Composition for alleviating fatty liver disease by using single or complex extract of sigesbeckia glabrescens and luffa cylindrica

Abstract

The present invention provides a composition for alleviating fatty liver disease by using a single or complex extract of Sigesbeckia glabrescens and Luffa cylindrica, the composition exhibiting the activities of inhibiting fat accumulation and inflammation in liver cells without showing toxicity to liver cells, inhibiting fat accumulation and inflammation in liver tissue in high-fat-diet animal models, and increasing the expression of glutathione, which is a strong antioxidant in liver tissue.

Description

Composition for improving fatty liver disease using single or combined extracts of Jindeukchal and Susemioi

[0001] The present invention relates to a composition for improving fatty liver using a single or combined extract of Sigesbeckia glabrescens and Luffa cylindrica.

[0002] The liver is the most crucial organ in the body, responsible for energy and lipid metabolism. It stores free fatty acids (FFAs), which are fundamental to energy metabolism, in the form of triglycerides (TG). These triglycerides, stored in the liver, are transported to other organs through the bloodstream through lipid metabolism. When an imbalance occurs in this lipid metabolism process, triglycerides accumulate within hepatocytes, leading to fatty liver disease.

[0003] Fatty liver disease is broadly divided into alcoholic fatty liver disease (AFLD) caused by alcohol and non-alcoholic fatty liver disease (NAFLD) related to metabolic diseases such as hyperlipidemia, diabetes, and obesity.

[0004] Alcoholic fatty liver disease (AFLD) is caused by excessive alcohol consumption, which promotes fat synthesis in the liver and disrupts energy metabolism. Nonalcoholic fatty liver disease (NAFLD) is reported to be associated with metabolic syndrome, but the cause and progression of the disease remain unclear.

[0005] Simple fatty liver disease caused by the accumulation of fat within liver cells can be improved with diet and appropriate exercise, but it often becomes chronic and can be a factor in causing hepatitis and liver fibrosis.

[0006] In particular, nonalcoholic fatty liver disease (NAFLD) consists of two clinical states: nonalcoholic fatty liver disease (NAFL), which is a simple steatosis stage as described above, and nonalcoholic steatohepatitis (NASH), which is accompanied by inflammation, and is explained by the 'two-hit' model. The 'first hit' is the simple steatosis (NAFL) stage, in which neutral fat is deposited in hepatocytes. The 'First hit' stage of simple steatosis in non-alcoholic fatty liver disease is caused by an imbalance between the inflow and outflow of neutral fat, and can be broadly explained by five factors: ① accumulation of neutral fat in hepatocytes due to impaired synthesis and excretion of very low-density lipoprotein (VLDL), ② errors in fatty acid oxidation due to mitochondrial dysfunction, ③ impairment of lipid metabolism due to deficiency of essential nutrients, ④ continuous inflow of excessive free fatty acids, and ⑤ promotion of excessive production of fatty acids in hepatocytes.

[0007] The "second hit" occurs when accumulated triglycerides, triggered by factors such as alcohol and oxidative stress, trigger the release of various inflammatory cytokines, leading to inflammation. This process, known as the "second hit," can further worsen into liver fibrosis, cirrhosis, and liver cancer. Because NAFLD is a disease characterized by both steatosis and inflammation, prevention and treatment should consider both steatosis inhibition and anti-inflammation.

[0008] The severity of NAFLD is being highlighted by the increasing mortality rate worldwide, but there are currently no approved treatments for it. NAFLD patients typically require long-term treatment, which can lead to various side effects. Therefore, the development of functional materials with superior efficacy and minimal side effects is essential.

[0009] Jindeukchal (Sigesbeckia glabrescens (Makino) Makino), also known as hee-ryeom, hee-cheom, si-cheom, and jeogog, is an annual herbaceous plant in the Asteraceae family. It is widely distributed throughout the country and can be commonly seen near fields and farms, and mainly grows in Asian countries such as China, Japan, and Taiwan. It grows to an average height of about 40 to 100 cm, and its leaves are opposite, triangular with a long central leaf width, 5 to 13 cm long and 3.5 to 11 cm wide, and the front has an irregularly serrated edge. The leaves narrow as they go up the stem, becoming oblong or linear and losing their petioles. The flowers are yellow, blooming in August and September, and are borne in corymbs at the ends of branches and main stems. There are three types of Jindeukchal in Korea: Jindeukchal, Hairy Jindeukchal, and Jeju Jindeukchal. Traditionally, it has been used in oriental medicine to eliminate dampness and treat arthritis, limb pain and paralysis, lower extremity weakness, hemiplegia due to stroke, boils, rashes, itchy skin, and eczema. It is also used to treat hypertension, headaches, dizziness, and acute hepatitis. It is known to have a blood pressure-lowering effect, so patients with hypertension can take it as tea. Recent studies have confirmed its anti-obesity effects, raising hopes for its efficacy in fatty liver disease. However, there is a dearth of research on non-alcoholic fatty liver disease.

