Anti-obesity composition comprising geranium wilfordii extract

Abstract

The present invention relates to an anti-obesity composition comprising a Geranium wilfordii extract. The extract according to the present invention exhibits an excellent body fat reduction effect by inhibiting lipid accumulation, and thus can be used in pharmaceutical and food fields as an anti-obesity composition.

Description

Anti-obesity composition containing extract of three-leafed rat's hand

[0001] The present invention relates to an anti-obesity composition comprising an extract of the three-leafed rattlesnake.

[0002] Obesity is a chronic physical condition characterized by excessive accumulation of adipose tissue, resulting from energy intake exceeding energy expenditure, ultimately leading to overweight. While the exact cause of obesity remains unclear, it is believed to be induced by energy imbalance, caused by excessive nutrient intake relative to energy expenditure over a long period of time. Furthermore, hormonal changes, genetics, mental health issues, and socioeconomic factors are complexly involved.

[0003] The biggest problem with obesity isn't simply the obesity itself, but rather the various metabolic diseases that arise from prolonged obesity, such as diabetes, hyperlipidemia, heart disease, stroke, arteriosclerosis, and fatty liver disease. Furthermore, obesity increases the risk of various diseases, including infertility, menstrual irregularities, degenerative arthritis, some cancers, sleep apnea, respiratory disorders, gallstones, and depression, and is recognized as a risk factor for adult diseases and a social problem.

[0004] To prevent and improve obesity and maintain a healthy weight, consistent management through a balanced diet and regular exercise is necessary. In particular, once fat cells are formed, their size can be reduced, but they cannot be eliminated naturally, remaining in the body permanently. Therefore, it's crucial to prevent obesity in the first place.

[0005] Current obesity treatments include fenfluramine, which inhibits the serotonergic nervous system; ephedrine and caffeine, which act through the noradrenergic nervous system; sibutramine, which acts simultaneously on both serotonergic and noradrenergic nervous systems; and orlistat, which inhibits lipase produced in the pancreas to reduce fat absorption. However, existing drugs such as fenfluramine have been banned due to side effects such as primary pulmonary hypertension and heart valve lesions. Other drugs also cause problems such as hypotension and lactic acidosis, making them unsuitable for patients with heart failure or renal disease.

[0006] Diet medications are categorized by their mechanism of action: either suppressing fat accumulation (appetite suppressants, lipogenesis inhibitors, etc.) or increasing fat activity (thermogenesis, lipolysis, etc.). While these medications can be prescribed after a medical examination, they can cause side effects such as digestive problems and insomnia, increasing the need for the development of natural alternatives.

[0007] Accordingly, the inventors of the present invention completed the present invention by developing an extract of natural materials with relatively few side effects and toxicity and excellent body fat reduction efficacy after much effort.

[0008] The purpose of the present invention is to provide an anti-obesity use of the extract of the three-leafed rat.

[0009] The present invention provides a pharmaceutical composition for preventing or treating obesity-related diseases containing an extract of the three-leafed rattlesnake.

[0010] At this time, the above three-leafed rat extract is preferably prepared by extracting at 30 to 70°C for 1 to 9 hours using water or ethanol as a solvent.

[0011] At this time, the obesity-related disease may be, for example, any one selected from the group consisting of obesity, diabetes, hyperlipidemia, heart disease, stroke, arteriosclerosis, kidney disease, muscle wasting, non-alcoholic fatty liver disease, and non-alcoholic steatohepatitis.

[0012] Meanwhile, the present invention provides a food composition for improving obesity containing an extract of three-leafed rat's tail.

[0013] Meanwhile, the present invention provides a body fat reduction supplement containing an extract of the three-leafed rat's hand.

[0014] The extract according to the present invention exhibits an excellent body fat reduction effect by exerting an effect of inhibiting fat accumulation, and thus can be utilized in the pharmaceutical and food fields as an anti-obesity composition.

[0015] Figure 1 shows the results of confirming the lipid accumulation inhibition effect of the extract of the three-leafed rat's hand by solvent (T1: water (distilled water), T2: 10% (v / v) ethanol aqueous solution, T3: 30% (v / v) ethanol aqueous solution, T4: 50% (v / v) ethanol aqueous solution, T5: 70% (v / v) ethanol aqueous solution).

[0016] Figure 2 shows the results of confirming the neutral fat level reduction efficacy of the extract of the three-leafed rat's hand by solvent (T1: water (distilled water), T2: 10% (v / v) ethanol aqueous solution, T3: 30% (v / v) ethanol aqueous solution, T4: 50% (v / v) ethanol aqueous solution, T5: 70% (v / v) ethanol aqueous solution).

[0017] Figure 3 shows the results of evaluating the effect of the extract of the three-leafed rat's paw on the expression of biomarkers related to fatty acid synthesis.

[0018] Figure 4 shows the results of evaluating the effect of administration of the extract of the three-leafed rat's paw on body weight change in a high-fat diet animal model (#p<0.05, ##p<0.01, ###p<0.001 G2_HFD vs. G1_ND; *p<0.05, **p<0.01, ***p<0.001 vs. G2_HFD).

