Composition for preventing, ameliorating or treating fatty liver comprising melon leaf extract

Abstract

This invention relates to melon leaf extract, and more particularly to the effects of melon leaf alcohol extract on the prevention, improvement, or treatment of fatty liver caused by metabolic disorders. The melon leaf extract can reduce liver weight and lipid accumulation in liver tissue, and can inhibit serum ALT activity, thereby providing preventive, ameliorative, or therapeutic effects on fatty liver. Furthermore, the extraction yield of the melon leaf extract of this invention is approximately 21%, making it an excellent material for the prevention, improvement, or treatment of fatty liver, and thus economically viable.

Description

Technical Field

[0001] This application claims priority to Korean Patent Application No. 10-2024-0141226, filed on October 16, 2024, the entire description of which is incorporated herein by reference.

[0002] This invention relates to melon leaf extract, and more particularly to the effects of melon leaf alcohol extract on the prevention, improvement, or treatment of metabolically abnormal fatty liver.

[0003] This invention was completed with the support of the Ministry of Science and ICT of the Republic of Korea, under project number RS-2023-00274576 (2710003021) and project number 2021R1A6A1A03039211 (RS-2021-NR060118, 2340006719). Background Technology

[0004] Muskmelon (Cucumis melo var. makuwa) belongs to the Cucurbitaceae family. Its origin is known to be India, and it has diversified into Oriental and Western varieties depending on its spread. Currently, it is mainly cultivated in Korea, China, and Japan. In Korea, it is speculated that muskmelon cultivation began before the Silla Dynasty, and many different local varieties have been cultivated in various regions. As a fruit and vegetable crop with a sweet taste and unique aroma, muskmelon's refreshing flavor appeals to people and has been widely consumed as a summer fruit since ancient times.

[0005] However, the processing of melon leaves after harvesting melons in melon farms is difficult and incurs costs during the disposal process. Therefore, it is necessary to conduct research on utilizing melon leaves generated during melon cultivation as a high-value-added resource.

[0006] Metabolic dysfunction-associated steatotic liver disease (MASLD) is a condition that, although unrelated to alcohol consumption, presents with histological features similar to alcoholic hepatitis. It belongs to a metabolic syndrome encompassing the progression from non-alcoholic fatty liver disease (NAFLD) to metabolic dysfunction-associated steatohepatitis (MASH), cirrhosis, and hepatocellular carcinoma. MASLD is showing a synchronous increasing trend with the rise in obesity and diabetes, with an annual incidence rate of approximately 16% in South Korea.

[0007] Currently, there is no established treatment for metabolic fatty liver disease, as it is associated with multiple factors. Therefore, ongoing research is needed to develop materials that can provide preventative, ameliorative, or therapeutic benefits for metabolic fatty liver disease in a non-invasive manner, derived from natural sources rather than synthetic compounds.

[0008] Existing technical documents

[0009] Patent documents

[0010] (Patent Document 1) KR 10-2013-0125282 (2013-10-21) Summary of the Invention

[0011] The technical problem that the invention aims to solve

[0012] The inventors conducted long-term research in order to provide materials from natural sources that can provide excellent preventive, improvement, or therapeutic effects for metabolically abnormal fatty liver disease. The results confirmed that melon leaf extract can reduce liver weight, liver fat, and ALT and AST levels, and can provide excellent preventive, improvement, or therapeutic effects for metabolically abnormal fatty liver disease. Based on this, the present invention was completed.

[0013] Therefore, the purpose of this invention is to provide the preventive, ameliorative, or therapeutic effects of melon leaf extract, especially melon leaf alcohol extract, on metabolically abnormal fatty liver disease.

[0014] means for solving problems

[0015] This invention provides a pharmaceutical composition for the prevention or treatment of fatty liver disease, comprising a melon (Cucumis melo var. makuwa) leaf extract.

[0016] According to a preferred embodiment of the present invention, the melon (Cucumis melo var. makuwa) leaf extract is prepared by the following steps: step i) extracting melon (Cucumis melo var. makuwa) leaf powder; step ii) after the extraction, separating the extract from the residue and re-extracting the residue; and step iii) after the re-extraction, allowing the solvent to evaporate.

[0017] According to a preferred embodiment of the present invention, the extract is a melon leaf alcohol extract.

[0018] According to a preferred embodiment of the present invention, the alcohol is 1-100% ethanol.

[0019] According to a preferred embodiment of the present invention, the fatty liver disease is a metabolic dysfunction-associated steatotic liver disease.

[0020] In addition, the present invention provides a health food composition for preventing or improving fatty liver disease, which contains melon (Cucumis melo var. makuwa) leaf extract.

[0021] According to a preferred embodiment of the present invention, the fatty liver disease is a metabolic dysfunction-associated steatotic liver disease.

[0022] In addition, the present invention provides a composition for improving liver function, which comprises melon (Cucumis melovar. makuwa) leaf extract.

[0023] Furthermore, the present invention provides a method for preparing a composition comprising a melon (Cucumis melo var. makuwa) leaf extract for the prevention, improvement or treatment of fatty liver, comprising the following steps: Step i) extracting melon (Cucumis melo var. makuwa) leaf powder;

[0024] Step ii) After the extraction, the extract is separated from the residue and the residue is extracted again; and

[0025] Step iii) After the re-extraction, allow the solvent to evaporate.

[0026] Furthermore, the present invention provides a composition for improving liver function, comprising a melon (Cucumis melo var. makuwa) leaf extract. Additionally, the present invention provides a treatment for fatty liver comprising the step of administering a melon (Cucumis melo var. makuwa) leaf extract to an individual in need of it.

[0027] According to a preferred embodiment of the present invention, the extract is a melon leaf alcohol extract.

[0028] According to a preferred embodiment of the present invention, the alcohol is 1-100% ethanol.

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

[0030] The present invention will now be described in further detail.

