Anti-Hyperlipidemic or Anti-Obesity Composition Using an Extract of Smilax sieboldii

Abstract

The present invention discloses an antihyperlipidemia or anti-obesity composition using a *Clematis terniflora* extract having activity that inhibits, in a concentration-dependent manner, the differentiation of mouse preadipocytes (3T3-L1) into adipocytes, which are used for candidate drug screening for hyperlipidemia, obesity, etc.

Description

Technology Field

[0001] The present invention relates to the blue thorn vine ( Smilax sieboldii This relates to a composition for improving hyperlipidemia or obesity using an extract. Background Technology

[0002] Due to the Westernization of dietary habits, increased consumption of processed foods, and lack of exercise resulting from the development of transportation, the number of patients with lifestyle diseases such as obesity, hyperlipidemia, diabetes, hypertension, and arteriosclerosis is increasing (Lee HJ. 2009. MS Thesis. Changwon University).

[0003] According to data released by the Ministry of Health and Welfare in 2019, the severely obese population in South Korea is projected to steadily increase and reach 9% of the total population by 2030, doubling from the current level. Furthermore, it was announced that among OECD countries, the obesity rate of male children and adolescents is higher than the OECD average, raising concerns that socio-economic problems caused by obesity will intensify in 10 years when they become adults. According to data on causes of death released by Statistics Korea in 2018, heart disease and cerebrovascular disease account for the first and second leading causes of death, excluding cancer, and hypertension and diabetes are on the rise due to population aging and changes in lifestyle.

[0004] Hyperlipidemia is one of the major risk factors for cardiovascular diseases, such as cerebrovascular disease, and refers to a condition in which lipids such as cholesterol, phospholipids, and triglycerides are abnormally increased in the blood. Hyperlipidemia can manifest in three forms: (i) hypercholesterolemia, (ii) hypertriglyceridemia, and (ii) a combination of these (hypercholesterolemia and hypertriglyceridemia). Although hyperlipidemia itself does not present with specific symptoms, high levels of lipids in the blood can adhere to the walls of blood vessels, causing atherosclerosis, and may lead to angina pectoris, myocardial infarction, cerebrovascular disease, and peripheral vascular occlusion (E. Falk et al., Circulation 92, 657-671, 1995).

[0005] Drugs used to treat hyperlipidemia include drugs that lower blood cholesterol, such as HMG-CoA reductase inhibitors and bile acid binding resins; drugs that lower blood triglycerides, such as fibrate derivatives and nicotinic acid derivatives; and drugs that promote lipid metabolism, such as heparin derivatives. However, these drugs have been reported to have side effects such as liver toxicity, gastrointestinal disorders, cholelithiasis, and carcinogenicity.

[0006] Therefore, it can be said that there is still a need to develop drugs that are highly safe and effective in treating hyperlipidemia.

[0007] Meanwhile, 3T3-L1, a preadipocyte derived from mouse embryos, is used in drug screening for obesity and hyperlipidemia, and in studies on the mechanisms of lipid metabolism disorders. In vitroPre-adipocytes, which have been widely used as models (Biotechnology and Bioprocess Engineering 20(1):157-167, 2015; Prev Nutr Food Sci (17):1~7, 2012; J. Antibiot. 58(10): 634-39, 2005), differentiate into adipocytes through a complex process involving morphological changes, hormone sensitivity, and the expression of genes and proteins. It is known that PPARγ (peroxisome proliferator-activated receptor γ) and the C / EBP family (CCAAT / enhancer binding proteins), C / EBRβ and C / EBRδ, play a pivotal role in early differentiation, while C / EBRα and PPARγ (peroxisome proliferator-activated receptor γ) play a pivotal role in late differentiation (Genes De 2000, 14(11) 1293~1307) (Physiol Rev 1998, 78(3):783~809; Annu Rev Biochem 2008, 77:289~312; Genes Dev 1996, 10:1096~1107). During this differentiation of adipocytes, triglyceride accumulation occurs and the expression of adipocyte-specific proteins (aP2, FAS, GLUT4, LPL, leptin) is induced.

