step of volatilizing the solvent after the above re-extraction;

[0099] A method for preparing a composition for preventing, improving, or treating fatty liver, comprising a Korean melon (Cucumis melo var. makuwa) leaf extract containing [the ingredient], can be provided.

[0100] In step i) above, the weight ratio of Korean melon leaf powder to extraction solvent may be 1:10 to 1:20, and preferably, the weight ratio of Korean melon leaf powder to extraction solvent may be 1:12 to 1:17.

[0101] In step ii) above, the weight ratio of the residue to the extraction solvent may be 1:5 to 1:15, and preferably, the weight ratio of the residue to the extraction solvent may be 1:7 to 1:12.

[0102] According to a preferred embodiment of the present invention, the extract may be an alcohol extract of Korean melon leaves.

[0103] According to a preferred embodiment of the present invention, the alcohol may be 1 to 100% ethanol. Preferably, the alcohol may be 50 to 80% ethanol.

[0104] According to a preferred embodiment of the present invention, the fatty liver may be a metabolic dysfunction-associated steatotic liver disease.

[0105]

[0106] In addition, the present invention may provide a method for treating fatty liver comprising the step of administering a leaf extract of Korean melon (Cucumis melo var. makuwa) to an individual in need.

[0107] According to a preferred embodiment of the present invention, the Korean melon leaf extract is,

[0108] i) Step of extracting Korean melon leaf powder;

[0109] ii) a step of separating the extract and the residue after the above extraction and re-extracting the residue; and

[0110] iii) A step of volatilizing the solvent after the above re-extraction;

[0111] It may be manufactured through

[0112] In step i) above, the weight ratio of Korean melon leaf powder to extraction solvent may be 1:10 to 1:20, and preferably, the weight ratio of Korean melon leaf powder to extraction solvent may be 1:12 to 1:17.

[0113] In step ii) above, the weight ratio of the residue to the extraction solvent may be 1:5 to 1:15, and preferably, the weight ratio of the residue to the extraction solvent may be 1:7 to 1:12.

[0114] According to a preferred embodiment of the present invention, the extract may be an alcohol extract of Korean melon leaves.

[0115] According to a preferred embodiment of the present invention, the alcohol may be 1 to 100% ethanol. Preferably, the alcohol may be 50 to 80% ethanol.

[0116] The above individuals may include normal individuals requiring improvement of liver function, recovery from liver damage, or maintenance of liver health, as well as individuals that have already developed or may develop liver disease. The above individuals may refer to all mammals, including but not limited to humans, dogs, cattle, horses, rabbits, mice, rats, or chickens.

[0117] Since the above administration is the same as the administration in the above pharmaceutical composition, the description is replaced by the description thereof.

[0118] According to a preferred embodiment of the present invention, the fatty liver disease may be a metabolic dysfunction-associated steatotic liver disease.

[0119]

[0120] The Korean melon leaf extract of the present invention can reduce liver weight and the accumulation of fat within liver tissue, and significantly inhibit blood ALT and AST activity, thereby providing effective effects for preventing, improving, or treating fatty liver. In addition, the Korean melon leaf extract of the present invention has a high yield of about 21%, so it may be highly economical as an excellent material for preventing, improving, or treating fatty liver.

[0121]

[0122] Figure 1 shows the effect of Korean melon leaf ethanol extract (CHY) on improving fatty liver.

[0123] Figure 2 shows the liver function improvement effect of Korean melon leaf ethanol extract (CHY) in liver tissue.

[0124] Figure 3 shows the liver lipid metabolism improvement and antioxidant effects of Korean melon leaf ethanol extract (CHY).

[0125] Figure 4 shows the inhibitory effect of Korean melon leaf ethanol extract (CHY) on fatty liver accumulation in primary liver cells.

[0126] Figure 5 shows the results of serum analysis following treatment with Korean melon leaf ethanol extract (CHY), illustrating the improvement effects on liver indicators and metabolism.