[0019] Figure 5 shows the results of evaluating the effect of administration of the extract of Se-leaf Jwison on changes in body composition in a high-fat diet animal model (#p<0.05, ###p<0.001 G2_HFD vs. G1_ND; *p<0.05, **p<0.01 vs. G2_HFD).

[0020] Figure 6 shows the results of evaluating the effect of administration of the extract of the three-leafed rat's paw on changes in anti-obesity indices in a high-fat diet animal model ((#p<0.05, ##p<0.01, ###p<0.001 G2_HFD vs. G1_ND; *p<0.05, ***p<0.001 vs. G2_HFD).

[0021] Figure 7 shows the results of evaluating the effect of administration of the extract of the three-leafed rat on changes in renal damage indicators in a high-fat diet animal model (###p<0.001 G2_HFD vs. G1_ND; *p<0.05, ***p<0.001 vs. G2_HFD).

[0022] Figure 8 shows the results of evaluating the effect of administration of the extract of the three-leafed rat's paw on changes in muscle damage indicators in a high-fat diet animal model (##p<0.01 G2_HFD vs. G1_ND; *p<0.05, **p<0.01 vs. G2_HFD).

[0023] Figure 9 shows the results of evaluating the effect of administration of the extract of Se-leaf Jwison on changes in fatty liver (salt) indicators in a high-fat diet animal model (##p<0.01, ###p<0.001 G2_HFD vs. G1_ND; **p<0.01, ***p<0.001 vs. G2_HFD).

[0024] Figure 10 shows the results of evaluating the effect of administration of the extract of the three-leafed rat's paw on tissue weight changes in a high-fat diet animal model (##p<0.01, ###p<0.001 G2_HFD vs. G1_ND; *p<0.05 vs. G2_HFD).

[0025] Figure 11 shows the results of evaluating the effect of administration of the extract of the three-leafed rat's paw on changes in the size of retroperitoneal fat cells in a high-fat diet animal model.

[0026] Figure 12 shows the results of evaluating the effect of administration of the extract of the three-leafed rat's paw on changes in the size of fat vacuoles in liver tissue in a high-fat diet animal model.

[0027] To avoid confusion due to overlapping content, the description of redundant content has been omitted below. In other words, the content of the invention is not limited to the content described below, and the content of the invention should be interpreted based on the overall content of the invention.

[0028]

[0029] In one embodiment of the present invention, the optimal extraction conditions for the extract of the three-leafed rat's-eye to exhibit anti-obesity efficacy were established, and it was confirmed that the extract produced thereby exhibited an excellent body fat reduction efficacy by exhibiting an effect of inhibiting fat accumulation.

[0030] Accordingly, the present invention aims to provide a pharmaceutical composition, a food composition and a body fat reduction supplement for the anti-obesity use of the extract of Se-leaf Jwison.

[0031] The three-leafed rattlesnake is a perennial herb of the rattlesnake family, rattlesnake order, and rattlesnake plant, growing in mountainous areas. It grows 40–80 cm tall, has thick nodes, long branches that grow obliquely. The leaves are opposite, deeply divided into three lobes, and have lying hairs on the veins on the upper and lower surfaces. The lobes are lanceolate, similar to diamonds, with pointed tips and irregularly deeply serrated edges. The stipules are narrow and separate. The whole plant, along with rattlesnake, is used as an antidiarrheal. It is distributed in Korea, Japan, China, and the Amur region.

[0032] The extract of Se-leaf Jwison according to the present invention is preferably prepared by extracting with water or ethanol as a solvent at 30 to 70°C for 1 to 9 hours. More preferably, it is prepared by extracting with 5 to 87% (v / v) ethanol as a solvent at 45 to 65°C for 3 to 8 hours, and most preferably, it is prepared by extracting with 30 to 70% (v / v) ethanol as a solvent at 55 to 65°C for 5 to 7 hours. At this time, the solvent is preferably added in an amount of 5 to 20 times the weight of Se-leaf Jwison, and more preferably in an amount of 8 to 15 times the weight of Se-leaf Jwison. This is because the content of useful ingredients for exhibiting significant anti-obesity efficacy under the given conditions can be maintained.

[0033] Additionally, the extract according to the present invention may be provided in the form of a dried substance or dried powder. The drying method can utilize any known method in the art, and is not limited to the form or conditions. However, as a preferred example, the drying may be either freeze-drying or spray-drying.

[0034]

[0035] Accordingly, one aspect of the present invention provides a pharmaceutical composition for preventing or treating obesity-related diseases containing an extract of the three-leafed rat's tail.

[0036] At this time, the obesity-related disease may be, for example, any one selected from the group consisting of obesity, diabetes, hyperlipidemia, heart disease, stroke, arteriosclerosis, kidney disease, muscle wasting, non-alcoholic fatty liver disease, and non-alcoholic steatohepatitis.