[0008] The present invention discloses the antihyperlipidemia and anti-obesity activities of *Clematis terniflora* extract based on the differentiation inhibitory activity and fat accumulation inhibitory activity of mouse-derived adipocyte progenitor cells 3T3-L1. The problem to be solved

[0009] The objective of the present invention is to provide a composition for antihyperlipidemia using an extract of *Clematis terniflora*.

[0010] Another objective of the present invention is to provide an anti-obesity composition using a blue thorn vine extract.

[0011] Another object or specific object of the present invention will be presented below. means of solving the problem

[0012] The present invention is completed by confirming that the extract of *Clematis terniflora* inhibits the differentiation of mouse adipocytes into adipocytes and the accumulation of fat in a concentration-dependent manner, as confirmed in the following examples and experimental examples.

[0013] Considering the foregoing, the present invention can be understood in one aspect as a composition for antihyperlipidemia having a blue thorn extract as an active ingredient, and in another aspect as a composition for anti-obesity having a blue thorn extract as an active ingredient.

[0014] In this specification, "Clematis extract" refers to an extract obtained by leaching the stem, leaf, fruit, flower, root, whole plant, above-ground parts, underground parts, or a mixture thereof of the *Clematis* plant to be extracted using water, a 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 mixture of these solvents, an extract obtained using a supercritical extraction solvent such as carbon dioxide or pentane, or a fraction obtained by fractionating the extract. The extraction method may be any method such as cold maceration, reflux, heating, ultrasonic radiation, or supercritical extraction, taking into account the polarity of the active substance, the degree of extraction, and the degree of preservation. The term "fractionated extract" includes fractions obtained by suspending an extract in a specific solvent and then mixing and settling it with a solvent of different polarity, as well as fractions obtained by adsorbing the crude extract onto a column packed with silica gel or the like and using a hydrophobic solvent, a hydrophilic solvent, or a mixture thereof as the mobile phase. Furthermore, the term "extract" includes concentrated liquid or solid extracts from which the extraction solvent has been removed by methods such as freeze-drying, vacuum drying, hot-air drying, or spray drying. Preferably, it refers to an extract obtained using water, ethanol, or a mixture thereof as the extraction solvent, and more preferably, an extract obtained using a mixture of water and ethanol as the extraction solvent.

[0015] In this specification, the term "active ingredient" means an ingredient that exhibits the intended activity alone or can exhibit activity together with a carrier that is inactive itself.

[0016] Also, in this specification, "antihyperlipidemia" means lowering the concentration of triglycerides in the blood or lowering the concentration of cholesterol in the blood, or lowering both the concentration of triglycerides and the concentration of cholesterol in the blood.

[0017] In addition, in this specification, "anti-obesity" means reducing body fat or reducing body weight. Therefore, it includes the prevention or treatment of obesity, and further includes reducing body weight / body fat for cosmetic or health purposes even when the body weight is not classified as obese or overweight.

[0018] In the composition for antihyperlipidemia or anti-obesity of the present invention (hereinafter referred to as the "composition of the present invention"), the active ingredient may be included in any amount (effective amount) depending on the use, formulation, etc., as long as it can exhibit an antihyperlipidemia effect, an anti-obesity effect, etc., and the typical effective amount will be determined within the range of 0.0001 weight % to 15 weight % based on the total weight of the composition. Here, "effective amount" refers to the amount of the active ingredient included in the composition of the present invention that can exhibit intended medical and pharmacological effects, such as an antihyperlipidemia effect or an anti-obesity effect, when the composition of the present invention is administered to mammals, preferably humans, the subjects of application, for a period of administration as recommended by medical professionals, etc. Such effective amount may be determined experimentally within the ordinary capacity of a person skilled in the art.

[0019] In addition to the active ingredient, the composition of the present invention may additionally include any compound or natural extract whose safety has already been verified and whose activity has been confirmed in the art, for the purpose of enhancing or reinforcing the anti-hyperlipidemia effect and anti-obesity effect, or for the convenience of taking or consuming through the addition of similar activities such as blood circulation improvement activity, blood sugar control activity, and blood pressure control activity.