[0010] Luffa cylindrica (L.) M. Roem., also known as Tianluo or Tianluosi, is a climbing herbaceous plant of the Cucurbitaceae family. It grows in tropical and subtropical climates and has been traditionally cultivated and used in China, Japan, and Korea. The stem can reach up to 12 m in length, and the leaves are 13-30 cm in length and width, with a rough texture and no hair. The flowers are unisexual and bloom yellow from August to September. The fruit is green, cylindrical, 30-60 cm in length, and has shallow ridges on the surface. Luffa cylindrica has a record of being used as a folk remedy for hemostasis, diuresis, detumescence, detoxification, phlegm resolution, arthralgia relief, blood circulation improvement, etc. Recent research has reported that it is an antioxidant material with excellent antibacterial and anti-inflammatory effects.

[0011] Although research results on the efficacy of the above two materials have been reported based on past experience of using them as folk remedies, there is no research on the improvement of non-alcoholic fatty liver disease (NAFLD) by the single or combined use of these two materials.

[0012] Therefore, in the present invention, the efficacy of improving fatty liver by the single or combined extract of Rheum palmatum and Luffa cylindrica was confirmed.

[0013] The object of the present invention is to provide a composition for improving fatty liver disease using the single or combined extract of Rheum palmatum and Luffa cylindrica.

[0014] Other objects or specific objects of the present invention will be presented below.

[0015] As confirmed in the following Examples and Experimental Examples, the extract of Rheum palmatum, the extract of Luffa cylindrica, and the extract of their combination do not show toxicity to liver cells, inhibit lipid accumulation and inflammation in liver cells, and also inhibit lipid accumulation and inflammation in liver tissues in a high-fat diet animal model and increase the expression of glutathione, a powerful antioxidant in liver tissues. Thus, the present invention is completed.

[0016] In consideration of the above, the present invention can be understood as a composition for improving fatty liver disease, which comprises, in one aspect, a jindeukchal extract, a susemioi extract, or an extract of a combination thereof as an effective ingredient, and in another aspect, it can be understood as a composition for preventing liver damage or improving liver function, which comprises, in another aspect, a jindeukchal extract, a susemioi extract, or an extract of a combination thereof as an effective ingredient.

[0017] In the present specification, the Jindeukchal extract refers to an extract obtained by leaching the whole plant, leaf, branch, stem, fruit, root, aerial part, underground part or mixture thereof of Jindeukchal using water, lower alcohol having 1 to 4 carbon atoms (methanol, ethanol, butanol, etc.), methylene chloride, ethylene, acetone, hexane, ether, chloroform, ethyl acetate, butyl acetate, N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 1,3-butylene glycol, propylene glycol or a mixed solvent thereof as an extraction solvent, an extract obtained by using a supercritical extraction solvent such as carbon dioxide or pentane, or a fraction obtained by fractionating the extract, and the extraction method may be any method such as cold steeping, reflux, heating, ultrasonic radiation, or supercritical extraction, taking into account the polarity, degree of extraction, and degree of preservation of the active substance. In the case of a fractionated extract, it means a fraction obtained by suspending the extract in a specific solvent and then mixing and allowing it to settle with a solvent of different polarity, and a fraction obtained by adsorbing the crude extract on a column filled with silica gel or the like and using a hydrophobic solvent, a hydrophilic solvent, or a mixed solvent thereof as a mobile phase. In addition, the meaning of the extract includes a concentrated liquid extract or solid extract from which the extraction solvent has been removed by a method such as freeze-drying, vacuum drying, hot air drying, or spray drying. Preferably, it means an extract obtained using water, an alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof as an extraction solvent, more preferably, an extract obtained using a mixed solvent of water and an alcohol having 1 to 4 carbon atoms, particularly an extract obtained using a mixed solvent of water and ethanol (ethanol content 50 to 99%) as an extraction solvent.

[0018] Also, in this specification, “active ingredient” means an ingredient that exhibits the desired activity alone or can exhibit the activity together with a carrier that is inactive in itself.

[0019] In addition, in this specification, “fatty liver disease” means fatty liver disease, which is simple steatosis, and steatohepatitis accompanied by inflammation, and particularly means non-alcoholic fatty liver disease.

[0020] In addition, in this specification, “improvement of fatty liver disease” means prevention and treatment of fatty liver disease and suppression of fat accumulation in liver tissue.

[0021] Also, in this specification, “prevention of liver damage or liver protection” means suppressing or recovering liver cell death or functional decline caused by high-fat diets, alcohol, drugs, heavy metals, carbon tetrachloride, reactive oxygen species, etc., which cause hepatocyte toxicity or lower liver function, including enhancing the function of normal hepatocytes.