[0127] Figures 6a and 6b show the results of RNA sequencing analysis for the analysis of the molecular mechanism following treatment with Korean melon leaf ethanol extract (CHY).

[0128] Figures 7a to 7f show the results of RNA sequencing analysis following treatment with Korean melon leaf ethanol extract (CHY), and represent the results of differential expression gene analysis.

[0129] Figure 8 shows the results of RNA sequencing-based molecular mechanism analysis following treatment with Korean melon leaf ethanol extract (CHY), indicating up- or down-regulated signaling pathways.

[0130] Figures 9a and 9b show the results of RNA sequencing-based molecular mechanism analysis following treatment with Korean melon leaf ethanol extract (CHY), representing comprehensive network analysis results.

[0131] Figures 10a and 10b show the results of single-substance analysis based on UPLC / Q-TOF Mass Spectrometry of Korean melon leaf ethanol extract (CHY).

[0132]

[0133] The present invention will be described in more detail below through examples. These examples are intended solely to illustrate the present invention, and it is obvious to those skilled in the art that the scope of the present invention is not to be interpreted as being limited by these examples.

[0134]

[0135] [Example 1]

[0136] Experiment preparation

[0137] <1-1> Preparation of Korean Melon Leaf Extract

[0138] 400 g of ground Korean melon leaf powder, obtained by separating leaves from completely dried Korean melon stems, was immersed in 6 L of 70% ethanol (weight ratio of leaf powder to solvent = 1:15) and extracted using a reflux extraction device at 76°C for 15 hours. After extraction, the extract and residue were separated; the extract was filtered and stored at 4°C, while the residue was immersed again in 4 L of 70% ethanol (weight ratio of residue to solvent = 1:10) and re-extracted under the same conditions, followed by filtration. The ethanol from the filtered extract was completely evaporated using a rotary vacuum evaporator, and the extract was freeze-dried for 72 hours to be used as a sample. The freeze-dried extract in powder form was stored at -20°C, yielding 84.88 g of the Korean melon leaf ethanol extract (CHY), representing a yield of approximately 21%.

[0139]

[0140] <1-2> Experimental Animals and Methods - Construction of a Fatty Liver Disease Model Due to Metabolic Disorders

[0141] Male C57BL / 6J mice (4 weeks old) were supplied by Hana Biotech and used in the experiment after acclimatizing them in the animal laboratory for one week. During the acclimatization and experimental period, sufficient feed and water were supplied, and the temperature was maintained at 25±2℃, humidity at 50±10%, and a 12-hour light-dark cycle. The experimental groups consisted of a standard diet group (CON), a 60% kcal high-fat diet group (HFD), a high-fat diet + 0.4% Korean melon leaf ethanol extract group (CHY 0.4%), a high-fat diet + 0.8% Korean melon leaf ethanol extract group (CHY 0.8%), and a 0.8% Garcinia extract group (GRC 0.8%, positive control), with 10 mice assigned to each group. The Korean melon leaf ethanol extract and Garcinia extract were mixed into the high-fat diet at 0.4% and 0.8%, respectively, and then fed to the mice after sterilization. Based on a daily feed intake (approx. 2.5 g), the daily intake of Korean melon leaf ethanol extract was 10 mg and 20 mg, respectively. Male C57BL / 6J mice were sacrificed using isoflurane after a total of 18 weeks of experimentation, from 4–5 weeks of age to 21–22 weeks of age. Blood, subcutaneous fat, epididymal fat, liver, muscle, and small intestine were collected upon sacrifice. The blood was centrifuged at 3000 rpm for 20 minutes to separate serum, and the serum was stored at -70°C. The subcutaneous fat, epididymal fat, liver, muscle, and small intestine were flash-frozen in liquid nitrogen and stored at -70°C.

[0142]

[0143] [Example 2]

[0144] Anti-fatty liver effect of Korean melon leaf extract_Liver tissue

[0145] We intended to confirm the anti-fatty liver effect of mice by administering the Korean melon leaf extract (CHY) prepared in <Example 1-1> above to C57BL / 6J mice that had obesity induced by a high-fat diet.