[0020] These compounds or extracts include compounds or extracts listed in pharmacopoeias of each country (in Korea, the “Korean Pharmacopoeia”), health functional food codes of each country (in Korea, the “Standards and Specifications for Health Functional Foods” notified by the Ministry of Food and Drug Safety), compounds or extracts for which product approval has been obtained 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 for which functionality has been individually 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”). For example, as individually recognized ingredients pursuant to the Korean Codex of Health Functional Foods (Ministry of Food and Drug Safety Notification "Standards and Specifications for Health Functional Foods") or the Korean "Act on Health Functional Foods," ingredients having body fat reduction functionality such as Garcinia cambogia peel extract, conjugated linolenic acid (free fatty acid), conjugated linolenic acid (triglyceride), green tea extract, chitosan, green mate extract, green coffee bean extract, perilla leaf extract, complex extract of soybean germ, etc., Gynostemma pentaphyllum leaf ethanol extract powder, lactoferrin (milk refined protein), mixed powder of lemon balm extract, hot water mate extract, or complex extract of seaweed (Xantigen), etc., or ingredients having blood cholesterol improvement functionality such as green tea extract or spirulina, bamboo leaf extract, barley beta-glucan extract, Pu-erh tea extract, plant stanol ester, Ecklonia cava ethanol extract, flaxseed, aloe complex extract or aloe extract, etc., or ingredients having blood triglyceride improvement functionality such as DHA concentrated oil, indigestible maltodextrin, Bamboo leaf extract, vegetable oil diglycerides, refined squid oil, globin hydrolysate or refined sardine oil, ginkgo leaf extract with blood circulation improving function, natto culture powder, natto culture, melon extract or cacao powder or red ginseng extract, etc. would be considered as such compounds or extracts.

[0021] One or more of these compounds or extracts may be included in the composition of the present invention together with the active ingredients.

[0022] The composition of the present invention can be understood as a food composition in specific embodiments.

[0023] The food composition of the present invention can be manufactured in any form, for example, as beverages such as tea, juice, carbonated drinks, and isotonic drinks; processed dairy products such as milk and yogurt; chewing gums; rice cakes, Korean confectionery, bread, cookies, and noodles; and health functional food preparations such as tablets, capsules, pills, granules, liquids, powders, flakes, pastes, syrups, gels, jellies, and bars.

[0024] In addition, the food composition of the present invention may have any product classification in terms of legal and functional classification, as long as it complies with the regulations in effect at the time of manufacture and distribution. For example, it may be a health functional food under the Korean "Act on Health Functional Foods," or a confectionery, legume, tea, beverage, special dietary food, etc., according to each food type in the Food Code of the Korean "Food Sanitation Act" (Notification of the Ministry of Food and Drug Safety "Standards and Specifications for Food").

[0025] The food composition of the present invention may include food additives in addition to its active ingredients. Food additives can generally be understood as substances added to, mixed with, or permeated into food during the manufacture, processing, or preservation of food; since they are consumed daily and over a long period of time along with food, their safety must be guaranteed. Food additive codes in accordance with national laws governing the manufacture and distribution of food (the "Food Sanitation Act" in Korea) restrictively define food additives with guaranteed safety in terms of composition or function. The Korean Food Additive Code (Notification of the Ministry of Food and Drug Safety, "Standards and Specifications for Food Additives") classifies food additives into chemically synthesized products, natural additives, and mixed preparations in terms of composition, and these food additives are classified into sweeteners, flavorings, preservatives, emulsifiers, acidulants, thickeners, etc., in terms of function.

[0026] Sweeteners are used to impart a suitable sweetness to food, and both natural and synthetic sweeteners can be used in the composition of the present invention. Preferably, natural sweeteners are used, and examples of natural sweeteners include corn syrup solids, honey, sucrose, fructose, lactose, maltose, etc.