[0037] According to one embodiment of the present invention, the extract according to the present invention was confirmed to exhibit excellent neutral fat reduction and lipid accumulation inhibition effects. Furthermore, in experiments using animal models, the extract according to the present invention exhibited anti-obesity effects, effectively suppressing obesity-related kidney disease, muscle loss, and non-alcoholic fatty liver disease.

[0038] Therefore, the term "prevention" used in the present invention means any action that suppresses or delays the onset of a disease affected by administration of the pharmaceutical composition according to the present invention, i.e., an obesity-related disease.

[0039] In addition, the term "treatment" used in the present invention means all acts in which the symptoms of an obesity-related disease are improved or beneficially changed by administering a pharmaceutical composition according to the present invention.

[0040] The above-described three-leafed rat extract according to the present invention may contain one or more effective ingredients exhibiting the same or similar function as the above-described ingredients.

[0041] The pharmaceutical composition of the present invention may additionally contain a pharmaceutically acceptable carrier in addition to the extract of the three-leafed rattlesnake according to the present invention.

[0042] The type of carrier that can be used in the present invention is not particularly limited, and any carrier commonly used in the relevant technical field can be used. Non-limiting examples of the carrier include lactose, dextrose, sucrose, sorbitol, mannitol, saline solution, sterile water, Ringer's solution, buffered saline, albumin injection solution, xylitol, erythritol, maltitol, maltodextrin, glycerol, ethanol, etc. These may be used alone or in combination of two or more.

[0043] In addition, the pharmaceutical composition of the present invention may be used by adding other pharmaceutically acceptable additives such as antioxidants, excipients, diluents, buffers or bacteriostatic agents, if necessary, and may be used by additionally adding surfactants, binders, fillers, bulking agents, wetting agents, disintegrants, dispersants or lubricants.

[0044] In the pharmaceutical composition of the present invention, the extract of the three-leafed rat according to the present invention may be included in an amount of 0.00001 wt% to 99.99 wt% based on the total weight of the pharmaceutical composition, preferably 0.1 wt% to 90 wt%, more preferably 0.1 wt% to 70 wt%, and even more preferably 0.1 wt% to 50 wt%, but is not limited thereto and may be variously changed depending on the condition of the administration subject, the type and degree of specific symptoms, etc. If necessary, it may also be included in the total content of the pharmaceutical composition.

[0045] That is, the pharmaceutically effective amount and effective dosage of the pharmaceutical composition of the present invention may vary depending on the method of formulating the pharmaceutical composition, the method of administration, the time of administration, and / or the route of administration, and may vary depending on various factors including the type and degree of the response to be achieved by administration of the pharmaceutical composition, the type, age, body weight, general health condition, symptoms or degree of the disease, sex, diet, excretion, drugs used simultaneously or simultaneously in the subject, other components of the composition, and similar factors well known in the medical field, and a person having ordinary skill in the art can easily determine and prescribe an effective dosage for the desired treatment. For example, the daily dosage of the pharmaceutical composition of the present invention is about 0.01 to 1,000 mg / kg, preferably 0.1 to 100 mg / kg, and may be administered once or several times a day in divided doses.

[0046] The pharmaceutical composition of the present invention may be administered once daily or divided into several doses. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with another therapeutic agent, and may be administered sequentially or simultaneously with conventional therapeutic agents. Taking all of the above factors into consideration, the dosage that achieves maximum efficacy with the minimum amount possible without causing side effects can be administered, and this amount can be readily determined by those skilled in the art.

[0047] The pharmaceutical composition of the present invention can be additionally used in combination with various methods such as hormone therapy and drug therapy to prevent or treat obesity.

[0048] The term "administration" used in the present invention means introducing the pharmaceutical composition of the present invention to a patient by any appropriate method, and the route and method of administration of the pharmaceutical composition of the present invention may be independent of each other, and any route and method of administration may be followed without particular limitation as long as the pharmaceutical composition can reach the desired site.

[0049] The above pharmaceutical composition can be administered orally or parenterally, and can be formulated into various suitable dosage forms for oral or parenteral administration.

[0050] Non-limiting examples of oral administration preparations using the pharmaceutical composition of the present invention include oily suspensions, troches, lozenges, tablets, aqueous suspensions, prepared powders, granules, emulsions, hard capsules, soft capsules, syrups, or elixirs.

[0051] In order to formulate the pharmaceutical composition of the present invention for oral administration, binders such as sorbitol, mannitol, starch, amylopectin, cellulose, lactose, saccharose or gelatin; lubricants such as magnesium stearate, calcium stearate, sodium stearyl fumarate or polyethylene glycol wax; excipients such as dicalcium phosphate; disintegrants such as corn starch or sweet potato starch; and fragrances, syrups, sweeteners, etc. can also be used. Furthermore, in the case of capsules, in addition to the above-mentioned substances, liquid carriers such as fatty oils can be additionally used.