[0146] When the livers of mice were visually observed at the end of the experiment, the livers of the control group fed a high-fat diet showed the typical color tone of fatty liver (light yellow or yellowish-brown), whereas the livers of the groups administered Korean melon leaf ethanol extract (0.4% and 0.8%) showed a reddish-brown color similar to that of the normal group (Fig. 1). Some improvement in liver color was also observed in the group administered 0.8% Garcinia extract, which was used as a positive control, but the effect was relatively minor compared to the group administered Korean melon leaf ethanol extract (Fig. 1).

[0147] Histological changes in liver tissue were observed through Hematoxylin & Eosin (H&E) staining, and it was confirmed that intracellular fat accumulation was reduced in the groups administered Korean melon leaf ethanol extract (0.4% and 0.8%) compared to the high-fat diet group, with the inhibitory effect on fat accumulation being more pronounced at the 0.4% concentration (Fig. 1). In addition, the group administered 0.4% Korean melon leaf ethanol extract showed a decrease in both liver weight and tissue fat accumulation levels compared to the high-fat diet group, and this effect was evaluated as superior to that of the positive control group administered Garcinia extract (0.8%) (Figs. 1 and 2).

[0148] In addition, the effect of the Korean melon leaf ethanol extract on liver function recovery was confirmed by checking blood ALT and AST activities using reagents for quantitative analysis of ALT and AST components in serum (Asan set GPT Assay kit (Manual GPT (ALT)) and Asan set GOT Assay kit (Manual GOT (AST)). As a result, ALT activity was significantly reduced in the Korean melon leaf ethanol extract group compared to the high-fat diet group (Fig. 2). These results indicated that the Korean melon leaf ethanol extract improves fatty liver and restores liver function.

[0149]

[0150] [Example 3]

[0151] Improvement of liver lipid metabolism and antioxidant effects of Korean melon leaf extract

[0152] <3-1> Analysis of Liver Triglycerides (TG) and Liver Total Cholesterol (TC)

[0153] To extract lipids from the liver, 100 mg of liver tissue was homogenized in 1 mL of DPBS, and 200 μL of the homogenized solution was mixed with 800 μL of chloroform (2:1, v:v) to separate the lipids. Subsequently, an equal volume of distilled water was added, and the mixture was centrifuged at 1000 g at 4°C for 5 minutes to collect the lower chloroform layer, which was then dried in a fume hood for 24 hours. The dried lipids were resuspended in 100 μL of methanol (9:1, v:v), and the concentrations of TG and TC were measured using a microplate reader (AMR-100, Allsheng, Hangzhou, China) according to the usage instructions of the respective analysis kits.

[0154] As a result, the HFD group showed a significant increase in liver triglyceride (TG) and total cholesterol (TC) levels compared to the CON group, but these lipid indicators were significantly reduced in the CHY 0.4% and CHY 0.8% treatment groups compared to the HFD group (Fig. 3 A, B). In particular, the CHY 0.4% treatment group reduced triglyceride and total cholesterol levels by 55.86% and 37.76%, respectively, compared to the HFD group. This suggests that CHY extract is effective in improving liver lipid metabolism and inhibiting fat accumulation.

[0155]

[0156] <3-2> ROS and ONOO - measurement

[0157] ROS and ONOO generation were measured using fluorescent probes. For ROS analysis, 10 μL of homogenized tissue sample was mixed with 190 μL of 50 mM phosphate buffer (PB), and the reaction was carried out for 30 minutes in a light-blocked environment using 0.125 μM DCFDA solution. Fluorescence intensity was recorded 7 times at 5-minute intervals over 30 minutes in a 96-well plate. For ONOO analysis, 10 μL of homogenized liver sample was mixed with a mixture consisting of 175.8 μL of Rhodamine, 4 μL of DTPA, and 0.2 μL of DHR123, and measurements were taken 7 times over 30 minutes at an excitation wavelength of 485 nm and an emission wavelength of 535 nm.

