Anti-obesity composition comprising as active ingredient compound isolated from alnus japonica extract

Abstract

The present invention provides an anti-obesity composition comprising, as an active ingredient, a compound isolated from an Alnus japonica extract. More particularly, the composition comprising, as active ingredients, oregonin, hirsutanonol, and / or hirsutenone compounds isolated from an Alnus japonica extract significantly reduces the accumulation of fat in adipocytes and significantly increases lipolysis, and thus may be useful as an anti-obesity pharmaceutical composition, a health functional food composition, or a feed composition.

Description

Anti-obesity composition comprising a compound isolated from an extract of the alder tree as an active ingredient

[0001] The present invention relates to an anti-obesity composition comprising a compound isolated from an extract of the alder tree as an active ingredient.

[0002] Obesity is one of the most common nutritional disorders worldwide. Obesity is a condition in which excess calories are consumed compared to the calories burned, resulting in the accumulation of excess calories in the body as fat.

[0003] Obesity is known to be caused by a variety of factors, including genetic influences, environmental influences due to a Westernized diet, and psychological stress. Obesity not only presents its own problems, including physical appearance, but is also closely linked to the development of various adult diseases. As obesity levels rise, the prevalence of diabetes, gallstones, hypertension, heart disease, and stroke increases.

[0004] For patients with obesity, treatment of obesity is essential in addition to disease management, so prevention and active management of obesity are essential. To control obesity, food intake must be reduced and energy expenditure, such as through exercise, must be increased to prevent fat accumulation in the body. While it is well known that appetite suppression through diet directly contributes to weight loss, this is often challenging, so obesity control through increased energy expenditure may be more effective.

[0005] Additionally, many modern people are treating obesity by taking drugs and dietary supplements that have an anti-obesity effect, and the market size of related foods, health functional foods, and pharmaceuticals for anti-obesity purposes is increasing every year.

[0006] However, most anti-obesity health functional foods only have appetite suppressing and digestive health benefits. Long-term consumption can lead to side effects such as indigestion, abdominal gas, nausea, dizziness, and hives. Therefore, the development of safe, natural anti-obesity food ingredients is increasingly urgent.

[0007] Against this backdrop, the inventors of the present invention conducted extensive research to discover natural active substances with anti-obesity effects, and as a result, confirmed that a composition containing oregonin, hirsutanonol and / or hirsutenone compounds isolated from an extract of the alder tree (Alnus japonica) as active ingredients significantly reduces fat accumulation in fat cells and significantly increases fat decomposition, thereby completing the present invention.

[0008] The purpose of the present invention is to provide a pharmaceutical composition for preventing or treating obesity, which comprises a compound isolated from an extract of an oregano tree as an active ingredient, wherein the compound is oregano.

[0009] Another object of the present invention is to provide a health functional food composition for preventing or improving obesity, which comprises a compound isolated from an extract of an oregano tree as an active ingredient, wherein the compound is oregano.

[0010] Another object of the present invention is to provide a feed composition for preventing or improving obesity, comprising a compound isolated from an extract of an oregano tree as an active ingredient, wherein the compound is oregano.

[0011] The advantages and features of the present invention, and the methods for achieving them, will become clearer with reference to the embodiments described in detail below together with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below and may be implemented in various different forms. These embodiments are provided solely to ensure that the disclosure of the present invention is complete and to fully inform those skilled in the art of the scope of the present invention, and the present invention is defined solely by the scope of the claims.

[0012] The terminology used herein is for the purpose of describing embodiments only and is not intended to limit the present invention. In this specification, the singular also includes the plural unless specifically stated otherwise. As used herein, the terms "comprises" and / or "comprising" do not exclude the presence or addition of one or more other components in addition to the mentioned components. Like reference numerals refer to like components throughout the specification, and "and / or" includes each and any combination of one or more of the mentioned components. Although "first", "second", etc. are used to describe various components, these components are not limited by these terms. These terms are only used to distinguish one component from another. Therefore, it should be understood that a first component mentioned below may also be a second component within the technical spirit of the present invention.

[0013] Unless otherwise defined, all terms (including technical and scientific terms) used herein may be used in their common sense to those skilled in the art to which the present invention pertains. Furthermore, terms defined in commonly used dictionaries are not to be interpreted ideally or excessively unless explicitly and specifically defined otherwise.

[0014] The present invention provides a pharmaceutical composition for preventing or treating obesity, comprising a compound isolated from an extract of an oregano tree as an active ingredient, wherein the compound is oregano.