[0052] As a parenteral administration method of the pharmaceutical composition of the present invention, intramuscular administration, transdermal administration, intravenous administration, intraperitoneal administration, or subcutaneous administration may be used, and a method of applying, spraying, or inhaling the composition to the diseased area may also be used, but is not limited thereto.

[0053] Non-limiting examples of parenteral preparations using the pharmaceutical composition of the present invention include injections, suppositories, ointments, powders for application, oils, powders for respiratory inhalation, aerosols for sprays, creams, etc.

[0054] In order to formulate the pharmaceutical composition of the present invention for parenteral administration, a sterile aqueous solution, a non-aqueous solvent, a suspension, an emulsion, a freeze-dried preparation, an external preparation, etc. can be used. As the non-aqueous solvent and suspension, vegetable oils such as olive oil, propylene glycol, polyethylene glycol, injectable esters such as ethyl oleate, etc. can be used.

[0055] When the pharmaceutical composition of the present invention is formulated as an injection, the pharmaceutical composition of the present invention may be prepared as a solution or suspension by mixing it in water with a stabilizer or buffer, and this may be formulated for unit dose in an ampoule or vial.

[0056] When the pharmaceutical composition of the present invention is formulated as an aerosol, the dispersed concentrate or wet powder may be mixed with an additive such as a propellant to ensure dispersion.

[0057] When the pharmaceutical composition of the present invention is formulated into an ointment, oil, cream, powder for application, external skin preparation, etc., it can be formulated using animal oil, vegetable oil, wax, paraffin, polyethylene glycol, silicone, bentonite, silica, talc, starch, tragacanth, cellulose derivative, zinc oxide, etc. as a carrier.

[0058]

[0059] Another aspect of the present invention provides a food composition for improving obesity comprising an extract of the three-leafed rat.

[0060] The term "improvement" of the present invention means any act in which obesity is improved or beneficially changed by administration of the composition of the present invention.

[0061] In the food composition for improving obesity of the present invention, the extract of the three-leafed rattlesnake according to the present invention is preferably included in an amount of 0.00001 to 50 wt% relative to the food composition. If it is less than 0.00001 wt%, the effect is minimal, and if it exceeds 50 wt%, the increase in effect relative to the amount used is minimal, making it uneconomical.

[0062] The food composition of the present invention may be, for example, any one selected from among noodles, gum, dairy products, ice cream, meat, grains, caffeinated beverages, general beverages, chocolate, bread, snacks, confectionery, candy, pizza, jelly, alcoholic beverages, alcohol, vitamin complexes, and other health supplements, but is not necessarily limited thereto.

[0063] When the food composition of the present invention is used as a food additive, it can be added as is or used together with other foods or food ingredients, and can be used appropriately according to a conventional method.

[0064]

[0065] Another aspect of the present invention provides a health functional food for improving obesity (reducing body fat) containing an extract of the plant, Se-leaf Jwison. The health functional food according to the present invention refers to a body fat reduction supplement.

[0066] The above “health functional food” refers to a food manufactured and processed using raw materials or ingredients with functionality useful to the human body as defined in Act No. 6727 on Health Functional Foods, and “functionality” refers to consumption for the purpose of obtaining a useful effect for health purposes, such as regulating nutrients for the structure and function of the human body or physiological effects.

[0067] The health functional food (supplement) of the present invention may typically include additional ingredients that can improve odor, taste, sight, etc. For example, it may include biotin, folate, pantothenic acid, vitamins A, C, D, E, B1, B2, B6, B12, niacin, etc. In addition, it may include minerals such as chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu), zinc (Zn), iron (Fe), calcium (Ca), etc. In addition, it may include amino acids such as cysteine, valine, lysine, and tryptophan. In addition, food additives such as preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), coloring agents (tar color, etc.), coloring agents (sodium nitrite, sodium nitrite, etc.), bleaching agents (sodium sulfite), bactericides (bleaching powder and high-purity bleaching powder, sodium hypochlorite, etc.), leavening agents (alum, D-potassium hydrogen tartrate, etc.), reinforcing agents, emulsifiers, thickeners (pasting agents), film-forming agents, antioxidants [butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), etc.], seasonings (MSG, monosodium glutamate, etc.), sweeteners (dulcin, cyclamate, saccharin, sodium, etc.), flavorings (vanillin, lactones, etc.), gum bases, antifoaming agents, solvents, and improvers can be added. The above additives can be selected depending on the type of food and used in an appropriate amount.

[0068] In the health functional food of the present invention, the content of the extract of the three-leafed rattlesnake according to the present invention is not particularly limited and may vary depending on the condition of the subject of administration, the type and degree of specific disease, etc. If necessary, it may also be included as the total content of the food.

[0069]

[0070] Hereinafter, the present invention will be described in more detail through the following examples or experimental studies. However, the scope of the present invention is not limited to the following examples or experimental studies, but also encompasses modifications of technical concepts equivalent thereto.

[0071]

[0072] [Example 1: Preparation of extract of three-leafed rattlesnake]

[0073] The purpose of this study was to prepare extracts of the three-leafed rat's hand by solvent.