[0027] Flavoring agents can be used to improve taste or aroma, and both natural and synthetic ones may be used. Preferably, natural ones are used. When natural ones are used, nutritional enhancement can be achieved in addition to flavor. Natural flavoring agents may be obtained from apples, lemons, citrus fruits, grapes, strawberries, peaches, etc., or from green tea leaves, Solomon's seal, bamboo leaves, cinnamon, chrysanthemum leaves, jasmine, etc. Additionally, those obtained from ginseng (red ginseng), bamboo shoots, aloe vera, ginkgo, etc., may be used. Natural flavoring agents may be liquid concentrates or solid extracts. In some cases, synthetic flavoring agents may be used, and synthetic flavoring agents may include esters, alcohols, aldehydes, terpenes, etc.

[0028] Calcium sodium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate, EDTA (ethylenediaminetetraacetic acid), etc. may be used as preservatives, and acacia gum, carboxymethylcellulose, xanthan gum, pectin, etc. may be used as emulsifiers, and acetic acid, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, phosphoric acid, etc. may be used as acidifiers. Acidifiers may be added to the food composition to achieve an appropriate acidity, in addition to for the purpose of enhancing flavor, for the purpose of inhibiting the growth of microorganisms.

[0029] As thickening agents, suspending agents, settling agents, gel-forming agents, puffing agents, etc. may be used.

[0030] 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 whose stability as food additives is guaranteed, for the purpose of supplementing and reinforcing functionality and nutritional value.

[0031] 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, dibenzoylthiamine, etc., and examples of minerals include calcium preparations such as calcium citrate, magnesium preparations such as magnesium stearate, iron preparations such as iron citrate, chromium chloride, potassium iodide, selenium, germanium, vanadium, zinc, etc.

[0032] The food composition of the present invention may include the food additives described above in an appropriate amount to achieve the purpose of their addition, depending on the product type.

[0033] Regarding other food additives that may be included in the food composition of the present invention, reference may be made to the food codes or food additive codes of each country.

[0034] The composition of the present invention may be understood as a pharmaceutical composition in other specific embodiments.

[0035] The pharmaceutical composition of the present invention may be prepared as an oral or parenteral formulation according to the route of administration by conventional methods known in the art, including a pharmaceutically acceptable carrier in addition to the active ingredient. The route of administration may be any suitable route including a local route, an oral route, an intravenous route, an intramuscular route, and direct absorption through mucosal tissues, and may also be used in combination of two or more routes. An example of a combination of two or more routes is a case where two or more formulations of drugs according to the route of administration are combined, for example, one drug is administered first via the intravenous route and another drug is administered second via the local route.

[0036] Pharmaceutically acceptable carriers are known in the art according to the route of administration or dosage form, and specifically, one may refer to the pharmacopoeias of each country, including the "Korean Pharmacopoeia."

[0037] When the pharmaceutical composition of the present invention is prepared as an oral formulation, it may be prepared in the form of powder, granules, tablets, pills, coated tablets, capsules, liquids, gels, syrups, suspensions, wafers, etc., in accordance with methods known in the art together with a suitable carrier. Examples of suitable carriers include sugars such as lactose, glucose, sucrose, dextrose, sorbitol, mannitol, and xylitol; starches such as corn starch, potato starch, and wheat starch; celluloses such as cellulose, methylcellulose, ethylcellulose, sodium carboxymethylcellulose, and hydroxypropylmethylcellulose; polyvinylpyrrolidone; water; methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate; mineral oil; malt; gelatin; talc; polyols; vegetable oils; ethanol; glycerol, etc. When formulating, appropriate binders, lubricants, disintegrants, coloring agents, diluents, etc. may be included as needed. Suitable binders include starch, magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone, glucose, corn sweeteners, sodium alginate, polyethylene glycol, wax, etc. Suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, silica, talcum, stearic acid, its magnesium and calcium salts, polyethylene glycol, etc. Examples of disintegrants include starch, methylcellulose, agar, bentonite, xanthan gum, starch, alginic acid, or its sodium salt, etc. Other diluents include lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, glycine, etc.