[0022] The composition for improving fatty liver disease or the composition for preventing liver damage of the present invention may contain an arbitrary amount (effective amount) of the effective ingredient depending on the intended use, formulation, mixing purpose, etc., as long as the effective ingredient can exhibit fatty liver improvement activity, liver damage prevention activity, etc., and a typical effective amount will be determined within the range of 0.001 wt % to 20.0 wt % based on the total weight of the composition. Here, the "effective amount" refers to the amount of the effective ingredient included in the composition of the present invention, which can exhibit the intended functional and pharmacological effects, such as fatty liver improvement effect, liver damage prevention effect, etc., when the composition of the present invention is administered to a mammal, preferably a human, which is the subject of application, for an administration period as recommended by a medical professional. Such an effective amount can be experimentally determined within the normal ability range of a person skilled in the art.

[0023] In addition to the effective ingredient, the composition of the present invention may additionally include any compound or natural extract that has already been verified as safe and known to have the corresponding activity in the art in order to enhance or reinforce the effect of improving fatty liver, or to improve the convenience of taking or ingesting through the addition of similar activities such as anti-obesity activity, blood pressure control activity, blood lipid improvement activity, and fatigue improvement activity.

[0024] These compounds or extracts include compounds or extracts listed in the pharmacopoeia of each country (in Korea, the “Korean Pharmacopoeia”), the code of health functional foods of each country (in Korea, the “Standards and Specifications for Health Functional Foods,” a notice issued by the Ministry of Food and Drug Safety), compounds or extracts approved for sale in accordance with the laws of each country regulating the manufacture and sale of pharmaceuticals (in Korea, the “Pharmaceutical Affairs Act”), and compounds or extracts whose functionality is recognized in accordance with the laws of each country regulating the manufacture and sale of health functional foods (in Korea, the “Act on Health Functional Foods”).

[0025] For example, according to the Korean 「Health Functional Food Act」, the liver health functionalities recognized include doraji extract, milk thistle extract, fermented turmeric, bokbunja extract (powder), broccoli sprout powder, shiitake mushroom mycelia extract (powder), shiitake mushroom mycelia, lactic acid bacteria fermented garlic extract, lactic acid bacteria fermented kelp extract, and oriental walnut fruit extract, as well as Garcinia cambogia peel extract, conjugated linolenic acid (free fatty acid), conjugated linolenic acid (triglyceride), green tea extract, chitosan, Lactobacillus gasseri BNR17, L-carnitine tartrate, green mate extract, green coffee bean extract, perilla leaf extract, soybean germ extract, and complexes such as Goljoe leaf ethanol extract powder, lactoferrin (milk purified protein), lemon balm extract mixed powder, mate hot water extract, and seaweed. Extract (Xanthigen), fermented vinegar pomegranate complex, pu-erh tea extract, sesame seed bean (mouse bean) peptide complex, vegetable oil diglyceride, wild mango seed extract, oil containing medium chain fatty acid (MCFA), Coleus forskohlii extract, chitooligosaccharide, fingerroot extract powder, hibiscus complex extract, etc., and L-glutamic acid-derived GABA-containing powder with blood pressure control functionality, bonito oligopeptide, natto culture powder, sesame seed peptide complex, salmon peptide, olive leaf extract, sardine peptide, casein hydrolysate, coenzyme Q10, grape seed enzyme-decomposed extract powder, seaweed oligopeptide, etc., and DHA concentrated oil with blood triglyceride improvement functionality, globin hydrolysate, indigestible maltodextrin, bamboo leaf extract, vegetable oil diglyceride, sardine refined fish oil, refined squid oil L-arabinose, nopal extract, cinnamon extract powder, guava leaf extract, indigestible maltodextrin, freeze-dried silkworm powder, hemp extract, banaba leaf extract, mulberry leaf extract, etc., which are recognized for their blood sugar control functionality, and fermented amino acid complexes with recognized fatigue-improving functionality.Examples of such compounds or extracts include extracts of the fruit of the Chinese juniper tree, extracts of Hong Kyung-Cheon, L-theanine, ashwagandha extract, hydrolyzed milk protein, and extracts of the leaves of the Chinese juniper tree, which have been recognized for their anti-stress functionality.

[0026] One or more of these compounds or extracts may be included in the composition of the present invention together with the active ingredient.

[0027] The composition of the present invention can be understood as a food composition in a specific aspect.

[0028] The food composition of the present invention can be manufactured in any form, for example, beverages such as tea, juice, carbonated beverages, and sports beverages; processed dairy products such as milk and yogurt; foods such as gums, rice cakes, Korean traditional sweets, bread, confectionery, and noodles; and health functional food preparations such as tablets, capsules, pills, granules, liquids, powders, flakes, pastes, syrups, gels, jellies, and bars. In addition, the food composition of the present invention can have any product classification as long as it complies with the laws and regulations in effect at the time of manufacturing and distribution in terms of legal and functional classification. For example, it can be a health functional food according to the Korean 「Act on Health Functional Foods」, or confectionery, beans, tea, beverages, special-purpose foods, etc. according to each food type according to the Food Code of the Korean 「Food Sanitation Act」 (the 「Standards and Specifications of Foods」 notified by the Ministry of Food and Drug Safety).