[0031] In this invention, "prevention" refers to all actions that inhibit fatty liver or fatty liver-related diseases or delay their onset due to the melon leaf alcohol extract of this invention.

[0032] In this invention, “improvement” or “treatment” refers to all actions that improve or change in a favorable direction the parameters related to fatty liver and even fatty liver-related diseases, such as the severity of symptoms, due to the melon leaf alcohol extract of this invention.

[0033] This invention relates to the preventive, ameliorative, or therapeutic effects of melon leaf ethanol extract on fatty liver caused by metabolic disorders, and even its liver health-promoting effects. Using C57BL / 6J mice induced by a high-fat diet to achieve obesity, the inventors confirmed the effects of melon leaf ethanol extract on fatty liver and related liver health indicators.

[0034] The muskmelon leaf alcohol extract of this invention significantly reduced liver weight and decreased fat accumulation in liver tissue compared to the high-fat diet group (0.8%). Furthermore, compared to the high-fat diet group, the activities of serum ALT and AST were significantly reduced in the muskmelon leaf alcohol extract group, confirming its liver function recovery effect.

[0035] Therefore, the present invention can provide a pharmaceutical composition comprising a melon (Cucumis melo var. makuwa) leaf extract for the prevention or treatment of fatty liver disease.

[0036] According to a preferred embodiment of the present invention, the melon (Cucumis melo var. makuwa) leaf extract is prepared by the following steps: step i) extracting melon (Cucumis melo var. makuwa) leaf powder; step ii) after the extraction, separating the extract from the residue and re-extracting the residue; and step iii) after the re-extraction, allowing the solvent to evaporate.

[0037] In step i), the weight ratio of melon leaf powder to extraction solvent can be 1:10 to 1:20, and preferably, the weight ratio of melon leaf powder to extraction solvent can be 1:12 to 1:17.

[0038] In step ii), the weight ratio of residue to extraction solvent can be 1:5 to 1:15, preferably 1:7 to 1:12.

[0039] According to a preferred embodiment of the present invention, the extract may be a melon leaf alcohol extract.

[0040] According to a preferred embodiment of the present invention, the alcohol is 1-100% ethanol. Preferably, the alcohol is 50-80% ethanol.

[0041] According to a preferred embodiment of the present invention, the fatty liver disease can be a metabolic dysfunction-associated steatotic liver disease.

[0042] The pharmaceutical compositions of the present invention can be in various dosage forms, both oral and non-oral. When formulating the compositions, one or more buffers (e.g., physiological saline or PBS), antioxidants, antibacterial agents, chelating agents (e.g., EDTA or glutathione), fillers, extenders, binders, adjuvants (e.g., aluminum hydroxide), suspending agents, thickeners, wetting agents, disintegrants or surfactants, diluents or excipients can be used.

[0043] Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc. These solid dosage forms are prepared by mixing one or more compounds with at least one excipient. The excipients include, for example, starch (including corn starch, wheat starch, rice starch, potato starch, etc.), calcium carbonate, sucrose, lactose, glucose, sorbitol, mannitol, xylitol, erythritol, maltitol, cellulose, methylcellulose, sodium carboxymethylcellulose, and hydroxypropyl methylcellulose or gelatin. For example, the active ingredient can be combined with a solid excipient, pulverized, and then appropriate excipients added and processed into a granular mixture to obtain tablets or sugar-coated tablets.

[0044] In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid formulations for oral administration include suspensions, oral liquids, emulsions, or syrups. Besides commonly used simple diluents such as water and liquid paraffin, they may contain various excipients, such as wetting agents, sweeteners, flavoring agents, or preservatives. Furthermore, depending on the circumstances, croscarmellose, agar, alginate, or sodium alginate may be added as disintegrants, and may further contain anti-caking agents, fragrances, emulsifiers, solubilizers, dispersants, flavoring agents, antioxidants, coating agents, pigments, and preservatives.

[0045] Formulations intended for non-oral administration include sterile aqueous solutions, non-aqueous solvents, suspension formulations, emulsions, lyophilized formulations, and suppositories. Non-aqueous solvents and suspension formulations may use propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As a base for suppositories, may use Wipedosol, polyethylene glycol, Tween 61, cocoa butter, lauryl acetate, glycerin, and gelatin.

[0046] The compositions of the present invention can be administered orally or non-orally. When administered non-orally, they can be formulated into preparations for intraperitoneal, rectal, intravenous, intramuscular, or subcutaneous administration according to methods known in the art.

[0047] In the case of the injectable preparation, sterilization is mandatory, and contamination by microorganisms such as bacteria and fungi must be prevented. Suitable carriers for the injectable preparation may include, but are not limited to, solvents or dispersion media composed of water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), mixtures thereof, and / or vegetable oils. More preferably, suitable carriers may be Hanks' solution, Ringer's solution, PBS (phosphate buffered saline) containing triethanolamine, or sterile water for injection, as well as isotonic solutions of 10% ethanol, 40% propylene glycol, and 5% glucose. To protect the injectable preparation from microbial contamination, it may further contain various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. Furthermore, in most cases, the injectable preparation may further contain isotonic agents such as sugars or sodium chloride.

[0048] The compositions of the present invention can be administered in pharmaceutically effective amounts. A pharmaceutically effective amount is defined as an amount sufficient to treat a disease with a reasonable benefit / risk ratio suitable for medical treatment. The effective dose level can be determined based on factors including the patient's disease type, severity, drug activity, drug sensitivity, timing of administration, route of administration and excretion rate, treatment duration, concurrent medications, and other factors known in the medical field. The compositions of the present invention can be administered as a single therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with existing therapeutic agents, and can be administered in a single dose or multiple doses. That is, the total effective amount of the compositions of the present invention can be administered to the patient as a single dose or through a fractionated treatment protocol administered as multiple doses over a longer period. Taking all the above factors into consideration, it is important to administer the minimum amount that yields the maximum effect without side effects, and this amount can be readily determined by those skilled in the art.