[0074] To this end, a solvent was added to the crushed three-leafed rattlesnake in an amount 10 times the weight of the three-leafed rattlesnake, and extraction was performed at 60°C for 6 hours to prepare a three-leafed rattlesnake extract. The solvents used were water (distilled water), 10% (v / v) ethanol aqueous solution, 30% (v / v) ethanol aqueous solution, 50% (v / v) ethanol aqueous solution, and 70% (v / v) ethanol aqueous solution.

[0075]

[0076] [Experimental Example 1: Evaluation of Body Fat Reduction Efficacy (in vitro)]

[0077] In all experiments, the spray-dried extract of the three-leafed rattlesnake prepared in Example 1 was used.

[0078]

[0079] 1. 3T3-L1 adipocyte culture and differentiation induction

[0080] 3T3-L1 preadipocytes were cultured in DMEM (10% FBS, 1% PEST) medium and passaged until sufficient cell numbers were secured. Afterwards, 3 × 10 5 The cells were seeded and the medium was changed every 48 hours. After the cells filled the culture dish, the medium was changed and cultured for 48 hours.

[0081] Afterwards, the culture medium was replaced with a culture medium containing 0.5 μM dexamethasone, 500 μM IBMX, and 10 ng / ml insulin, and cultured for 48 hours to induce differentiation. Afterwards, the culture medium was replaced with a culture medium containing 10 ng / ml insulin, and cultured for another 48 hours to maintain differentiation induction. Afterwards, the general medium (10% FBS, 1% PEST in DMEM) was replaced twice at 48-hour intervals to complete differentiation induction.

[0082]

[0083] 2. Measurement of lipid accumulation

[0084] In the above 3T3-L1 preadipocyte differentiation induction process, after 3T3-L1 preadipocyte differentiation induction, the samples were diluted by concentration and treated when exchanging the culture medium (0 day). After 48 hours, the culture medium was exchanged with 10 ng / ㎖ of insulin, and differentiation induction was maintained by culturing for 48 hours, and the samples were diluted by concentration and treated again when exchanging the culture medium (2 days). After that, the general medium (10% FBS, 1% PEST in DMEM) was exchanged twice at 48-hour intervals to complete differentiation induction, and the samples were diluted by concentration and treated again at each medium exchange (4-6 days).

[0085] Eight days after initial sample treatment, cells were washed with PBS, treated with 10% formalin, and fixed for 1 hour at 4°C (day 8). After fixation, cells were washed with PBS and stained with 60% oil red O solution for 1 hour at room temperature. Cells were then washed with PBS, and oil red O dye was extracted with isopropanol. The absorbance was measured at 520 nm and compared with that of the control group (adipocytes).

[0086] As a result of the experiment, all experimental groups using ethanol aqueous solution of 30% (v / v) or more as a solvent showed significant lipid accumulation inhibition efficacy (Fig. 1). Fig. 1 shows the results of confirming the lipid accumulation inhibition efficacy of the extract of Se-leaf Jwison-i by solvent.

[0087]

[0088] 3. Measurement of triglyceride levels

[0089] In the above 3T3-L1 preadipocyte differentiation induction process, after 3T3-L1 preadipocyte differentiation induction, the samples were diluted by concentration and treated when exchanging the culture medium (0 day). After 48 hours, the culture medium was exchanged with 10 ng / ㎖ of insulin, and differentiation induction was maintained by culturing for 48 hours, and the samples were diluted by concentration and treated again when exchanging the culture medium (2 days). After that, the general medium (10% FBS, 1% PEST in DMEM) was exchanged twice at 48-hour intervals to complete differentiation induction, and the samples were diluted by concentration and treated again at each medium exchange (4-6 days).

[0090] Eight days after the initial sample treatment, the cells were washed with PBS, 200 μl of PBS was added to each well, the cells were harvested, and TG levels were measured using the EZ-Triglyceride Quantification Assay Kit (8 day).

[0091] The experimental results confirmed a significant neutral fat-reducing effect in all solvent samples (water and ethanol solutions) (Fig. 2). Fig. 2 shows the results confirming the neutral fat-reducing effect of the three-leafed lily extract in each solvent.

[0092] Therefore, in the following experiment, among the extracts of Saposhnikovia japonica in ethanol solution at a concentration of 30% (v / v) or higher, which commonly showed high body fat reduction efficacy in the results of fat accumulation analysis and neutral fat level measurement, the extract of Saposhnikovia japonica in ethanol solution at a concentration of 30% (v / v) or higher was used for economic reasons.

[0093]

[0094] 4. Protein expression measurement

[0095] In this experiment, we aimed to analyze the effect of the extract of Sativa japonica on the expression of biomarkers that regulate lipogenesis.

[0096] LKB1 encodes a serine / threonine kinase that directly phosphorylates and activates AMPK, a central metabolic sensor. AMPK acutely inhibits fatty acid and cholesterol synthesis through direct phosphorylation of the metabolic enzymes acetyl-CoA carboxylase (ACC) and HMG-CoA reductase (HMGR).