[0029] The food composition of the present invention may contain food additives in addition to its effective ingredients. Food additives can generally be understood as substances added to, mixed with, or infiltrated into food during the manufacturing, processing, or preservation of food. Since they are consumed daily and over a long period of time with food, their safety must be guaranteed. The Food Additive Codex, which is based on the laws of each country that regulate the manufacturing and distribution of food (in Korea, the “Food Sanitation Act”), provides limited regulations on food additives with guaranteed safety in terms of ingredients or functions. The Korean Food Additive Codex (Ministry of Food and Drug Safety Notice “Food Additive Standards and Specifications”) classifies food additives into chemically synthesized products, natural additives, and mixed preparations in terms of ingredients. These food additives are classified into sweeteners, flavoring agents, preservatives, emulsifiers, acidulants, and thickeners in terms of functions.

[0030] Sweeteners are used to impart an appropriate sweetness to foods, and both natural and synthetic sweeteners can be used in the food composition of the present invention. Preferably, a natural sweetener is used. Examples of natural sweeteners include sugar sweeteners such as corn syrup solids, honey, sucrose, fructose, lactose, and maltose.

[0031] Flavoring agents are used to enhance taste or aroma, and both natural and synthetic flavors can be used. Natural flavoring agents are preferred. When using natural flavoring agents, they can also serve the purpose of enhancing nutrition in addition to flavor. Natural flavoring agents can be obtained from apples, lemons, tangerines, grapes, strawberries, peaches, etc., or from green tea leaves, Polygonum multiflorum, bamboo leaves, cinnamon, chrysanthemum leaves, jasmine, etc. Also, flavoring agents obtained from ginseng (red ginseng), bamboo shoots, aloe vera, ginkgo biloba, etc. can be used. Natural flavoring agents can be liquid concentrates or solid extracts. In some cases, synthetic flavoring agents can be used, such as esters, alcohols, aldehydes, and terpenes.

[0032] Preservatives that can be used include calcium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate, EDTA (ethylenediaminetetraacetic acid), etc.; emulsifiers that can be used include acacia gum, carboxymethylcellulose, xanthan gum, pectin, etc.; and acidulants that can be used include citric acid, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, phosphoric acid, etc. In addition to the purpose of enhancing taste, acidulants can be added to ensure that the food composition has an appropriate acidity for the purpose of inhibiting the growth of microorganisms. Thickeners that can be used include suspending agents, sedimentation agents, gel-forming agents, and puffing agents.

[0033] In addition to the food additives described above, the food composition of the present invention may include physiologically active substances or minerals known in the art and guaranteed to be safe as food additives for the purpose of supplementing and reinforcing functionality and nutrition.

[0034] Examples of such physiologically active substances include catechins contained in green tea, vitamins such as vitamin B1, vitamin C, vitamin E, and vitamin B12, tocopherol, and dibenzoylthiamine, and examples of minerals include calcium preparations such as calcium citrate, magnesium preparations such as magnesium stearate, iron preparations such as ferrous citrate, chromium chloride, potassium iodide, selenium, germanium, vanadium, and zinc.

[0035] The food composition of the present invention may include the aforementioned food additives in an appropriate amount that can achieve the purpose of addition depending on the product type.

[0036] With regard to other food additives that may be included in the food composition of the present invention, reference may be made to the food code or food additive code according to the laws of each country.

[0037] The composition of the present invention may be considered as a pharmaceutical composition in other specific embodiments.

[0038] The pharmaceutical composition of the present invention may be prepared as an oral or parenteral formulation, depending on the route of administration, by a conventional method known in the art, including a pharmaceutically acceptable carrier in addition to the active ingredient. "Pharmaceutically acceptable" herein means that the composition does not inhibit the activity of the active ingredient and does not exhibit toxicity exceeding that tolerated by the intended subject.

[0039] When the pharmaceutical composition of the present invention is prepared as an oral dosage form, it can be prepared in the form of powder, granules, tablets, pills, dragees, capsules, liquids, gels, syrups, suspensions, wafers, etc., using a suitable carrier and a method known in the art. At this time, examples of suitable pharmaceutically acceptable carriers include sugars such as lactose, glucose, sucrose, dextrose, sorbitol, mannitol, xylitol, etc.; starches such as corn starch, potato starch, and wheat starch; celluloses such as cellulose, methylcellulose, ethylcellulose, sodium carboxymethylcellulose, and hydroxypropylmethylcellulose; polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, mineral oil, malt, gelatin, talc, polyols, and vegetable oils. In case of formulation, the formulation may include diluents and / or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants, as needed.