[0158] As a result, in the CHY treatment group, liver ROS and ONOO were lower compared to the HFD group. - The values ​​decreased significantly (Fig. 3 C, D). This suggests that CHY treatment contributes to the reduction of oxidative stress in the liver and has a hepatoprotective effect.

[0159]

[0160] [Example 4]

[0161] Anti-fatty liver effect of Korean melon leaf extract_Liver cells

[0162] <4-1> Primary hepatocyte isolation and lipid accumulation inhibitory effect

[0163] In fatty liver experiments using cell lines, the reactivity related to fat accumulation and related signal transduction differ depending on the cell model used, so primary hepatocytes were isolated and confirmed from a mouse model in which animal experiments were conducted.

[0164] Specifically, hepatocytes derived from C57BL / 6J mice were isolated from mice using a two-step collagenase perfusion method and a Percol density gradient. Briefly, after anesthetizing mice with Isoplan, the liver was perfused for 5 minutes via the inferior vena cava with Hank's balance solution containing 25 mM HEPES and 0.5 mM EDTA, which is free of calcium and magnesium, followed by an additional 5 minutes of perfusion with Hank's balance solution containing 25 mM HEPES and 25 μg / mL Liberase, which is free of calcium and magnesium. The isolated hepatocytes were seeded at a density of 2.5 × 10^5 cells per well in collagen-coated 12-well plates (SPL, Korea) using DMEM supplemented with 5% FBS and 1% P / S. After 6 hours, cell attachment was confirmed, and CHY was treated at different concentrations (100, 200 ppm) in Williams E medium containing 2 mM glutamine and 1% P / S. One hour later, free fatty acids (FFA) were added to reach a final concentration of 1 mM to induce intracellular lipid accumulation. After confirming that lipid accumulation occurred effectively in response to fatty acid treatment, the experiment proceeded.

[0165]

[0166] <4-2> Oil Red O Staining and Intracellular Triglyceride Measurement

[0167] C57BL / 6J mouse-derived hepatocytes were incubated in 10% neutral buffered formalin for 10 minutes, and after suction removal, fixed in 10% neutral buffered formalin for 1 hour. After washing twice with 60% isopropanol at room temperature and drying completely, the cells were stained with 60% Oil Red O working solution for 30 minutes. After washing with triple-distilled water, intracellular stained lipid droplets were observed under a light microscope, and the area of ​​the stained lipids was quantified and graphed using ImageJ. To measure intracellular triglyceride (TG) content, CHY-treated mouse hepatocytes were washed twice with PBS, the cells were scraped, and centrifuged at 300g for 5 minutes. The cell pellet was dissolved in 1% Triton X-100 / PBS, and intracellular triglyceride concentrations were measured according to the manufacturer's instructions (Asan Set Reagent for Triglyceride Measurement, Asan Pharmaceutical Co., Ltd., Seoul, Korea) and standardized to protein concentration.

[0168]

[0169] <4-3> Confirmation of the effects of Korean melon leaf ethanol extract on hepatocytes with fatty liver induced by FFAs

[0170] Treatment of hepatocytes with 1 mM FFA resulted in a significant increase in the amount of accumulated lipids, as confirmed by Oil Red O staining. Optical microscopy revealed that a large number of lipid droplets were stained in the FFA-treated cells. When CHY was applied at concentrations of 100 and 200 ppm, a decrease in lipid accumulation was observed, with the most pronounced reduction occurring at 200 ppm (Fig. 4A). Quantification of the stained lipid area using ImageJ showed that lipid accumulation increased significantly in the FFA-treated group compared to CON, and upon CHY treatment, lipid accumulation decreased in a concentration-dependent manner, with significant reductions confirmed at both 100 and 200 ppm concentrations (Fig. 4B). In addition, when intracellular triglyceride (TG) content was measured after treating hepatocytes with FFA, it was confirmed that while TG content significantly increased in the FFA-treated group, TG content significantly decreased upon treatment with 200 ppm CHY (Fig. 4C). In particular, treatment with 200 ppm of CHY reduced the triglyceride concentration in primary hepatocytes by approximately 91%.