[0015] The Alnus genus plant of the present invention is a deciduous tree belonging to the Betulaceae family, and more than 17 species are known to grow natively in Korea, including Alnus japonica, Alnus hirasuta, and Alnus borealis koidzumi.

[0016] The above-mentioned alder tree contains components such as lupenone, glutenol, taraxerol, sitosterol, and heptacosane, and is known to be effective in treating diseases such as enteritis, diarrhea, and external bleeding.

[0017] The compound isolated from the above extract of the orchard tree may additionally include hersutanol and hersutanone.

[0018] The composition of the present invention may include as an active ingredient at least one selected from the group consisting of oregonine, hersutanol, hersutanone, and combinations thereof.

[0019] The extraction solvent of the above-mentioned duckweed extract may be ethanol, and the ethanol may be 50 to 70% (v / v), preferably 60% (v / v).

[0020] The above oregonine may be obtained by solvent fractionation of the supercritical extract of the ore tree, and the solvent may be, but is not limited to, ethyl acetate.

[0021] The above-mentioned hersutanol and hersutanone may be obtained by enzymatic decomposition of an extract of the ethanol of the alder tree. The enzyme may be a mixture of Pectinex Ultra pulp and Pectinex SP-L, but is not limited thereto.

[0022] The composition of the present invention can have an excellent anti-obesity effect by reducing fat accumulation and neutral fat content in fat cells and increasing lipolysis in fat cells.

[0023] Prevention of the present invention means any action that suppresses or delays obesity or fat accumulation.

[0024] Treatment of the present invention means suppression of occurrence or recurrence of a disease, alleviation of symptoms, reduction of direct or indirect pathological consequences of the disease, reduction of the rate of disease progression, improvement, alleviation, palliation or improved prognosis of the disease state.

[0025] The term pharmaceutical composition of the present invention may be described interchangeably with pharmaceutical composition.

[0026] The pharmaceutical composition of the present invention may include as an active ingredient at least one selected from the group consisting of oregonine, hersutanol, hersutanone, and combinations thereof, which are compounds isolated from an extract of the alder tree, but may additionally include pharmaceutically acceptable carriers, excipients, diluents, etc., depending on the formulation and intended use.

[0027] The pharmaceutical composition of the present invention may further include conventional fillers, bulking agents, binders, disintegrants, surfactants, anticoagulants, lubricants, wetting agents, flavoring agents, emulsifiers, etc., and may be used both parenterally and orally. In addition, it may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., as well as external preparations, suppositories, and sterile injection solutions according to conventional methods. Carriers, excipients and diluents that may be included include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxy benzoate, propyl hydroxy benzoate, talc, magnesium stearate, mineral oil and the like.

[0028] When formulating the above composition, it can be prepared using diluents or excipients such as commonly used fillers, bulking agents, binders, wetting agents, disintegrating agents, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, and capsules, and these solid preparations can be prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose, lactose, and gelatin, with the above composition. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups, and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives can be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solutions and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include witepsol, macrogol, Tween 61, cacao butter, laurin, and glycerogelatin.

[0029] Administration of the present invention means providing the composition of the present invention to a subject by any suitable method.

[0030] The pharmaceutical composition of the present invention can be administered in a therapeutically effective amount, which is an amount of the active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue, animal, or human, i.e., an amount that induces alleviation of the symptoms of the disease or disorder being treated. It will be apparent to those skilled in the art that the therapeutically effective dosage and frequency of administration of the pharmaceutical composition of the present invention will vary depending on the desired effect. Therefore, the optimal dosage to be administered can be readily determined by those skilled in the art and can be adjusted according to various factors, including the type of disease, the severity of the disease, the content of the active ingredient and other ingredients contained in the composition, the type of formulation, and the patient's age, weight, general health, sex, and diet, the time of administration, the route of administration, and the excretion rate of the composition, the treatment period, and concurrently used drugs. For a desirable effect, the pharmaceutical composition of the present invention may be administered in an amount of 1 to 1,000 mg / kg / day, preferably 1 to 200 mg / kg / day, more preferably 1 to 100 mg / kg / day, and may be administered once a day or divided into several doses.

[0031] The dosage of the pharmaceutical composition for preventing or treating obesity according to the present invention can be appropriately selected by those skilled in the art, taking into account the administration method, the age, sex, and weight of the recipient, and the severity of the disease. For example, the pharmaceutical composition for preventing or treating disease according to the present invention can be administered at a dosage of 0.0001 mg / kg to 1000 mg / kg, and for greater effectiveness, at a dosage of 0.01 mg / kg to 100 mg / kg. Administration can be administered once a day or divided into several doses. The above dosage does not limit the scope of the present invention.