[0049] The dosage of the pharmaceutical composition of the present invention can vary depending on the patient's weight, age, sex, health status, diet, administration time, administration method, excretion rate, and severity of disease.

[0050] The compositions of the present invention can be used alone or in combination with surgery, radiation therapy, hormone therapy, chemotherapy, and methods using biological response modifiers.

[0051] In addition, the present invention can provide a health food composition for the prevention or improvement of fatty liver disease, which contains melon (Cucumis melo var. makuwa) leaf extract.

[0052] According to a preferred embodiment of the present invention, the melon (Cucumis melo var. makuwa) leaf extract is prepared by the following steps: step i) extracting melon (Cucumis melo var. makuwa) leaf powder; step ii) after the extraction, separating the extract from the residue and re-extracting the residue; and step iii) after the re-extraction, allowing the solvent to evaporate.

[0053] In step i), the weight ratio of melon leaf powder to extraction solvent can be 1:10 to 1:20, and preferably, the weight ratio of melon leaf powder to extraction solvent can be 1:12 to 1:17.

[0054] In step ii), the weight ratio of residue to extraction solvent can be 1:5 to 1:15, preferably 1:7 to 1:12.

[0055] According to a preferred embodiment of the present invention, the extract may be a melon leaf alcohol extract.

[0056] According to a preferred embodiment of the present invention, the alcohol may be 1-100% ethanol. Preferably, the alcohol may be 50-80% ethanol.

[0057] According to a preferred embodiment of the present invention, the fatty liver disease can be a metabolic dysfunction-associated steatotic liver disease.

[0058] The food compositions of the present invention can be prepared in various forms according to conventional methods known in the art. As general food, not limited to these, they can be prepared by adding the melon leaf alcohol extract of the present invention to beverages (alcoholic beverages), fruits and their processed foods (e.g., canned fruits, bottled foods, jams, marmalade, etc.), fish, meat and their processed foods (e.g., ham, sausages, corned beef, etc.), breads and pasta (e.g., udon noodles, soba noodles, ramen, pasta, macaroni, etc.), fruit juices, various beverages, biscuits, syrups, dairy products (e.g., butter, cheese, etc.), edible vegetable oils, magarin, plant proteins, canned foods, frozen foods, and various seasonings (e.g., miso, soy sauce, sauces, etc.). Furthermore, as a nutritional supplement, not limited to these, it can be prepared by adding the melon leaf alcohol extract of the present invention to capsules, tablets, pills, etc. Furthermore, as a health food, not limited to these, for example, the melon leaf alcohol extract of the present invention can be made into tea, fruit juice, and beverages for consumption (health drinks), and ingested after liquefaction, granulation, encapsulation, or powdering. Furthermore, in order to use the melon leaf alcohol extract of the present invention as a food additive, it can be prepared as a powder or a concentrated liquid. Additionally, the melon leaf alcohol extract of the present invention can be mixed with known active ingredients known to have preventive or ameliorative effects on fatty liver to prepare a composition.

[0059] Furthermore, when the melon leaf alcohol extract of the present invention is used as a health beverage, the health beverage composition may, like ordinary beverages, further include various flavoring agents or natural carbohydrates as additional ingredients. The aforementioned natural carbohydrates may be monosaccharides (e.g., glucose, fructose); disaccharides (e.g., maltose, sucrose); polysaccharides (e.g., dextrin, cyclodextrin); and sugar alcohols (e.g., xylitol, sorbitol, erythritol). The sweeteners may be natural sweeteners, such as thaumatin, stevia extract; and synthetic sweeteners, such as saccharin, aspartame, etc. The proportion of the natural carbohydrates is typically about 0.01 to 0.04 g per 100 mL of the composition of the present invention, preferably about 0.02 to 0.03 g.

[0060] Furthermore, the melon leaf alcohol extract of the present invention can be included as an effective ingredient in a health food composition for preventing or improving fatty liver. Its content is not particularly limited, as long as it is an effective amount capable of achieving the preventive or improving effect on fatty liver, but preferably from 0.01 to 100% by weight relative to the total weight of the entire composition. The health food composition of the present invention can be prepared by mixing the melon leaf alcohol extract with other known active ingredients effective against fatty liver.

[0061] Furthermore, the health food products of the present invention may also contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid, salts of pectic acid, alginic acid, salts of alginic acid, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, or carbonating agents, etc. In addition, the health food products of the present invention may also contain fruit pulp used in the preparation of natural fruit juices, fruit juice beverages, or vegetable beverages. These ingredients may be used alone or in combination.

[0062] In addition, the present invention provides a composition for improving liver function, which comprises melon (Cucumis melovar. makuwa) leaf extract.

[0063] According to a preferred embodiment of the present invention, the melon (Cucumis melo var. makuwa) leaf extract is prepared by the following steps: step i) extracting melon (Cucumis melo var. makuwa) leaf powder; step ii) after the extraction, separating the extract from the residue and re-extracting the residue; and step iii) after the re-extraction, allowing the solvent to evaporate.

[0064] In step i), the weight ratio of melon leaf powder to extraction solvent can be 1:10 to 1:20, and preferably, the weight ratio of melon leaf powder to extraction solvent can be 1:12 to 1:17.

[0065] In step ii), the weight ratio of residue to extraction solvent can be 1:5 to 1:15, preferably 1:7 to 1:12.

[0066] According to a preferred embodiment of the present invention, the extract may be a melon leaf alcohol extract.

[0067] According to a preferred embodiment of the present invention, the alcohol may be 1-100% ethanol. Preferably, the alcohol may be 50-80% ethanol.

[0068] Furthermore, the present invention provides a method for preparing a composition containing melon (Cucumis melo var. makuwa) leaf extract for the prevention, improvement or treatment of fatty liver, comprising the following steps: step i) extracting melon (Cucumis melo var. makuwa) leaf powder; step ii) after the extraction, separating the extract from the residue and re-extracting the residue; and step iii) after the re-extraction, evaporating the solvent.