[0171] The results of this experiment showed that CHY directly acts on primary hepatocytes derived from C57BL / 6J mice induced by FFA to effectively inhibit lipid accumulation and triglyceride content. These results suggest that CHY may be useful for the treatment of metabolic dyslipidemia (MASLD) and related metabolic diseases.

[0172]

[0173] [Example 5]

[0174] Serum Analysis and Metabolic Improvement Effects Following Treatment with Korean Melon Leaf Extract

[0175] Serum analysis was performed using a free fatty acid (NEFA) assay kit (NEFA assay reagent, Wako Pure Chemical Industries, Osaka, Japan) and an LDL-cholesterol assay kit (LDL-Cholesterol assay reagent, Wako Pure Chemical Industries, Osaka, Japan). Kits for the analysis of the remaining serum components were purchased from Asan Pharmaceutical (Seoul, Korea) for the experiment. Additionally, alkaline phosphatase (ALP) and lipase (LIPA) levels were measured using Exdia PT10V (Precision Biosensor, Korea).

[0176] Serum analysis revealed that cholesterol levels decreased by an average of 14.72% in the CHY 0.4% treatment group and by 9.08% in the CHY 0.8% treatment group compared to the high-fat diet (HFD) mouse group (p < 0.05, Fig. 5A). However, no significant differences were observed between the low-fat diet (CON) group and the HFD group regarding triglyceride (Fig. 5B), blood glucose (Fig. 5C), and free fatty acid (NEFA) levels (Fig. 5D), indicating no further changes were observed in the CHY treatment group. ALP levels showed a significant difference, decreasing by 15.10% in the CHY 0.4% treatment group compared to the HFD group (p < 0.05, Fig. 5E), while LIPA (Lipase) levels increased in the HFD group, decreasing by an average of 14.16% in the CHY 0.4% treatment group (Fig. 5F). These results suggest that CHY treatment is effective in reducing cholesterol, ALP, and LIPA levels, and that metabolic improvement and liver health protection effects were particularly evident in the CHY 0.4% treatment group.

[0177]

[0178] [Example 6]

[0179] Molecular mechanism analysis and gene expression analysis

[0180] <6-1> Analysis of Molecular Mechanisms via RNA Sequencing

[0181] To investigate the effect of CHY treatment on the improvement of metabolic dyslipidemia, RNA-Seq (transcriptome analysis) was performed on primary hepatocytes in which lipid accumulation was induced by fatty acid treatment after treatment with CHY. During the RNA purification process, only RNA with a RIN value of 7.0 or higher was used for RNA library construction. Libraries were independently constructed using the Illumina TruSeq Stranded mRNA Sample Prep Kit (Illumina, Inc., San Diego, CA, USA, #RS-122-2101) with 1 μg of total RNA per sample. The constructed libraries were quantified using the KAPA Library Quantification Kit for Illumina Sequencing Platforms (KAPA BIOSYSTEMS, #KK4854), and quality control was performed using TapeStation D1000 ScreenTape (Agilent Technologies, #5067-5582). The indexed library was sequenced using the Illumina NovaSeqX platform (Illumina, Inc., San Diego, CA, USA) in a paired-end (2 × 100 bp) manner. Sequencing reads were mapped to a reference genome using HISAT2 (version 2.2.1), and read counts were calculated using featureCounts (version subread-2.0.8).

[0182]

[0183] <6-2> Analysis of Differentially Expressed Genes

[0184] Differential expression gene analysis was performed using TMM normalization with the edgeR package (version 4.0.16). The No Replicate method described in the “Analysis of Sequence Read Count Data User's Guide” was used for TMM normalization, and a square root variance value of 0.1 was applied. Differential expression genes were filtered based on log2 fold change ≥ 1, log2 fold change ≤ -1, and FDR < 0.01, and the filtered genes were used for Gene Ontology analysis.