[0097] ATGL (adipose triglyceride lipase) catalyzes the initial step of triglyceride breakdown, converting triglycerides into diacylglycerol (DG).

[0098] G0S2 (G0G1 switch protein 2) is found in highest concentrations in adipose tissue and liver, and G0S2 expression is very low in adipose tissue during fasting but increases after a meal.

[0099] Perilipin is a lipid droplet envelope protein and a major adipocyte protein that increases triacylglyceride (TAG) storage by decreasing the rate of TAG hydrolysis.

[0100] For the experiment, the medium of the differentiated 3T3-L1 preadipocytes was replaced with serum-free medium (containing only 1% PEST) and cultured for 18 hours (serum starvation). Samples were then diluted to various concentrations and cultured for 24 hours. The culture medium was then removed, the cells were washed with PBS, lysed with RIPA buffer, and centrifuged at 8,000 rpm for 10 minutes.

[0101] After recovering the supernatant, the protein content was quantified to 20 μg using the Bradford method. The mixture was then mixed with sample buffer at a 3:1 ratio, heated at 100°C for 10 minutes, and cooled. The protein sample was electrophoresed and transferred to a PVDF membrane, then blocked with 8% skim milk (in TBST) for 1 hour. After washing three times with TBST, the membrane was treated with antibodies and incubated at 4°C for 18 hours. After washing three more times with TBST, the membrane was treated with secondary antibodies and incubated at 4°C for 1 hour. After washing three times with TBST, the membrane was treated with ECL solution, film developed, and the bands were quantified using the ImageJ program and compared with the control group (adipocytes).

[0102] Experimental results showed that the extract of Sativa japonica stimulated the expression of LKB1 and AMPK, which are involved in the inhibition of fatty acid and cholesterol synthesis, and ATGL, which is involved in the breakdown of neutral fat, and suppressed the expression of perilipin, which is involved in the storage of G0S2 and TAG, which are found in large quantities in fat and liver, by reducing their hydrolysis rate (Fig. 3). Fig. 3 shows the results of evaluating the effect of Sativa japonica extract on the expression of biomarkers related to fatty acid synthesis.

[0103] Therefore, it was determined that the extract of the three-leafed rat was effective in reducing body fat.

[0104]

[0105] [Experimental Example 2: Evaluation of Body Fat Reduction Efficacy (in vivo)]

[0106] The purpose of this study was to evaluate the anti-obesity effect of the extract of Sativa japonica L. in a 12-week high-fat diet-induced obesity model using male SD rats. The extract of Sativa japonica L. was used as an extract (NG-GT-3TL) prepared with a 30% (v / v) ethanol aqueous solution.

[0107] For this purpose, 48 6-week-old male SD rats free of specific pathogens were purchased and used as experimental animals. After a one-week quarantine and acclimatization period, healthy animals without weight loss were selected for use in the experiment. The experimental animals were housed in a SPF facility under the following conditions: temperature 23±3℃, relative humidity 50±20%, ventilation rate 10–15 times / hour, lighting time 12 hours, and light intensity 150–200 Lux. During the acclimatization and testing periods, the animals were housed three per cage made of polysulfone with PG3 beta chips (lavendi PG-3, LASvendi, Germanry) in an SPF facility. During the acclimatization period, the experimental animals were fed a dedicated feed (Teklad global 18% protein, Cat No. 2018C, Enviog) sterilized by UV irradiation and had free access to tap water sterilized by an autoclave.

[0108] For the high-fat diet group including the test substance treatment group, obesity was induced using a 60% kcal high fat feed (D12492, Rodent Diet With 60 kcal%, Research Diets, USA) for a 12-week test period.

[0109] Meanwhile, for the test group and test substance administration, after a one-week adaptation period, healthy animals were selected and classified into G1_ND (normal feed group), G2_HFD (high-fat feed group), G3_HFD_HCA (positive control group), G4_HFD (low dose of test substance (30 mg / kg)), G5_HFD (medium dose of test substance (60 mg / kg)), and G6_HFD (high dose of test substance (90 mg / kg)) administration groups based on the egg block method. The specific test group classification and diet are shown in Table 1 below.

[0110] Test groupTest substanceAdministration dose(mg / kg)Administration amount(ml / kg)Administration cycleAdministration routeNumber of animalsG1_ND(normal feed group)0.1% MC-5QD / 12WPO8G2_HFD(high-fat feed group)0.1% MC-8G3_HFD_HCA(positive control group)HCA2008G4_HFD(low dose of test substance)NG-GT-3TL308G5_HFD(medium dose of test substance)NG-GT-3TL608G6_HFD(high dose of test substance)NG-GT-3TL908

[0111]

[0112] All test data in the following experiments are expressed as mean±SD. If an individual's data exceeded the mean±SD, it was excluded as an outlier. Statistical significance was tested for test data using ANOVA using the acquired data. Post hoc tests were performed using the Dunnett or Newman-Keuls method. If the variances were not equal, ANOVA on ranks was used. Statistical methods were implemented using GraphPad Prism 8.4.3, a widely used commercial statistical package.