[0040] When the pharmaceutical composition of the present invention is prepared as a parenteral dosage form, it can be formulated in the form of eye drops, injections, transdermal administration agents, nasal inhalants, or suppositories using a suitable carrier according to a method known in the art. When formulated as eye drops, suitable carriers include sterile water, saline, isotonic solutions such as 5% dextrose, and, if necessary, benzalkonium chloride, mephylparaben, ethylparaben, etc. can be added for preservative purposes. When formulated as an injection, suitable carriers include sterile water, ethanol, polyols such as glycerol or propylene glycol, or mixtures thereof, and preferably, Ringer's solution, PBS (phosphate buffered saline) containing triethanolamine, sterile water for injection, isotonic solutions such as 5% dextrose, etc. When formulated as a transdermal agent, it can be formulated in the form of ointments, creams, lotions, gels, external solutions, pastes, liniments, aerosols, etc. In the case of nasal inhalation, it can be formulated in the form of an aerosol spray using a suitable propellant such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, etc. When formulated as a suppository, the base can be witepsol, tween 61, polyethylene glycols, cacao butter, laurin butter, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, sorbitan fatty acid esters, etc.

[0041] Specific formulations of pharmaceutical compositions are known in the art and can be found, for example, in Remington's Pharmaceutical Sciences (19th ed., 1995), which is incorporated herein by reference.

[0042] The preferred dosage of the pharmaceutical composition of the present invention may range from 0.001 mg / kg to 10 g / kg per day, preferably from 0.001 mg / kg to 1 g / kg, depending on the patient's condition, weight, sex, age, severity of the condition, and route of administration. Administration may be administered once daily or divided into several doses. Such dosage should not be construed as limiting the scope of the present invention in any way.

[0043] As described above, the present invention provides a composition for improving fatty liver disease using a single or combined extract of Jindeukchal and Susemioi. The composition for improving fatty liver disease of the present invention can be commercialized as a functional food, pharmaceutical, or other product.

[0044] Figures 1 to 3 show the results of evaluating the degree of toxicity of a single extract of Jindeukchal leaves, a single extract of Susemioi fruit, and a combined extract of these on human liver cancer cells, HepG2 cells, based on cell viability.

[0045] Figures 4 to 6 show the results of evaluating the degree of inhibition of fat accumulation in HepG2 cells induced to accumulate fat with free fatty acids (FFAs) according to treatment with a single extract of Jindeukchal leaves, a single extract of Susemioi fruit, and a combination of these extracts.

[0046] Figures 7 to 12 show the results of measuring the degree of inhibition of the production of pro-inflammatory cytokines TNF-α and IL-6 according to treatment with a single extract of Jindeukchal leaves, a single extract of Susemioi fruit, and a combined extract of these in HepG2 cells induced with inflammation by free fatty acids (FFAs).

[0047] Figure 13 shows the results of measuring the AST and ALT contents in the blood following administration of a single extract of Jindeukchal leaves, a single extract of Susemioi fruit, and a combined extract of these to an animal model fed a high-fat diet.

[0048] Figure 14 shows the results of histopathological examination of liver tissue following administration of a single extract of Jindeukchal leaves, a single extract of Susemioi fruit, and a combined extract of these to an animal model fed a high-fat diet.

[0049] Figure 15 shows the results of a test evaluating the degree to which the accumulation of neutral fat in liver tissue was suppressed by administering a single extract of Jindeukchal leaves, a single extract of Susemioi fruit, and a combined extract of these to an animal model fed a high-fat diet.

[0050] Figure 16 shows the results of measuring the degree of inhibition of the production of pro-inflammatory cytokines TNF-α and IL-6 in liver tissue following administration of a single extract of Jindeukchal leaves, a single extract of Susemioi fruit, and a combined extract of these to an animal model fed a high-fat diet.

[0051] Figure 17 shows the results of measuring the expression level of glutathione, a powerful antioxidant, in liver tissue following administration of a single extract of Jindeukchal leaves, a single extract of Susemioi fruit, and a combined extract of these to an animal model fed a high-fat diet.

[0052] The present invention is described below with reference to examples and experimental examples. However, the scope of the present invention is not limited to these examples and experimental examples.

[0053]

[0054] <Example> Improvement of fatty liver disease by extracts of Jindeukchal and Susemioi, either alone or in combination

[0055] 1. Preparation of single or combined extracts of Jindeukchal and Susemioi

[0056] In this example, single extracts of Siegesbeckia glabrescens (Makino) Makino's leaf, hereinafter "SGL") and Luffa cylindrica (L.) M. Roem.'s fruit, hereinafter "LCF"), and their combined extracts ("(SGL+LCF)70E" or "SL") were prepared as follows. As shown in Table 1, 70% ethanol in an amount 10 times the dry weight was added to dried slices of Siegesbeckia glabrescens (Makino) Makino's leaf, dried slices of Luffa cylindrica (L.) M. Roem.'s fruit, and their mixtures at different ratios (original dry weight ratio), and then extraction was performed at 50°C for 6 hours using a reflux condenser. The extract was filtered using a filter paper, concentrated using a rotary vacuum evaporator, and dried using a freeze dryer to obtain a solid extract.