[0069] In step i), the weight ratio of melon leaf powder to extraction solvent can be 1:10 to 1:20, and preferably, the weight ratio of melon leaf powder to extraction solvent can be 1:12 to 1:17.

[0070] In step ii), the weight ratio of residue to extraction solvent can be 1:5 to 1:15, preferably 1:7 to 1:12.

[0071] According to a preferred embodiment of the present invention, the extract may be a melon leaf alcohol extract.

[0072] According to a preferred embodiment of the present invention, the alcohol may be 1-100% ethanol. Preferably, the alcohol may be 50-80% ethanol.

[0073] According to a preferred embodiment of the present invention, the fatty liver can be a metabolic dysfunction-associated steatotic liver disease.

[0074] In addition, the present invention can provide a treatment for fatty liver, which includes the step of applying a melon (Cucumis melovar. makuwa) leaf extract to an individual in need of it.

[0075] According to a preferred embodiment of the present invention, the melon (Cucumis melo var. makuwa) leaf extract is prepared by the following steps: step i) extracting melon (Cucumis melo var. makuwa) leaf powder; step ii) after the extraction, separating the extract from the residue and re-extracting the residue; and step iii) after the re-extraction, allowing the solvent to evaporate.

[0076] In step i), the weight ratio of melon leaf powder to extraction solvent can be 1:10 to 1:20, and preferably, the weight ratio of melon leaf powder to extraction solvent can be 1:12 to 1:17.

[0077] In step ii), the weight ratio of residue to extraction solvent can be 1:5 to 1:15, preferably 1:7 to 1:12.

[0078] According to a preferred embodiment of the present invention, the extract may be a melon leaf alcohol extract.

[0079] According to a preferred embodiment of the present invention, the alcohol may be 1-100% ethanol. Preferably, the alcohol may be 50-80% ethanol.

[0080] The individuals mentioned may include normal individuals who need to improve liver function, recover from liver damage, or maintain liver health, as well as individuals who have already developed or may develop liver disease. The individuals may refer to all mammals, including but not limited to, such as humans, dogs, cattle, horses, rabbits, mice, rats, or chickens.

[0081] The application is the same as that in the above-described pharmaceutical composition, and therefore its description is replaced by this record.

[0082] According to a preferred embodiment of the present invention, the fatty liver disease can be a metabolic dysfunction-associated steatotic liver disease.

[0083] The effects of the invention

[0084] The melon leaf extract can reduce liver weight and lipid accumulation in liver tissue, and inhibit the activity of ALT and AST in serum, thus providing preventive, ameliorative, or therapeutic effects for fatty liver. Furthermore, the melon leaf extract of this invention has an extraction yield of approximately 21%, making it an excellent material for the prevention, ameliorative, or therapeutic use of fatty liver, and it is economically viable. Attached Figure Description

[0085] Figure 1 A graph illustrating the effect of melon leaf alcohol extract CHY on improving fatty liver.

[0086] Figure 2 A figure illustrating the liver function improvement effect of melon leaf alcohol extract CHY in liver tissue.

[0087] Figure 3 A figure illustrating the effects of melon leaf alcohol extract CHY on improving liver lipid metabolism and its antioxidant properties.

[0088] Figure 4 A figure illustrating the inhibitory effect of melon leaf extract CHY on fatty liver accumulation in primary hepatocytes.

[0089] Figure 5 To illustrate the serum analysis results after treatment with melon leaf alcohol extract CHY, a graph showing the improvement in liver indicators and metabolism is presented.

[0090] Figure 6a and Figure 6b This figure illustrates the results of RNA sequencing analysis used to analyze the molecular mechanisms induced by CHY treatment of melon leaf extract.

[0091] Figures 7a-7fThe graph used to illustrate the results of differentially expressed gene analysis is based on RNA sequencing analysis induced by treatment with the ethanol extract CHY from melon leaves.

[0092] Figure 8 The results of molecular mechanism analysis based on RNA sequencing induced by treatment with melon leaf alcohol extract CHY are used to illustrate the upregulated or downregulated signal transduction systems.

[0093] Figure 9a and Figure 9b The results of molecular mechanism analysis based on the treatment of melon leaf alcohol extract CHY induced RNA sequencing are used to illustrate the results of comprehensive network analysis.

[0094] Figure 10a and Figure 10b A figure showing the results of single-substance analysis of the muskmelon leaf alcohol extract CHY based on UPLC / Q-TOF Mass Spectrometry. Detailed Implementation

[0095] The present invention will be further described in detail below through embodiments. These embodiments are only for illustrating the present invention, and it is obvious to those skilled in the art that the scope of the present invention should not be construed as being limited to these embodiments.

[0096]

Example 1

[0097] Experimental preparation

[0098] <1-1> Preparation of Melon Leaf Extract

[0099] Leaves separated from completely dried melon stems were pulverized to obtain 400g of melon leaf powder, which was then impregnated in 6L of 70% ethanol (melon leaf powder to solvent weight ratio = 1:15) and extracted at 76°C using a reflux condenser for 15 hours. After extraction, the extract was separated from the residue, filtered, and stored at 4°C. The residue was impregnated again in 4L of 70% ethanol (residue to solvent weight ratio = 1:10) and extracted again under the same conditions, followed by filtration. Subsequently, all ethanol in the filtered extract was evaporated using a rotary vacuum evaporator, and the extract was lyophilized for 72 hours for use as a sample. The lyophilized powder extract was stored at −20°C to obtain 84.88g of melon leaf ethanol extract CHY, showing a yield of approximately 21%.