[0113] 1. Weight measurement

[0114] Initial body weights of test animals were measured upon separation from each group, and again twice weekly (Mondays and Thursdays). Furthermore, fasting body weights were measured after 10 hours of fasting before necropsy.

[0115] As a result of the experiment, a significant weight loss was confirmed in the control group administered HCA 200 mg / kg and the groups administered all doses of the three-leafed rat extract compared to the high-fat diet-fed group (Fig. 4). Fig. 4 shows the results of evaluating the effect of administration of the three-leafed rat extract on body weight changes in a high-fat diet animal model.

[0116]

[0117] 2. Echo-MRI body composition analysis

[0118] Body composition was analyzed using Echo-MRI before autopsy.

[0119] As a result of the experiment, fat mass was significantly reduced in the groups administered medium-dose (60 mg / kg) and high-dose (90 mg / kg) of the extract of Se-leaf Jwison compared to the high-fat feed group, but no significant reduction was observed in the group administered HCA 200 mg / kg.

[0120] Considering that there was no change in feed intake in the HCA and Sae-yeop-jwi-son-i extract administration groups compared to the high-fat diet feeding group, this anti-obesity effect can be judged to be due to an increase in energy expenditure (Fig. 5). Fig. 5 shows the results of evaluating the effect of Sae-yeop-jwi-son-i extract administration on body composition changes in a high-fat diet animal model (#p<0.05, ###p<0.001 G2_HFD vs. G1_ND; *p<0.05, **p<0.01 vs. G2_HFD).

[0121]

[0122] 3. Serum biochemical analysis

[0123] During necropsy, the test animals were anesthetized with 2% isoflurane, and the abdomen was opened to collect blood from the abdominal aorta. Blood collected in SST tubes (367957, BD STT II vacutainer, BD, USA) was centrifuged at 5,000 rpm for 10 minutes to obtain serum, which was then placed in tubes labeled with the animal number and stored in an ultra-low temperature freezer (-80°C).

[0124] Afterwards, the levels of total cholesterol, low density lipoprotein (LDL), triglyceride (TG), free fatty acid (FFA), blood urea nitrogen (BUN), creatinine, lactate dehydrogenase (LDH), creatine kinase (CK), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) were measured using an automatic serum biochemical analyzer (AU680, BECKMAN COULTER).

[0125] 1) Anti-obesity indicators

[0126] Total cholesterol (CHOL) and low-density lipoprotein (LDL) levels, which are blood biochemical evaluation indices associated with obesity, increased only in the high-fat diet feeding group. Meanwhile, significant decreases in triglyceride (TG) and free fatty acid (FFA) levels were confirmed in both the positive control HCA and the three-leafed licorice extract administration groups (Fig. 6). Fig. 6 shows the results of evaluating the effect of three-leafed licorice extract administration on changes in anti-obesity indices in a high-fat diet animal model ((#p<0.05, ##p<0.01, ###p<0.001 G2_HFD vs. G1_ND; *p<0.05, ***p<0.001 vs. G2_HFD).

[0127] Therefore, it was determined that the extract of the three-leafed rat was effective in preventing obesity.

[0128]

[0129] 2) Kidney damage indicators

[0130] The levels of blood urea nitrogen (BUN), creatinine, and lactate dehydrogenase (LDH), which are indicators of renal damage, were significantly reduced in both the HCA administration group and the Saenippus japonica extract administration group compared to the high-fat diet feeding group (Fig. 7). Fig. 7 shows the results of evaluating the effect of Saenippus japonica extract administration on changes in renal damage indicators in a high-fat diet animal model (###p<0.001 G2_HFD vs. G1_ND; *p<0.05, ***p<0.001 vs. G2_HFD).

[0131] Therefore, it was determined that the extract of the three-leafed rat's tail could reduce the possibility of developing kidney disease caused by obesity.

[0132]

[0133] 3) Muscle damage indicators

[0134] In the case of creatine kinase (CK), which is an indicator of muscle damage such as sarcopenia, a significant decrease was confirmed in both the HCA administration group and the Sativa japonica extract administration group compared to the high-fat diet feeding group (Fig. 8). Fig. 8 shows the results of evaluating the effect of Sativa japonica extract administration on changes in muscle damage indicators in a high-fat diet animal model (##p<0.01 G2_HFD vs. G1_ND; *p<0.05, **p<0.01 vs. G2_HFD).

[0135] Therefore, it was determined that the extract of the three-leafed rat was able to suppress the occurrence of muscle loss that occurs in obesity.