[0032] The pharmaceutical composition of the present invention can be administered to a subject via various routes. Administration may be, for example, oral, rectal, intravenous, intramuscular, subcutaneous, intrauterine, or intracerebrovascular injection, but is not limited thereto.

[0033] The pharmaceutical composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, or biological response modifiers for the prevention or treatment of obesity or its complications.

[0034] In addition, the present invention provides a health functional food composition for preventing or improving obesity, which comprises a compound isolated from an extract of an oregano tree as an active ingredient, wherein the compound is oregano.

[0035] The compound isolated from the above extract of the orchard tree may additionally include hersutanol and hersutanone.

[0036] The composition of the present invention may include as an active ingredient at least one selected from the group consisting of oregonine, hersutanol, hersutanone, and combinations thereof.

[0037] The amount of the compound isolated from the extract of the alder tree of the present invention may be suitably changed depending on the intended use, and the compound may be included in an amount of 0.01 to 20 wt%, preferably 1 to 10 wt%, based on the total weight of the food composition, but is not limited thereto. If the content of the compound is less than 0.01 wt%, the efficacy of administration may be reduced, and if it exceeds 20 wt%, there may be difficulties in formulation.

[0038] As a specific example, when manufacturing health functional foods or beverages, the compound of the present invention may be added in an amount of 15 wt% or less, preferably 10 wt% or less, relative to the raw material. However, when consumed for long-term purposes for health and hygiene or for health control, the compound may be added in an amount below the above range, and since there are no safety issues, the active ingredient may be used in an amount exceeding the above range.

[0039] There is no particular limitation on the types of the above health functional foods. Examples of foods to which the compound of the present invention can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, vitamin complexes, etc., and include all health foods in the conventional sense.

[0040] When the health functional food composition of the present invention is manufactured into a beverage, it may contain various flavoring agents or additional ingredients such as natural carbohydrates, as in conventional beverages. The natural carbohydrates may include monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; natural sweeteners such as dextrin and cyclodextrin; and synthetic sweeteners such as saccharin and aspartame. The natural carbohydrates may be included in an amount of 0.01 to 10 wt%, preferably 0.01 to 0.1 wt%, based on the total weight of the health functional food composition of the present invention.

[0041] In addition to the above, the health functional food composition of the present invention may include various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the health functional food composition of the present invention may include fruit pulp for the production of natural fruit juice, fruit juice drinks, and vegetable drinks. These components may be used independently or in combination, and may be included in an amount of 0.01 to 0.1 wt% based on the total weight of the health functional food composition of the present invention.

[0042] Improvement of the present invention means any action that at least reduces a parameter related to the condition being treated, for example the severity of a symptom.

[0043] In addition, the present invention provides a feed composition for preventing or improving obesity, comprising a compound isolated from an extract of an oregano tree as an active ingredient, wherein the compound is oregano.

[0044] The compound isolated from the above extract of the orchard tree may additionally include hersutanol and hersutanone.

[0045] The composition of the present invention may include as an active ingredient at least one selected from the group consisting of oregonine, hersutanol, hersutanone, and combinations thereof.

[0046] The compound of the present invention can have an excellent anti-obesity effect by reducing fat accumulation and neutral fat content in fat cells and increasing fat decomposition in fat cells, and therefore can be included in a feed composition for preventing and improving disease occurrence in animals or livestock.

[0047] Specifically, the feed containing the compound of the present invention as an active ingredient can be manufactured into various types of feed known in the art, and preferably may include a concentrate feed, roughage feed, or special feed.

[0048] Concentrated feed includes seeds and fruits of grains such as wheat, oats, and corn; bran, which is a by-product obtained by refining grains such as rice bran, wheat bran, and barley bran; sesame cake, which is a by-product obtained by extracting soybeans, sesame seeds, linseed, and coconut oil; residual starch, which is the main component of starch residue after removing starch from sweet potatoes and potatoes; animal feed such as fish meal, fish waste, fish soluble, which is a concentrate of fresh liquid obtained from fish; meat meal, blood meal, feather meal, skim milk powder, dried whey, which is the residue obtained when manufacturing cheese from milk or casein from skim milk; yeast, chlorella, and seaweed.