[0185]

[0186] <6-3> Results

[0187] A total of 17,415 genes showed statistically significant differential expression (p < 0.01), and the CHY treatment group was found to have 2,541 upregulated genes and 2,649 downregulated genes compared to the FFA alone group.

[0188] An analysis was performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database to identify the associated signaling pathways among the upregulated and downregulated genes. The upregulated genes were found to be primarily involved in pathways related to maintaining cell structure and tissue repair. The motor protein pathway plays a crucial role in cell migration, intracellular material transport, and maintaining structural stability, exhibiting particularly high activity. Furthermore, the upregulation of the ECM-receptor interaction and focal adhesion pathways indicates improved ECM remodeling, cell adhesion, and intracellular signaling, suggesting that signaling systems essential for tissue repair and cell survival were activated by CHY processing (Fig. 6a). Conversely, signaling systems associated with inflammation and oxidative stress were downregulated. The cytokine-cytokine receptor interaction pathway and IL-17 signaling pathway reflect a decrease in inflammatory cytokine signaling, while the downregulation of the non-alcoholic fatty liver disease pathway indicates an improvement in overall signaling associated with fatty liver disease (Fig. 6b).

[0189] To gain a deeper understanding of functional changes, Gene Ontology (GO) analysis was performed on differentially expressed genes (DEGs) in the categories of biological processes (BP), molecular functions (MF), and cellular components (CC) (Figs. 7a–7f). The BP analysis showed significant increases in pathways related to membrane potential (GO:0042391), lipid transport (GO:0006869), ECM organization (GO:0030198), glucose homeostasis (GO:0042593), hormone metabolism (GO:0042445), and regeneration (GO:0031099) (Fig. 8A). These results suggest that CHY treatment promotes processes involved in lipid metabolism activation, energy homeostasis restoration, cell repair, and regeneration. On the other hand, pathways related to lipopolysaccharide response (GO:0032496), cytokine-mediated signaling pathway (GO:0019221), regulation of inflammatory response (GO:0050727), leukocyte migration (GO:0050900), myeloid leukocyte migration (GO:0097529), and chemotaxis (GO:0006935) were significantly inhibited (Fig. 8). This indicates a reduction in inflammation and immune cell tissue infiltration, reflecting the anti-inflammatory effect of CHY treatment.

[0190] CHY treatment in primary hepatocytes with induced lipid accumulation exhibited unique gene expression patterns related to lipid transport, glucose homeostasis, cytokine signaling, and the regulation of inflammatory responses. Accordingly, a comprehensive network analysis was performed to identify detailed genetic changes, and specifically, genes associated with lipid transport, inflammatory responses, and major regulatory pathways were analyzed in CHY-treated hepatocytes, as well as genes related to the improvement of metabolic disorders and fatty liver disease.

[0191] In the GO analysis, the "regulation of membrane potential" category included 21 genes, and significant changes in expression were observed in Cacna1g, Chrne, Edn1, Flna, Gabrr2, and Gja5. In the "regeneration" category, significant changes were identified in Adm, Dysf, Igf1r, and Tnc. In the "lipid transport" category (containing 19 genes), high changes in expression were observed in Abca6 and Abca9. Additionally, in the "hormone metabolic process" category, Adm, Corin, Duox2, and Igf1r showed high changes in expression, and in the "glucose homeostasis" category, significant changes in expression were observed in Csrp3, Hk1, Igf1r, Myt1, and Myh9. Finally, in the extracellular matrix organization category, prominent changes in expression were observed in Axin2, Ccn2, Crispld2, Ihh, and Loxl4 (Fig. 9a).