[0100] <1-2> Experimental Animals and Methods—Establishment of a Metabolic Abnormal Fatty Liver Disease Model

[0101] Four-week-old male C57BL / 6J mice, provided by Hana Biotech, were acclimatized in the animal laboratory for one week before being used in the experiment. During the acclimatization and experimental periods, adequate food and water were provided, and environmental conditions were maintained at a temperature of 25±2℃, humidity of 50±10%, and a 12-hour light-dark cycle. Experimental groups included a standard diet group (CON), a 60% kcal high-fat diet group (HFD), a high-fat diet plus 0.4% melon leaf extract group (CHY 0.4%), a high-fat diet plus 0.8% melon leaf extract group (CHY 0.8%), and a 0.8% Garcinia extract group (GRC 0.8%, positive control), with 10 animals assigned to each group. Melon leaf extract and Garcinia extract were mixed in the high-fat diet at ratios of 0.4% and 0.8%, respectively, and administered to the mice after sterilization. Based on a daily feed intake of approximately 2.5g, the daily intake of melon leaf extract was 10mg and 20mg, respectively. Male C57BL / 6J mice aged 4–5 weeks to 21–22 weeks were subjected to an 18-week experiment and then euthanized with isoflurane. Blood, subcutaneous fat, epididymal fat, liver, muscle, and small intestine were collected at euthanasia. Blood was centrifuged at 3000 rpm for 20 min to separate serum and then stored at -70°C. Subcutaneous fat, epididymal fat, liver, muscle, and small intestine were rapidly frozen in liquid nitrogen and then stored at -70°C.

[0102]

Example 2

[0103] Anti-fatty liver effect of melon leaf extract – liver tissue

[0104] The melon leaf extract (CHY) prepared in Example 1-1 above was administered to C57BL / 6J mice that were obese due to a high-fat diet to confirm its anti-fatty liver effect.

[0105] At the end of the experiment, macroscopic observation of the mouse livers showed that in the control group given a high-fat diet, the livers exhibited the typical color of fatty liver (light yellow or yellowish-brown), while in the groups treated with melon leaf alcohol extract (0.4% and 0.8%), the livers showed a reddish-brown color similar to the normal group. Figure 1 In the 0.8% Garcinia Cambogia extract group, which served as a positive control, partial improvement in liver tone was also observed, but the effect was relatively weaker compared to the melon leaf alcohol extract group. Figure 1 ).

[0106] Histological changes in liver tissue observed by Hematoxylin & Eosin (H&E) staining showed that, compared with the high-fat diet group, intracellular fat accumulation was reduced in the melon leaf extract treatment groups (0.4% and 0.8%), and the inhibitory effect on fat accumulation was more significant at the 0.4% concentration. Figure 1 Furthermore, compared to the high-fat diet group, the 0.4% melon leaf extract group showed reduced liver weight and intra-tissue fat accumulation levels, and this effect was superior to the Garcinia Cambogia extract (0.8%) group, which served as the positive control. Figure 1 and Figure 2 ).

[0107] In addition, the activities of serum ALT and AST were measured using reagents for quantitative detection of serum ALT and AST (Asan set GPT Assay kit (Manual GPT (ALT))) and Assan set GOT Assay kit (Manual GOT (AST))) to evaluate the effect of melon leaf ethanol extract on liver function recovery. The results showed that compared with the high-fat diet group, the ALT activity in the melon leaf ethanol extract group was significantly reduced. Figure 2 The above results indicate that melon leaf alcohol extract improves fatty liver and restores liver function.

[0108]

Example 3

[0109] Improvement of liver lipid metabolism and antioxidant effects of melon leaf extract

[0110] <3-1> Analysis of liver triglycerides (TG) and total liver cholesterol (TC)

[0111] To extract lipids from the liver, 100 mg of liver tissue was homogenized in 1 mL of DPBS. 200 μL of the homogenate was then mixed with 800 μL of chloroform (2:1, v:v) to separate the lipids. An equal volume of distilled water was added, and the mixture was centrifuged at 1000 g and 4 °C for 5 min. The lower chloroform layer was collected and 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 triglycerides (TG) and total cholesterol (TC) were determined using a microplate reader (AMR-100, Allsheng, Hangzhou, China) according to the instructions of each analytical kit.

[0112] The results showed that, compared with the CON group, the levels of liver triglycerides (TG) and total cholesterol (TC) were significantly increased in the HFD group, while in the CHY 0.4% and CHY 0.8% administration groups, these lipid indicators were significantly lower than those in the HFD group. Figure 3 (A and 3B). In particular, compared with the HFD group, the CHY 0.4% administration group showed a 55.86% reduction in triglyceride and a 37.76% reduction in total cholesterol levels. This indicates that CHY extract helps improve hepatic lipid metabolism and inhibit lipid accumulation.

[0113] <3-2> ROS and ONOO⁻ determination

[0114] The formation of ROS and ONOO was determined using fluorescent probes. For ROS analysis, 10 μL of homogenized tissue sample was mixed with 190 μL of 50 mM phosphate buffer (PB) and reacted with 0.125 μM DCFDA solution under light-protected conditions for 30 min. Fluorescence intensity was recorded 7 times at 5-min intervals in a 96-well plate for a total measurement time of 30 min. For ONOO analysis, 10 μL of homogenized liver sample was mixed with a mixture of 175.8 μL Rhodamine, 4 μL DTPA, and 0.2 μL DHR123 and measured 7 times within 30 min under excitation and emission wavelengths of 485 nm and 535 nm, respectively.

[0115] The results showed that, compared with the HFD group, the CHY group had lower levels of intrahepatic ROS and ONOO. - The value decreased significantly ( Figure 3 (C, 3D). This indicates that CHY treatment helps reduce intrahepatic oxidative stress and has a hepatoprotective effect.

[0116]

Example 4

[0117] Anti-fatty liver effect of melon leaf extract – liver cells

[0118] <4-1> Isolation of primary hepatocytes and their effect on inhibiting lipid accumulation

[0119] Because different cell models can lead to differences in lipid accumulation-related reactivity and signal transduction when using cell lines for fatty liver experiments, primary hepatocytes are isolated from mouse models used in animal experiments for confirmation.