[0049] Forage includes raw forage such as wild grass, pasture, and green grass; root vegetables such as forage turnips, forage beets, and a type of turnip called rutabaga; silage, which is stored forage made by filling a silo with raw grass, green grass crops, and grain and fermenting it with lactic acid; hay made by cutting and drying wild grass and pasture; straw from crops for breeding; and leaves of legumes.

[0050] Special feeds include mineral feeds such as shells and rock salt, urea feeds such as urea or its derivative diuretic isobutane, feed additives that are added in small amounts to compound feeds to supplement ingredients that are likely to be lacking when only natural feed ingredients are mixed, or to increase the storability of the feed, and dietary supplements.

[0051] The feed composition of the present invention may include various feed additives.

[0052] The feed additive of the present invention corresponds to a supplementary feed under the Feed Management Act, and may additionally include mineral preparations such as sodium bicarbonate (sodium bicarbonate), bentonite, magnesium oxide, and complex minerals; mineral preparations that are trace minerals such as zinc, copper, cobalt, and selenium; vitamins such as carotene, vitamin E, vitamin A, D, E, nicotinic acid, and vitamin B complex; protected amino acids such as methionine and lysate; protected fatty acids such as fatty acid calcium salts; live bacteria such as probiotics (lactic acid bacteria), yeast cultures, and mold fermentations; and yeast agents.

[0053] The feed composition of the present invention has an effect of preventing or improving obesity, so it is applicable to any subject for the purpose of preventing or improving disease occurrence in animals or livestock, without particular limitation. The subject may refer to mammals, including animals, for example, non-primates (e.g., cows, pigs, horses, cats, dogs, rats, and mice) and primates (e.g., monkeys, such as cynomolgous monkeys and chimpanzees).

[0054] The dosage of the feed composition of the present invention will depend on a number of factors, such as the animal's species, size, weight, and age. Typically, a typical dosage range may be, but is not limited to, 0.001 to 1,000 μg / kg per animal / day.

[0055] The present invention provides an anti-obesity composition comprising a compound isolated from an extract of the alder tree as an active ingredient, and more specifically, a composition comprising oregonin, hirsutanonol and / or hirsutenone compounds isolated from an extract of the alder tree as an active ingredient significantly reduces fat accumulation in fat cells and significantly increases fat decomposition, and thus can be usefully used as an anti-obesity pharmaceutical composition, a health functional food composition or a feed composition.

[0056] Figure 1 is a diagram showing the results of evaluating the effect of treatment with oregonine (ORE) on fat accumulation in adipocytes.

[0057] Figure 2 is a diagram showing the HPLC chromatography analysis results of compounds oregonine, hersutanol, and hersutanone isolated from an extract of the orchard tree.

[0058] Hereinafter, the present invention will be described in more detail through the following examples and experimental examples. However, the scope of the present invention is not limited to the following examples and experimental examples, but includes modifications of technical concepts equivalent thereto.

[0059] Example 1. Preparation of materials and compounds

[0060] 15.25 kg of alder roots were obtained from the experimental forest of the Korea Forest Research Institute (921-2 Jinae-ri, Chuncheon-si, Gangwon-do), located in Chuncheon-si, Gangwon-do, Republic of Korea. AJ60E was obtained by 60% (v / v) ethanol extraction of 14.25 kg of alder roots. AJRF1, an EA solvent fraction, was obtained by 60% ethanol extraction of the supercritical extract residue, a by-product of supercritical extraction of 1 kg of alder roots. AJRF2 was obtained through enzymatic digestion using AJ60E, a 60% ethanol extract of alder roots, and a mixture of Pectinex Ultra pulp and Pectinex SP-L (Novozymes Co. Ltd, Bagsvaerd, Denmark) was used as the enzyme. In addition, AJRF3 was obtained through enzymatic digestion using AJRF1, an EA solvent fraction of a 60% ethanol extract of alder roots.

[0061] Afterwards, oregonine (compound 1), hustanonol (compound 2), and hustanone (compound 3) were obtained through separation and purification, and LC-MS / MS and NMR analyses were performed at the Kangwon National University Joint Laboratory Practice Center.

[0062] 1-1. Extraction of duckweed

[0063] 14.25 kg of Alnus japonica roots were soaked in 60% edible alcohol and 40% distilled water at 25°C for 1 week, extracted three times, and then filtered through filter paper measuring 594 mm × 941 mm (Hyundai Micro, Seoul, Korea).