[0192] Regarding downregulated genes in the GO analysis, the "response to lipopolysaccharide" category showed a significant decrease in the genes Cd14, Tlr4, Il1a, Il1b, and Tnf, indicating a reduction in the inflammatory response. In the "regulation of inflammatory response" category, significant inhibition of Il1b, Il6, Tnf, Ccl2, and Cxcl2 was observed, indicating a decrease in cytokine signaling. In the "myeloid leukocyte migration" category, the decreased expression of Ccr1, Ccr2, Cxcl1, Cxcl2, and Il1b suggests reduced tissue infiltration by myeloid immune cells. Similarly, in the "leukocyte migration" category, the genes Ccl2, Ccr5, Itgam, Cd44, and Sele were inhibited, also demonstrating a decrease in the migration and tissue infiltration of various immune cells. In addition, in the "cytokine-mediated signaling pathway" category, the genes Il1b, Il6, Tnf, Ccl2, and Cxcl1 were significantly reduced, confirming that pro-inflammatory cytokine signaling was suppressed. Finally, in the "chemotaxis" category, the expression of Ccl2, Ccl5, Cxcl1, Cxcl2, and Il8 was reduced, showing that immune cell motility and inflammatory responses were decreased (Fig. 9b).

[0193] In summary, mechanistic analysis via RNA sequencing indicates that CHY treatment inhibits metabolic disorders of fatty liver disease and exhibits hepatoprotective effects, suggesting a close association with the inhibition of inflammatory and oxidative stress signaling systems in hepatocytes, the activation of cellular metabolism, and the regulation of signaling pathways related to hepatocyte structural recovery.

[0194]

[0195] [Example 7]

[0196] Single-substance analysis of Korean melon leaf extract

[0197] <7-1> Sample Preparation for UPLC-MS Analysis

[0198] The ethanol extract (1 g) of Korean melon leaves of Example <1-1> was precipitated by adding 10 times the amount of a solution containing 1% formic acid to methanol:water (80:20, v / v). This mixture was sonicated for 30 minutes, stored overnight at 4°C, and then centrifuged at 12,000 RPM for 10 minutes. The finally obtained supernatant (120 μL) was recovered and stored at 4°C until MS analysis.

[0199]

[0200] <7-2> Ultra-high Performance Liquid Chromatography (UPLC)

[0201] Chromatographic analysis was performed using a UPLC system (Bruker, Massachusetts, USA). Precipitated CML samples were injected into a BEH C18 column (100 mm × 2.1 mm, 1.7 μm, 1 / pk; Waters, Massachusetts, USA), and the column temperature was maintained at 40°C. The flow rate was set to 300 μL / min. The mobile phase consisted of water containing 0.1% formic acid (Phase A) and acetonitrile containing 0.1% formic acid (Phase B). The UPLC automatic sampler was maintained at 10°C, and the injection volume for each sample was 1 μL.

[0202]

[0203] <7-3> Phytochemical Analysis Using UPLC / Q-TOF Mass Spectrometry

[0204] After separation by UPLC, mass spectrometry was performed using a Q-TOF Premier (Bruker, Massachusetts, USA) equipped with an Electron Spray Ionization (ESI) source (Waters, Massachusetts, USA). The settings in negative mode were as follows: capillary voltage: 2 kV, cone voltage: 40 V, source temperature: 100 ℃, desolvator temperature: 250 ℃, desolvator gas flow: 600 L / h, cone gas flow: 50 L / h, and data acquisition at 100–1300 m / z.

[0205]

[0206] <7-4> Data Processing and Statistical Analysis

[0207] Screened data were included in the analysis after correcting for personal bias using QC and blank data. Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA), Projected Weight Variable (VIP), and coefficient analysis by VIP score were performed using EZinfo 3.0 software controlled by Progenesis QI 3.0 software (Waters, Massachusetts, USA).

[0208]

[0209] <7-5> Results

[0210] LC-MS analysis was performed to identify plant compounds contained in the Korean melon leaf extract (Figs. 10a, 10b). The analysis was conducted in negative ionization mode, and a total of 438 compounds were detected. Among these, the top 20 compounds were selected based on low raw abundance values ​​and CV% values. Detailed information such as the retention time (R. Time), base m / z values, and names of each compound is provided in [Table 1] and [Table 2] below.