[0057] The obtained extracts are as shown in Table 1, consisting of one single extract of Jindeukchal, one single extract of Susemioi, and five combined extracts of two types, for a total of seven types.

[0058]

[0059] 2. Cytotoxicity Evaluation (1) MTT Assay

[0060] To determine the cytotoxicity of single or combined extracts of Jindeukchal and Susemioi on human hepatoma cell lines (HepG2 cells), an MTT assay was performed. HepG2 cells were cultured in 96-well plates (Corning), treated with the above extracts at various concentrations, and then cultured for 24 hours. MTT solution was reacted for 4 hours at 37°C under 5% CO2 conditions at a concentration of 5 mg / mL. After treating with MTT reagent and reacting for 4 hours, formazan produced in DMSO was sufficiently dissolved and measured at 540 nm to compare cell viability. The average value and standard deviation were obtained from three measurements.

[0061] The results are shown in Figs. 1 to 3. Fig. 1 shows the cytotoxicity results of the single extract of Jindeukchal leaves, the single extract of Susemi-oi fruit, and the combined extracts in the ratios of 1:1 and 1:2, Fig. 2 shows the cytotoxicity results of the single extract of Jindeukchal leaves, the single extract of Susemi-oi fruit, and the combined extracts in the ratios of 1:3 and 2:1, and Fig. 3 shows the cytotoxicity results of the single extract of Jindeukchal leaves, the single extract of Susemi-oi fruit, and the combined extract in the ratio of 3:1. The results of Figs. 1 to 3 show that the single extract of Jindeukchal leaves, the single extract of Susemi-oi fruit, and the combined extract did not exhibit cytotoxicity over the entire concentration range. It also shows that the combined extract did not exhibit cytotoxicity at any ratio.

[0062] 3. Cell-level efficacy evaluation (in vitro)

[0063] (1) Fat accumulation inhibition effect

[0064] To confirm the inhibitory efficacy of the extract of Jindeukchal alone, Susemioi alone, or their combined extracts on lipid accumulation in hepatocytes, human hepatoma cell line HepG2 cells were cultured in 12-well plates and pretreated with 5-100 μg / mL of the above extracts and 10 μM silibinin as a positive control for 2 hours. After pretreatment, 0.5 mM free fatty acid mixture (FFAs mix) containing palmitic acid and oleic acid in a 1:2 ratio was treated to induce lipid accumulation in hepatocytes. After treating with Oil red O reagent to stain lipids in hepatocytes, the degree of lipid accumulation inhibition was compared and measured at 492 nm absorbance. Silibinin, used as a positive control, is a component isolated from milk thistle extract and is known to have excellent liver disease improvement activity (World J Gastroenterol. 2011 May 14; 17(18): 2288-2301).

[0065] The results are shown in Figures 4 to 6, and the results in Figures 4 to 6 show that the single extract of Jindeukchal leaves, the single extract of Susemioi fruit, and the combined extract inhibit lipid accumulation in hepatocytes at concentrations of 50 to 100 μg / mL, comparable to the positive control level. In addition, the combined extracts at a ratio of 3:1 show particularly excellent lipid accumulation inhibition ability.

[0066] (2) Measurement of proinflammatory cytokines (TNF-α, IL-6)

[0067] HepG2 cells, a human hepatoma cell line, were cultured in 12-well plates, and the extracts were treated at 10-100 μg / mL and silibinin, a positive control, at a concentration of 10 μM, and incubated for 2 hours. After treating with 0.5 mM FFAs (containing oleate, palmitate in proportions of 2:1) and incubating for an additional 24 hours, the culture medium was centrifuged (600×g, 5 min) and the proinflammatory cytokine content of the supernatant obtained was measured. Proinflammatory cytokines were measured using RayBio 　 Human IL-6 ELISA Kit (RayBiotech, GA, USA), RayBio 　 The amount of TNF-α was quantified using a Human TNF-α ELISA Kit (RayBiotech, GA, USA) and the degree of inhibition of proinflammatory cytokines was compared by measuring the absorbance at 450 nm.

[0068] The results are shown in Figs. 7 to 12. Figs. 7 to 9 show the measurement results of TNF-α among pro-inflammatory cytokines. At concentrations of 50 to 100 μg / mL, the extract of Jindeukchal leaves alone, the extract of Susemi cucumber fruit alone, and the extract of their combination all show anti-inflammatory efficacy superior to the positive control group, and there is no difference according to the ratio of the complex extracts. Figs. 10 to 12 show the measurement results of IL-6 among pro-inflammatory cytokines, and at concentrations of 50 to 100 μg / mL, the extract of Jindeukchal leaves alone, the extract of Susemi cucumber fruit alone, and the extract of their combination all show anti-inflammatory efficacy superior to the positive control group. In terms of the ratio of the complex extracts, the complex extract with a ratio of 3:1 at a concentration of 100 μg / mL shows the best anti-inflammatory efficacy. By synthesizing the results of the above Figures 1 to 12, the optimal composite ratio of the composite extract was determined to be 3:1, and the composite with a ratio of 3:1 was used as a sample in subsequent animal experiments.