[0120] Specifically, a two-step collagenase perfusion method combined with a Percoll density gradient was used to isolate hepatocytes from C57BL / 6J mice. Briefly, mice were anesthetized with isoflurane, and the livers were perfused for 5 min via the inferior vena cava using Hank's balanced salt solution containing 25 mM HEPES and 0.5 mM EDTA (calcium and magnesium-free). This was followed by another 5 min of perfusion with Hank's balanced salt solution containing 25 mM HEPES, 25 μg / mL Liberase, and 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 adhesion was confirmed, and CHY was treated with different concentrations (100, 200 ppm) of Williams E medium containing 2 mM glutamine and 1% P / S. One hour later, free fatty acids (FFA) were added to a final concentration of 1 mM to induce intracellular lipid accumulation. After confirming that fatty acid treatment could effectively induce lipid accumulation, subsequent experiments were conducted.

[0121] <4-2> Oil Red O staining and intracellular triglyceride measurement

[0122] Hepatocytes derived from C57BL / 6J mice were treated in 10% neutral buffered formalin for 10 min, the fixative was discarded, and the cells were fixed in 10% neutral buffered formalin for 1 hour. After washing twice with 60% isopropanol and drying completely at room temperature, they were stained with 60% Oil Red O working solution for 30 min. After washing with distilled water three times, the stained lipid droplets in the cells were observed using an optical microscope, and the area of ​​the stained lipids was quantified and plotted using ImageJ. To determine the intracellular triglyceride (TG) content, CHY-treated mouse hepatocytes were washed twice with PBS, cells were scraped, and centrifuged at 300g for 5 min. The cell pellet was dissolved in 1% Triton X-100 / PBS, and the intracellular triglyceride concentration was determined according to the manufacturer's instructions (Asan Triglyceride Assay Kit, Asan Pharmaceutical Co., Ltd., Seoul, Korea), and normalized to protein concentration.

[0123] <4-3> Confirmation of the effect of melon leaf alcohol extract on hepatocytes with FFA-induced fatty liver

[0124] Treatment of hepatocytes with 1 mM FFA resulted in a significant increase in accumulated lipid mass, as confirmed by Oil Red O staining. Optical microscopy confirmed the presence of numerous lipid droplets stained in FFA-treated cells. Treatment with CHY at 100 ppm and 200 ppm resulted in decreased lipid accumulation, with the most significant reduction observed at 200 ppm. Figure 4 A). Quantitative analysis of the stained lipid area using ImageJ showed that, compared with the CON group, lipid accumulation was significantly increased in the FFA-treated group, while lipid accumulation decreased in a concentration-dependent manner under CHY treatment, with significant reductions observed at both 100 ppm and 200 ppm concentrations. Figure 4 B). Furthermore, the results of measuring intracellular triglyceride (TG) levels in hepatocytes after FFA treatment showed a significant increase in TG levels in the FFA-treated group, while a significant decrease in TG levels was observed after CHY treatment at 200 ppm. Figure 4 C). In particular, treatment with 200 ppm CHY reduced the concentration of neutral lipids in primary hepatocytes by approximately 91%.

[0125] The results of this experiment indicate that CHY can directly act on primary hepatocytes derived from FFA-induced C57BL / 6J mice, inhibiting lipid accumulation and triglyceride content. These results suggest that CHY has the potential for the treatment of metabolically abnormal fatty liver disease (MASLD) and related metabolic disorders.

[0126]

Example 5

[0127] Serum analysis and metabolic improvement effects after treatment with melon leaf extract

[0128] Serum analyses were performed using a free fatty acid (NEFA) assay kit (NEFA assay reagent, Wako Pure Chemical Industries, Osaka, Japan) and a low-density lipoprotein cholesterol assay kit (LDL-Cholesterol assay reagent, Wako Pure Chemical Industries, Osaka, Japan). Other kits used for serum component analysis were purchased from Asan Pharmaceutical Co., Ltd. (Seoul, Korea) and were used accordingly. Additionally, alkaline phosphatase (ALP) and lipase (LIPA) levels were measured using an Exdia PT10V (Precision Biosensor, Korea).

[0129] Serum analysis showed that, compared with the high-fat diet (HFD) mouse group, the average cholesterol level in the CHY 0.4% treatment group was reduced by 14.72%, and the average cholesterol level in the CHY 0.8% treatment group was reduced by 9.08% (p<0.05). Figure 5 A). However, in triglycerides ( Figure 5 B) Blood glucose ( Figure 5 C) and free fatty acid (NEFA) values ​​( Figure 5 Regarding aspect D), no significant differences were observed between the low-fat diet (CON) group and the HFD group, therefore no further changes were observed in the CHY treatment group. ALP levels were 15.10% lower in the CHY 0.4% treatment group compared to the HFD group, and this difference was statistically significant (p<0.05). Figure 5 E); while LIPA (Lipase) values ​​increased in the HFD group, compared to an average decrease of 14.16% in the CHY 0.4% treatment group (E). Figure 5 F). These results indicate that CHY treatment helps reduce cholesterol, ALP, and LIPA levels, with particularly significant improvements in metabolism and liver health protection observed in the CHY 0.4% treatment group.

[0130]

Example 6

[0131] Molecular mechanism analysis and gene expression analysis

[0132] <6-1> Molecular Mechanism Analysis Through RNA Sequencing

[0133] To investigate the effect of CHY treatment on the improvement of metabolically abnormal fatty liver disease, RNA-Seq (transcriptome analysis) was performed on primary hepatocytes induced by fatty acid treatment to induce lipid accumulation. During RNA purification, only RNA with a RIN value ≥7.0 was used for RNA library construction. 1 μg of total RNA was used for each sample, and libraries were independently constructed using the Illumina TruSeq Stranded mRNA Sample Prep Kit (Illumina, Inc., San Diego, CA, USA, #RS-122-2101). The constructed libraries were quantified using the KAPA Library Quantification Kit for Illumina Sequencing Platforms (KAPA BIOSYSTEMS, #KK4854) and quality-checked using TapeStation D1000 ScreenTape (Agilent Technologies, #5067-5582). The indexed library was sequenced using the Illumina NovaSeqX platform (Illumina, Inc., San Diego, CA, USA) with paired ends (2×100bp). Sequencing reads were aligned to the reference genome using HISAT2 (version 2.2.1), and read counts were then calculated using featureCounts (version subread-2.0.8).