[0136]

[0137] 4) Fatty liver indicators

[0138] The levels of AST, ALT, and ALP, which are indicators associated with hepatic fat accumulation and inflammatory damage, were significantly reduced in both the HCA administration group and the Saenipjusoni extract administration group compared to the high-fat diet feeding group (Fig. 9). Fig. 9 shows the results of evaluating the effect of Saenipjusoni extract administration on changes in fatty liver (salt) indicators in a high-fat diet animal model (##p<0.01, ###p<0.001 G2_HFD vs. G1_ND; **p<0.01, ***p<0.001 vs. G2_HFD).

[0139] Therefore, it was determined that the extract of Se-leaf Jwison showed a therapeutic effect on non-alcoholic fatty liver disease or non-alcoholic steatohepatitis.

[0140]

[0141] 4. Histopathological analysis and interpretation

[0142] During necropsy, test animals were anesthetized with 2% isoflurane, laparotomy was performed, and blood was collected from the abdominal aorta. After blood collection, the abdominal aorta and vein were incised to exsanguineate the animals, and the weights of the liver, retroperitoneal fat, epididymal fat, and visceral fat were measured and fixed in 10% neutral formalin.

[0143] At autopsy, fixed liver and retroperitoneal fat tissue were dehydrated, transparent, and infiltrated using a tissue processor (Tissue-Tek VIP 5 Jr, SAKURA Fineteck, Japan). Then, paraffin-embedded in an embedding center (LEICA Eg1150H, LEICA, Germany), 5 ㎛-thick sections were prepared using a rotary microtome (HM 340E, Thermo scientific, USA), and then deparaffinized, hydrated, and stained with hematoxyline and eosin (H&E).

[0144] Image analysis was performed under a microscope using the DIXI eXcope image analysis program to measure the lipid droplet area in stained liver tissue to evaluate steatosis (% area), and at least 10 adipocytes were measured in the retroperitoneal fat to determine the average size (㎛).

[0145] 1) Histopathological analysis

[0146] As a result of histopathological evaluation of the liver, the area of ​​fat globules accumulated in the liver was significantly reduced in the group administered the extract of Se-leaf Jwison-i compared to the high-fat diet feeding group, but this effect was not observed in the positive control group administered the HCA (Fig. 10). Fig. 10 shows the results of evaluating the effect of administration of the extract of Se-leaf Jwison-i on tissue weight changes in a high-fat diet animal model (##p<0.01, ###p<0.001 G2_HFD vs. G1_ND; *p<0.05 vs. G2_HFD).

[0147] Therefore, it was determined that the extract of Se-leaf-Jwi-Son-I showed a therapeutic effect on non-alcoholic fatty liver disease.

[0148]

[0149] 2) Histopathological interpretation

[0150] In H&E staining of retroperitoneal adipose tissue, the size of adipocytes was significantly reduced in a dose-dependent manner in all groups (Groups 4-6) administered the extract of Se-leaf-ju-soni compared to the high-fat diet group (Group 2). In particular, in the groups administered medium or higher doses of Se-leaf-ju-soni extract (Groups 5-6), the size of adipocytes was observed to be similar to that of the positive control group, HCA-administered group (Group 3) (Fig. 11). Fig. 11 shows the results of evaluating the effect of administration of Se-leaf-ju-soni extract on changes in the size of retroperitoneal adipocytes in a high-fat diet animal model.

[0151] Meanwhile, the area occupied by fat vacuoles in H&E staining of liver tissues significantly increased in the high-fat diet-fed group (Group 2), and the area of ​​fat vacuoles in the positive control HCA-administered group (Group 3) also increased significantly, as expected. On the other hand, the area of ​​fat vacuoles was significantly reduced in the groups administered medium or higher doses of the Sativa japonica extract (Groups 5-6) compared to the high-fat diet-fed group (Group 2) (Fig. 12). Fig. 12 shows the results of evaluating the effect of the administration of Sativa japonica extract on the change in the size of fat vacuoles in liver tissues in a high-fat diet animal model.

[0152] In summary of the above results, the efficacy of the extract of Se-leaf Jwison-i was confirmed in reducing the size of retroperitoneal fat cells and fat vacuole deposition in liver tissue, and it was determined that the extract of Se-leaf Jwison-i was effective in treating obesity and fatty liver.

Claims

1. A pharmaceutical composition for preventing or treating obesity-related diseases containing an extract of the three-leafed rat's hand.

2. In paragraph 1, The above three-leafed rat extract is, A pharmaceutical composition using water or ethanol as a solvent.

3. In paragraph 2, The above three-leafed rat extract is, A pharmaceutical composition prepared by extraction at 30 to 70°C for 1 to 9 hours.

4. In paragraph 1, The above obesity-related diseases are: A pharmaceutical composition, wherein the pharmaceutical composition is selected from the group consisting of obesity, diabetes, hyperlipidemia, heart disease, stroke, arteriosclerosis, kidney disease, muscle wasting, non-alcoholic fatty liver disease and non-alcoholic steatohepatitis.

5. A food composition for improving obesity containing an extract of the three-leafed rat's hand.

6. A fat reduction supplement containing the extract of the three-leafed rat's hand.

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