[0069] 4. Animal-level efficacy evaluation (in vivo)

[0070] (1) Laboratory animal breeding environment

[0071] Five-week-old male ICR mice were purchased from Daehan Biolink Co., Ltd. (Eumseong, Korea). After acclimating to the environment for one week, they were fed an experimental diet. The experimental environment was maintained at a temperature of 22±1℃ and a humidity of 50±5%, and the light / dark cycle was regulated to 12 h. Animal experiments were conducted with the approval of the Animal Experiment Ethics Committee of Cho-A Pharmaceutical Co., Ltd. in accordance with the operating regulations of the Animal Experiment Ethics Committee of the Cho-A Yangbyung Natural Products Research Institute (CHOA-IACUC-24-001).

[0072] (2) Efficacy evaluation

[0073] Efficacy in animal models was evaluated using a 3:1 ratio of a compound extract of Jindeukchal leaves and Susemi-oi fruit, which showed the highest activity in cell models. To confirm the synergistic effects of the Jindeukchal and Susemi-oi compound extracts, each extract was also included as a sample. Five-week-old male ICR mice were acclimated for one week and then grouped to ensure similar average body weights. The mice were divided into eight groups: normal diet group (N), 0.1% methionine-containing / choline-deficient high-fat diet control group (CDAHFD), 100 mg / kg Jindeukchal leaf extract only administration group (SGL-100), 300 mg / kg Jindeukchal leaf extract only administration group (SGL-300), 100 mg / kg Susemi cucumber fruit extract only administration group (LCF-100), 300 mg / kg Susemi cucumber fruit extract only administration group (LCF-300), 100 mg / kg complex extract administration group (SL-100), and 300 mg / kg complex extract administration group (SL-300). The normal diet group was fed 5L79 general feed containing 4% kcal fat, and the CDAHFD high-fat diet control group and sample administration groups were fed a 60% kcal fat, 0.1% methionine-containing / choline-deficient high-fat diet. The sample was administered orally, and the normal diet group and the CDAHFD high-fat diet control group were administered water orally instead of the extract. The efficacy evaluation was conducted over a period of 6 weeks.

[0074] (3) Blood biochemistry test

[0075] After the experiment, the animals were anesthetized, and blood samples were collected from the abdominal aorta. The blood samples were centrifuged at 3,000 rpm for 15 minutes, and serum was recovered. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were measured by T&P Bio (Gwangju, Korea).

[0076] The results are shown in Fig. 13. As a result of measuring AST and ALT, which are inflammation indicators of liver tissue, when the extracts of Jindeukchal leaves and Susemi cucumber fruit were administered alone, the AST and ALT levels showed statistically weak significance (p<0.05) or no significance compared to the CDAHFD high-fat diet control group, but in the group administered the complex extract of Jindeukchal leaves and Susemi cucumber fruit (mixing ratio 3:1), they were very significantly reduced (p<0.01 or p<0.001), confirming that the complex extract of Jindeukchal leaves and Susemi cucumber fruit had a synergistic effect compared to their individual extracts.

[0077] (4) Histopathological examination

[0078] To examine the condition of the liver tissue, the experimental animals that had completed oral administration for 6 weeks were anesthetized and the liver tissue was extracted. The extracted liver tissue was fixed in 10% neutral formalin, and the fixed liver tissue was stained with hematoxylin and eosin (H&E) to analyze the size and shape of fat globules and the degree of inflammation using an optical microscope. The histopathological examination and interpretation were performed by T&P Bio (Gwangju, Korea). As shown in Table 2, the NASH Clinical Research Network (CRN) scored the degree of inflammation, the degree of steatosis, and the degree of fat ballooning based on the method developed by David E. Kleiner et al. in 2005. Each score was combined to calculate the nonalcoholic fatty liver disease (NAFLD) activity score. The fat granule area (Area Fraction) was calculated by binary identifying fat granules using NIS-Elements AR 5.30.02 64-bit in photographs taken at a 200x field of view and calculating the % of fat granules compared to the total area.

[0079] The results are shown in Fig. 14 as a graph showing the excision photos, H&E staining photos, and the degree of inflammation.

[0080] Morphological observations of liver tissues revealed that the liver tissues of the negative control group (CADHFD) were enlarged and turbid, but this was significantly restored when administered the 3:1 ratio of the combined extract. Hematoxylin & E (H&E) staining revealed that the administration of the 3:1 ratio of the combined extract improved steatosis.