[0134] <6-2> Differentially expressed gene analysis

[0135] Differentially expressed genes were analyzed using TMM normalization performed with the edgeR software package (version 4.0.16). TMM normalization employed the No Replicate method described in the "Analysis of Sequence Read Count Data User's Guide" and applied a square root dispersion of 0.1. Differentially expressed genes were screened according to the criteria of log2 fold change ≥ 1, log2 fold change ≤ -1, and FDR < 0.01. The screened genes were then used for gene ontology analysis.

[0136] Results of <6-3>

[0137] Statistical analysis showed that a total of 17,415 genes exhibited statistically significant differential expression (p<0.01). Compared with the group treated with FFA only, 2,541 genes were upregulated and 2,649 genes were downregulated in the CHY treatment group.

[0138] To identify the associated signaling pathways in upregulated and downregulated genes, the Kyoto Encyclopedia of Genes and Genomes (KEGG) database was used for analysis. Upregulated genes were mainly involved in pathways related to cell structure maintenance and tissue repair. The motility protein pathway plays an important role in maintaining cell migration, intracellular substance transport, and structural stability, and exhibited high activity. Furthermore, the ECM-receptor interaction pathway and focal adhesion pathway were upregulated, indicating improved ECM remodeling, cell adhesion, and intracellular signaling. This suggests that signaling systems closely related to tissue repair and cell survival are activated under CHY treatment. Figure 6a Conversely, signaling pathways associated with inflammation and oxidative stress were downregulated. Downregulation of the cytokine-cytokine receptor interaction pathway and the IL-17 signaling pathway reflected reduced inflammatory cytokine signaling, while downregulation of pathways associated with non-alcoholic fatty liver disease indicated improved overall signaling associated with fatty liver disease. Figure 6b ).

[0139] To gain a deeper understanding of functional changes, GO (Gene Ontology) analysis was performed on differentially expressed genes (DEGs) across the categories of biological processes (BP), molecular functions (MF), and cellular components (CC). Figures 7a-7f In the BP analysis, pathways related to membrane potential (GO:0042391), lipid transport (GO:0006869), ECM tissueing (GO:0030198), glucose homeostasis (GO:0042593), hormone metabolism (GO:0042445), and regeneration (GO:0031099) showed a significant increase. Figure 8 A). The above results suggest that CHY treatment promotes processes related to lipid metabolism activation, energy homeostasis restoration, and cell repair and regeneration. Conversely, pathways related to lipopolysaccharide response (GO:0032496), cytokine-mediated signaling pathways (GO:0019221), regulation of inflammatory responses (GO:0050727), leukocyte migration (GO:0050900), myeloid leukocyte migration (GO:0097529), and chemotaxis (GO:0006935) were all significantly inhibited. Figure 8 This indicates a reduction in inflammation and immune cell infiltration into the tissue, reflecting the anti-inflammatory effect of CHY treatment.

[0140] In primary hepatocytes induced to accumulate lipids, CHY treatment exhibited unique gene expression patterns associated with lipid transport, glucose homeostasis, cytokine signaling, and inflammatory response regulation. Therefore, to confirm specific gene changes, a comprehensive network analysis was performed, focusing on genes in CHY-treated hepatocytes that were not only associated with the improvement of metabolically abnormal fatty liver disease, but also those related to lipid transport, inflammatory responses, and key regulatory pathways.

[0141] In the GO analysis, the "regulation of membrane potential" category included 21 genes, with significant expression changes observed in Cacna1g, Chrne, Edn1, Flna, Gabrr2, and Gja5. In the "regeneration" category, significant changes were confirmed in Adm, Dysf, Igf1r, and Tnc. In the "lipid transport" category (containing 19 genes), Abca6 and Abca9 showed significant expression changes. Furthermore, in the "hormone metabolic process" category, Adm, Corin, Duox2, and Igf1r showed high expression changes, while in the "glucose homeostasis" category, significant expression changes were observed in Csrp3, Hk1, Igf1r, Myt1, and Myh9. Finally, in the "extracellular matrix organization" category, significant expression changes were identified in Axin2, Ccn2, Crispld2, Ihh, and Loxl4. Figure 9a ).

[0142] In the GO analysis, for downregulated genes, the expression of Cd14, Tlr4, Il1a, Il1b, and Tnf genes was significantly reduced in the "response tolipopolysaccharide" category, indicating a weakened inflammatory response. In the "regulation of inflammatory response" category, significant inhibition of Il1b, Il6, Tnf, Ccl2, and Cxcl2 was observed, suggesting reduced cytokine signaling. In the "myeloid leukocyte migration" category, decreased expression of Ccr1, Ccr2, Cxcl1, Cxcl2, and Il1b suggested reduced infiltration of myeloid immune cells into tissues. Similarly, in the "leukocyte migration" category, the inhibition of Ccl2, Ccr5, Itgam, Cd44, and Sele genes also indicated reduced migration and infiltration of various immune cells into tissues. Furthermore, in the "cytokine-mediated signaling pathway" category, the genes Il1b, Il6, Tnf, Ccl2, and Cxcl1 were significantly downregulated, confirming the suppression of pro-inflammatory cytokine signaling. Finally, in the "chemotaxis" category, the expression of Ccl2, Ccl5, Cxcl1, Cxcl2, and Il8 was decreased, indicating a reduction in both immune cell migration and inflammatory response. Figure 9b ).