[0064] The water bath was RE-501 (Lanphan, Henan, China), and the low-temperature coolant circulation pump was DLSB-5L / -20℃ (Lanphan, Henan, China) for concentration. Then, a freeze dryer (Ilshin, Gyeonggi-do, Korea) was set to -80℃, and the concentrated 60% alcohol extract of the roots of the alder tree was freeze-dried for 48 hours to obtain a final weight of 428.32 g (yield 3.01%, AJ60E).

[0065] In addition, for supercritical extraction, 1 kg of dried alder roots were placed using supercritical extraction research equipment, ISA-SEFE-0500-0700-080 (Ilsin, Daejeon, Korea), and carbon dioxide gas was maintained at an isobaric pressure, and the temperature of the extraction tank was set to 50 ℃. Then, a pressure of 400 bar was applied using a high-pressure pump, and 180 mL of edible alcohol, ethanol, was injected into the bottom of the extraction tank at a rate of 3 mL / min for 1 hour.

[0066] After extracting carbon dioxide using a high-pressure pump for 30 minutes while maintaining the temperature and pressure, 970.2 g of the remaining sample of the supercritical extract of the alder tree was dried in the shade, extracted with 60% alcohol at room temperature, filtered, and concentrated to obtain a final yield of 17.27 g (1.78% AJRF0).

[0067] 1-2. Isolation of extracts

[0068] Of the 482.32 g of powder obtained through extracting the roots of the alder tree, 200 g was subjected to solvent fractionation with ethyl acetate using a seperatory funnel. The solvent fraction of the extract was filtered through filter paper (Hyundai Micro, Seoul, Korea) measuring 297 mm × 420 mm and concentrated using a vacuum evaporator. Finally, 74.39 g (yield 37.1%, AJRF1) was obtained in the form of a brown powder through lyophilization.

[0069] Among 17.2 g of powder obtained through AJRF0, a 60% ethanol extract of the supercritical extract of the root of the alder tree, 10 g was subjected to solvent fractionation with ethyl acetate using a seperatory funnel. The solvent fraction of the extract was filtered through filter paper (Hyundai Micro, Seoul, Korea) measuring 297 mm × 420 mm, concentrated in a reduced pressure evaporator, and freeze-dried to obtain 1.72 g (yield 17.2%, AJRF0-1) in the form of a brown powder.

[0070] 1-3. HPLC separation and purification

[0071] Of the 74.39 g of the 60% alcohol extract of the root of the alder tree, 10 g was dissolved in methyl alcohol at a concentration of 200 mg / mL and purified using MPLC AI-580S HR-Premium (YAMAZEN, Osaka, Japan). The first purification condition was UNIVERSAL from YAMAZEN. TMA Premium Silica Gel 2L 3 cm × 20 cm column and a Silica gel L size inject column were used. The mobile phase solvent conditions were a mixture of chloroform: methanol: water in a ratio of 70:30:4, and the mobile phase flow rate was 20 mL / min for analysis. The wavelengths were 254 nm and 280 nm, the injection volume was 5 mL, and the total analysis time was 30 min.

[0072] For the secondary purification conditions to secure a high-purity single compound, a UNIVERSALTM ODS-SM Gel L 3 cm × 16.5 cm column and a silica gel L sized inject column from YAMAZEN were used. Methyl alcohol and water were used as mobile phase solvents, and the flow rate was 15 mL / min for analysis. The wavelength was analyzed at 254 nm and 280 nm, and the injection volume was 2 mL and the analysis time was 40 min. The 2.42 g brown powder obtained by separation and purification was designated as compound 1.

[0073] The molecular weight of the obtained compound 1 was analyzed through LC-MS / MS, and the structure was confirmed through NMR analysis to be oregonine.

[0074] In addition, AJRF2 was obtained through enzymatic digestion using AJ60E, a 60% ethanol extract of alder root, and the enzyme was a mixture of Pectinex Ultra pulp and Pectinex SP-L (Novozymes Co. Ltd, Bagsvaerd, Denmark). In addition, AJRF3 was obtained through enzymatic digestion using AJRF1, an EA solvent fraction of a 60% ethanol extract of alder root.

[0075] Afterwards, through the above separation and purification, hustanonol (compound 2) and hustanone (compound 3) were obtained (Fig. 2).