[0081] Additionally, H&E staining images show a significant reduction in fat vacuoles when the complex extract was administered in a 3:1 ratio compared to the negative control group (CADHFD).

[0082] The graph scoring the degree of inflammation, etc. also shows that all items were significantly improved when the complex extract was administered in a 3:1 ratio, and a more outstanding effect on improving fatty liver was shown at a dose of 300 mg / kg.

[0083]

[0084] (5) Measurement of neutral fat (TG)

[0085] Approximately 100 mg of liver tissue was divided, weighed, and homogenized with 0.9% NaCl solution using a homogenizer. Then, approximately 20 ml of Folch solution (chloroform: methanol = 2:1) was added, shaken, and left at 4°C for 12 hours to extract lipid components. The solution was centrifuged at 3000 rpm for 20 minutes at 4°C to separate the layers, and only the chloroform layer was collected and concentrated under reduced pressure to obtain lipid solids. The solids were weighed, dissolved again in a mixed solution (triton-x 100: ethanol = 1:3), and TG analysis was performed using enzyme-linked immunosorbent assay (ELISA).

[0086] The results are shown in Figure 15. The inhibitory effect on triglyceride accumulation in hepatocytes was confirmed. The single extract of Jindeukchal leaves, the single extract of Susemioi fruit, and their combined extracts all demonstrated inhibitory effects on triglyceride accumulation. In particular, administration of the combined extract at a 3:1 ratio demonstrated the greatest inhibitory effect on triglyceride accumulation.

[0087] (6) Measurement of proinflammatory cytokines

[0088] Approximately 100 mg of liver tissue was homogenized with 0.9% NaCl solution using a homogenizer. After homogenization, the layers were separated by centrifugation at 3,000 rpm for 20 minutes at 4°C, and the middle layer was used as a sample. Proinflammatory cytokines were measured using RayBio 　 Human IL-6 ELISA Kit (RayBiotech, GA, USA), RayBio 　 The amount of TNF-α was quantified using a Human TNF-α ELISA Kit (RayBiotech, GA, USA) and the degree of inhibition of proinflammatory cytokines was compared by measuring the absorbance at 450 nm.

[0089] The results are shown in Figure 16. Histopathological examination results confirmed significant inflammation in the liver tissue. TNF-α and IL-6, representative inflammation-regulating markers among pro-inflammatory cytokines, were identified in the liver tissue. The measurement results showed that the single extract of Jindeukchal leaf, the single extract of Susemi-oi fruit, and their combined extracts all exhibited excellent anti-inflammatory efficacy, and in particular, the ability to suppress the expression of inflammation-regulating cytokines was excellent when the combined extract was administered in a 3:1 ratio.

[0090] (7) Glutathione measurement

[0091] The glutathione content of each tissue was measured by adding 0.3 ml of triple-distilled water and 0.5 ml of 0.4% sulfosalicylic acid to 0.2 ml of the tissue homogenate fraction, mixing them, centrifuging them, and adding 5,5'-dithios (2-nitrobenzoic acid) (DTNB) chromogenic reagent to 0.3 ml of the supernatant, and measuring the absorbance at 412 nm using enzyme-linked immunosorbent assay (ELISA).

[0092] The results are shown in Figure 17. Glutathione is a powerful antioxidant and has been reported to have excellent detoxification effects, as well as anti-aging and immune-boosting effects. Modulating glutathione expression in liver tissue may have a positive effect on liver protection. Administration of a single extract of Jindeukchal leaves and a 3:1 ratio of the combined extracts significantly increased glutathione expression in liver tissue.

[0093] (8) Statistical analysis

[0094] All experimental results were expressed as mean and standard deviation, and comparisons between each group were performed using Student's t-test. Statistically significant results were considered when *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.

[0095] The present invention can be commercialized as a functional food, drug, etc. for the purpose of improving fatty liver disease and used industrially.

Claims

1. A composition for improving fatty liver disease, comprising an extract of Jindeukchal, an extract of Susemioi, or a combination of these extracts as an active ingredient.

2. In paragraph 1, The above Jindeukchal extract is obtained by extracting Jindeukchal leaves with water, alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof. The above-mentioned Susemi-oi fruit extract is a composition obtained by extracting Susemi-oi fruit with water, alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof.

3. In paragraph 1, A composition characterized in that the fatty liver disease is non-alcoholic fatty liver disease.

4. In paragraph 1 or 2, The above composition is a food composition.

5. In paragraph 1 or 2, The above composition is a pharmaceutical composition.

6. A food composition for preventing liver damage or improving liver function, comprising an extract of Jindeukchal, an extract of Susemioi, or a combined extract thereof as an active ingredient.

7. In paragraph 5, The above Jindeukchal extract is obtained by extracting Jindeukchal leaves with water, alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof. The above-mentioned Susemi-oi fruit extract is a composition obtained by extracting Susemi-oi fruit with water, alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof.

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