[0143] In summary, the mechanistic analysis results obtained through RNA sequencing indicate that CHY treatment exhibits inhibitory and hepatoprotective effects against metabolically abnormal fatty liver disease. This is closely related to 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.

[0144]

Example 7

[0145] Single component analysis of melon leaf extract

[0146] <7-1> Sample preparation for UPLC-MS analysis

[0147] The melon leaf alcohol extract (1 g) from Example <1-1> was added to 10 volumes of a methanol:water (80:20, v / v) solution containing 1% formic acid to precipitate the sample. The mixture was sonicated for 30 min and stored overnight at 4°C, followed by centrifugation at 12,000 rpm for 10 min. The resulting supernatant (120 μL) was recovered and stored at 4°C until MS analysis.

[0148] <7-2> Ultra-high performance liquid chromatography (UPLC)

[0149] 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 autosampler was maintained at 10 °C, and the injection volume for each sample was 1 μL.

[0150] <7-3> Phytochemical analysis using UPLC / Q-TOF mass spectrometry

[0151] After UPLC separation, mass spectrometry analysis was performed using a Q-TOF Premier (Bruker, Massachusetts, USA) equipped with an electrospray ionization (ESI) source (Waters, Massachusetts, USA). The settings in negative ion mode were as follows: capillary voltage: 2 kV, cone voltage: 40 V, ion source temperature: 100 °C, desolvation temperature: 250 °C, desolvation gas flow rate: 600 L / h, cone gas flow rate: 50 L / h, and data acquisition range: 100–1300 m / z.

[0152] <7-4> Data Processing and Statistical Analysis

[0153] After screening, the data were corrected for individual bias using QC samples and blank data before being included in the analysis. Orthogonal partial least squares discriminant analysis (OPLS-DA), projected importance variable (VIP) analysis, and coefficient analysis based on VIP scores were performed using EZinfo 3.0 software under the control of Progenesis QI 3.0 software (Waters, Massachusetts, USA).

[0154] Results of <7-5>

[0155] To identify the plant compounds contained in the melon leaf extract, LC-MS analysis was performed. Figure 10a , Figure 10b The analysis was conducted in negative ionization mode, and a total of 438 compounds were detected. The top 20 compounds were selected based on their high raw abundance values ​​and low CV% values. Detailed information regarding the retention time (R. Time), base m / z values, and compound names for each compound is provided in Tables 1 and 2 below.

[0156] Table 1

[0157]

[0158]

[0159]

[0160] Table 2

[0161]

[0162]

[0163] Of the identified compounds, most are not yet clearly characterized or are under-studied, but Soyacerebroside (retention time: 26.73, basic m / z: 712.5358), a glycosphingolipid with reported anti-inflammatory properties, is of interest. This compound plays an important role in maintaining cell membrane structure and promoting signal transduction, and may have functions in regulating lipid metabolism and signal transduction pathways. Furthermore, changes in sphingolipid metabolism are associated with insulin resistance and obesity, suggesting that this compound may contribute to metabolic regulation. In addition to Soyacerebroside, a tripeptide, Glu-Glu-Arg (L-α-glutamyl-L-α-glutamyl-L-arginine), was also identified in the CHY ethanol extract and is indexed 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 functions further highlight the diversity of bioactive compounds in CHY extracts. Figure 10a The LC-MS chromatogram is displayed. Figure 10b The relatively abundant compounds are highlighted, visually demonstrating the importance of phytochemicals.

[0164] In summary, CHY ethanol extract contains a variety of physiologically active compounds, such as soyacerebroside and Glu-Glu-Arg, suggesting that these components may help regulate lipid metabolism and reduce metabolically abnormal fatty liver disease.

[0165] [Industrial Applicability]

[0166] The melon leaf extract of this invention can reduce liver weight and fat accumulation in liver tissue, and significantly inhibit the activity of ALT and AST in the blood, thereby providing effective prevention, improvement, or treatment of fatty liver. Furthermore, the melon leaf extract of this invention has an extraction yield of approximately 21%, making it an excellent raw material for the prevention, improvement, or treatment of fatty liver, and possessing high economic value, thus demonstrating potential for industrial application.

Claims

1. A pharmaceutical composition for the prevention or treatment of fatty liver disease, comprising an extract of melon (Cucumis melo var. makuwa) leaves.

2. The pharmaceutical composition of claim 1, wherein, The extract is melon leaf alcohol extract.

3. The pharmaceutical composition of claim 2, wherein, The alcohol is 1-100% ethanol.

4. The pharmaceutical composition of claim 1, wherein, The fatty liver disease mentioned is metabolic dysfunction-associated steatotic liver disease.

5. A health food composition for the prevention or improvement of fatty liver disease, comprising a melon (Cucumis melo var. makuwa) leaf extract.

6. The health food composition according to claim 5, characterized by, The fatty liver disease mentioned is metabolic dysfunction-associated steatotic liver disease.

7. A composition for improving liver function, comprising a melon (Cucumis melo var. makuwa) leaf extract.

8. A method for preparing a composition comprising a melon (Cucumis melo var. makuwa) leaf extract for the prevention, improvement or treatment of fatty liver, comprising the following steps: step i) extracting melon (Cucumis melo var. makuwa) leaf powder; Step ii) After the extraction, the extract is separated from the residue and the residue is extracted again; and Step iii) After the re-extraction, allow the solvent to evaporate.

9. The production method according to claim 8, characterized by, The fatty liver mentioned is a metabolic dysfunction-associated steatotic liver disease.

10. A treatment method for fatty liver, comprising the following steps: Apply the leaf extract of melon (Cucumis melo var. makuwa) to individuals in need.

11. The method of treatment according to claim 10, wherein, The extract is melon leaf alcohol extract.

12. The method of treatment according to claim 10, wherein, The alcohol is 1-100% ethanol.

13. The method of treatment according to claim 10, wherein, The fatty liver disease mentioned is metabolic dysfunction-associated steatotic liver disease.

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