[0211]

[0212]

[0213]

[0214]

[0215]

[0216] While most of the identified compounds have not yet been clearly characterized or are under-studied, Soyacerebroside (retention time: 26.73, baseline m / z: 712.5358) is a noteworthy substance as a glycosphingolipid reported to possess anti-inflammatory properties. This compound plays a crucial role in maintaining cell membrane structure and promoting signal transduction, and it is likely to possess functions that regulate lipid metabolism and signaling pathways. Furthermore, as changes in sphingolipid metabolism are associated with insulin resistance and obesity, the possibility is raised that this compound contributes to metabolic regulation. In addition to Soyacerebroside, a tripeptid named Glu-Glu-Arg (L-α-glutamyl-L-α-glutamyl-L-arginine) was identified in the CHY ethanol extract; this compound is listed in the ChEBI database. This compound is expected to play an important role in protein signaling and metabolic pathways. The identification of Glu-Glu-Arg and its potential regulatory function are considered factors that further emphasize the diversity of bioactive compounds in the CHY extract. [Figure 10a] shows the LC-MS chromatogram, and [Figure 10b] visually presents the importance of phytochemicals by highlighting compounds that show relatively high abundance.

[0217] Taken together, these results suggest that the CHY ethanol extract contains various bioactive compounds such as Soyacerebroside and Glu-Glu-Arg, and that these components may contribute to the regulation of lipid metabolism and the reduction of metabolic dyslipidemia.

[0218]

[0219] The Korean melon leaf extract of the present invention can reduce liver weight and the accumulation of fat within liver tissue, and significantly inhibit blood ALT and AST activity, thereby providing effective effects for preventing, improving, or treating fatty liver. In addition, the Korean melon leaf extract of the present invention has a high yield of approximately 21%, making it economically viable as an excellent material for preventing, improving, or treating fatty liver, thus having industrial applicability.

Claims

1. A pharmaceutical composition for the prevention or treatment of fatty liver disease comprising a leaf extract of Korean melon (Cucumis melo var. makuwa).

2. A pharmaceutical composition according to claim 1, characterized in that the extract is an alcohol extract of Korean melon leaves.

3. A pharmaceutical composition according to paragraph 2, characterized in that the alcohol is 1 to 100% ethanol.

4. A pharmaceutical composition according to claim 1, characterized in that the fatty liver disease is a metabolic dysfunction-associated steatotic liver disease.

5. A health functional food composition for preventing or improving fatty liver disease comprising a leaf extract of Korean melon (Cucumis melo var. makuwa).

6. A health functional food composition characterized in that, in claim 5, the fatty liver disease is a metabolic dysfunction-associated steatotic liver disease.

7. A composition for improving liver function comprising a leaf extract of Korean melon (Cucumis melo var. makuwa). 8.i) Step of extracting Korean melon (Cucumis melo var. makuwa) leaf powder; ii) a step of separating the extract and the residue after the above extraction and re-extracting the residue; and iii) A step of volatilizing the solvent after the above re-extraction; A method for preparing a composition for preventing, improving, or treating fatty liver, comprising a Korean melon (Cucumis melo var. makuwa) leaf extract.

9. A manufacturing method according to claim 8, characterized in that the fatty liver is a metabolic dysfunction-associated steatotic liver disease.

10. A method for treating fatty liver comprising the step of administering a Korean melon (Cucumis melo var. makuwa) leaf extract to an individual in need.

11. A treatment method according to claim 10, characterized in that the extract is an alcohol extract of Korean melon leaves.

12. A treatment method according to claim 10, characterized in that the alcohol is 1 to 100% ethanol.

13. A treatment method according to claim 10, characterized in that the fatty liver disease is a metabolic dysfunction-associated steatotic liver disease.

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