[0038] When the pharmaceutical composition of the present invention is prepared as a parenteral formulation, it may be formulated in the form of an injectable, transdermal, nasal inhalant, and suppository according to methods known in the art with a suitable carrier. When formulated as an injectable, an aqueous isotonic solution or suspension may be used as a suitable carrier; specifically, isotonic solutions such as PBS (phosphate buffered saline) containing triethanolamine, sterile water for injection, or 5% dextrose may be used. When formulated as a transdermal formulation, it may be formulated in the form of an ointment, cream, lotion, gel, topical solution, paste, liniment, aerosol, etc. In the case of nasal inhalers, they can be formulated in the form of an aerosol spray using suitable propellants such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, and carbon dioxide, and when formulated as suppositories, the carriers may include Witepsol, Tween 61, polyethylene glycols, cocoa starch, laurin starch, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearate, and sorbitan fatty acid esters.

[0039] Specific formulations of pharmaceutical compositions are known in the art, and reference may be made to literature such as [Remington's Pharmaceutical Sciences (19th ed., 1995)]. The said literature is to be considered part of this specification.

[0040] A preferred dosage of the pharmaceutical composition of the present invention may be in the range of 0.001 mg / kg to 10 g / kg per day, preferably 0.001 mg / kg to 1 g / kg, depending on the patient's condition, body weight, gender, age, severity of the patient, and route of administration. Administration may be administered once a day or divided into several doses. Such dosages shall not be construed as limiting the scope of the present invention in any aspect. Effects of the invention

[0041] As described above, according to the present invention, an antihyperlipidemia composition and an anti-obesity composition using a *Clematis terniflora* extract can be provided. The antihyperlipidemia composition and the anti-obesity composition of the present invention can be commercialized into products such as pharmaceuticals and functional foods. Brief explanation of the drawing

[0042] Figure 1 shows the results demonstrating that the extract of *Clematis terniflora* has concentration-dependent inhibitory activity on fat accumulation in adipocytes. Specific details for implementing the invention

[0043] The present invention will be explained below with reference to examples and experimental examples. However, the scope of the present invention is not limited to these examples and experimental examples.

[0044] <Example> Preparation of Blueberry Extract

[0045] Blue thorn vine Smilax sieboldii, 10 times the weight of 70% ethanol was added to the dried powder (extraction part, above-ground part) and steeped at room temperature for 24 hours, then filtered through Whatman paper No. 2, and the filtrate was concentrated under reduced pressure and freeze-dried to prepare a solid extract of *Clematis terniflora*.

[0046] < Experimental example > Blueberry extract of Antihyperlipidemia or Anti-obesity active

[0047] Mouse preadipocytes 3T3-L1 (Zenbio SP-L1-F) were cultured in 3T3-L1 Preadipocyte Medium (Zenbio PM-1-L1) at 37°C and 5% CO2. 5 × 10⁶ 3T3-L1 preadipocytes were placed in a 24-well plate. 4After seeding at the cell count per well, the cells were maintained for an additional 2 days once 100% confluency was reached. Adipocyte differentiation was induced for 2 days using 3T3-L1 Differentiation Medium (Zenbio DM-2-L1) containing MDI (Isobutylmethylxanthine, dexamethasone, insulin). After 48 hours of culture, the medium was replaced with 3T3-L1 Adipocyte Medium (Zenbio AM-1-L1) every 2 days for 6 days. During the induction of adipocyte differentiation, samples were added to each medium at various concentrations, and the degree of differentiation was observed on the 8th day, when differentiation was complete. The degree of adipocyte differentiation was primarily confirmed by microscopy using Oil Red O staining, and the staining intensity was measured by determining the amount of differentiated adipocytes at 510 nm absorbance.

[0048] The results are shown in Figure 1. Referring to Figure 1, it can be seen that the extract of *Clematis terniflora* has concentration-dependent inhibitory activity on the differentiation of adipocytes and fat accumulation.

Claims

Claim 1 An anti-obesity composition comprising, as an active ingredient, an extract of the above-ground parts of *Clematis terniflora* in water, ethanol, or a mixture thereof. Claim 2 A composition according to claim 1, wherein the extract is a 70% ethanol extract. Claim 3 In claim 1 or 2, the composition is a food composition. Claim 4 In claim 1 or 2, the composition is a pharmaceutical composition. Claim 5 delete Claim 6 delete

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