[0076] Example 2. Cell culture

[0077] 3T3-L1 cells, which are preadipocytes derived from mice, were purchased from the Korea Cell Line Bank. 3T3-L1 cells were cultured in a humidified CO2 incubator (5% CO2 / 95% air) at 37°C in complete DMEM medium (Welgene) supplemented with 10% bovine calf serum (BCF), 100 units / mL penicillin, and 100 μg / mL streptomycin. When the cells reached approximately 80% confluence in the culture dish, the cell monolayer was washed with phosphate-buffered saline (PBS, pH 7.4), and then trypsin-2.65 mM EDTA was added to detach the cells and subculture them. The medium was changed every 2 days.

[0078] Example 3. Measurement of cell viability

[0079] 3T3-L1 cells were cultured at 3 × 10 4 The cells were seeded into 24-well plates at a density of 10 cells / well and cultured for 24 hours. After 24 hours of culture, the culture medium containing various concentrations of test substances (compounds 1 to 3 and combinations thereof) was replaced with the cell culture medium, and the cells were cultured for 72 hours. After 72 hours of culture, the MTT assay was performed to measure cell viability. The MTT assay method is based on the principle that mitochondrial dehydrogenase reduces MTT to produce formazan, a blue substance. In this test, formazan was dissolved in isopropanol and the absorbance was measured at 570 nm.

[0080] Example 4. Induction of cell differentiation and treatment with test substances

[0081] 3T3-L1 cells were cultured at 1 × 10 5 Cells were seeded into a 24-well plate at a concentration of 10 cells / well. After the cells reached confluence, the cell culture medium was sequentially replaced with three types of differentiation-inducing medium (DM) to induce adipocyte differentiation. That is, the cell culture medium was replaced with a differentiation-inducing medium containing DMEM medium containing 10% FBS and DMI (1 μM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 5 μg / mL insulin), and differentiation was stimulated for 2 days.

[0082] After 2 days, the differentiation-inducing medium was replaced with a new differentiation-inducing medium containing 5 μg / mL insulin in DMEM containing 10% FBS, and differentiation was stimulated for another 2 days. After stimulating differentiation for a total of 4 days, the cells were maintained in DMEM containing 10% FBS for approximately 4 to 8 days to induce differentiation into adipocytes, and the cell culture medium was replaced every 2 days during this period.

[0083] To investigate the effect of compounds 1 to 3 and their combinations as test substances on adipocyte differentiation, various concentrations of test substances were added to the differentiation-inducing medium and treated on cells.

[0084] Example 5. Measurement of adipocyte differentiation (Oil red O staining and quantification)

[0085] 3T3-L1 cells were differentiated and treated with various concentrations of test substances. The cells were rinsed with DPBS (Welgene) and fixed with 4% paraformaldehyde (PFA, Biosesang) for 1 hour at room temperature. After fixation, the cells were stained with Oil Red O (Sigma-Aldrich) solution for 1 to 2 hours at room temperature. After observing the degree of adipocyte staining with the naked eye, the cells were rinsed with distilled water and observed under a microscope. To quantitatively analyze the degree of adipocyte differentiation, the cells were dissolved in isopropanol after staining with Oil Red O and the absorbance was measured at 490 nm.

[0086] Example 6. Measurement of neutral fat content in adipocytes

[0087] 3T3-L1 cells were cultured at 3 × 10 5 Cells were seeded into 6-well plates at a density of 10 cells / well, treated with test substances to induce differentiation, and cultured. The cell culture medium was removed and the cells were rinsed with DPBS (Welgene). 300 μL of distilled water was added to the cells, and the cells were collected in a 1.5 mL tube using a cell scraper and homogenized with a homogenizer. 900 μL of a chloroform: methanaol = 2:1 (v / v) solution was added to the homogenate, mixed well, and incubated at 37°C for 30 minutes.

[0088] After 30 minutes, centrifugation was performed for 10 minutes (5000 rpm), and the lipid-containing supernatant was transferred to a new 1.5 mL tube. Centrifugation was performed for 2 hours using a high-efficiency centrifuge to evaporate the chloroform to dryness to obtain a pellet. 20 μL of chloroform was added to this pellet to re-dissolve the lipids, and neutral fat was measured using a triglyceride assay kit (Biomax, Korea) according to the manufacturer's instructions.

[0089] Example 7. Measurement of glycerol content in adipocyte culture medium

[0090] 3T3-L1 cells were differentiated and treated with various concentrations of test substances. The cell culture medium was collected. The content of glycerol, a lipolysis product, in the cell culture medium was measured using a free glycerol assay kit (Biomax, Korea) as recommended by the manufacturer.

[0091] Example 8. Statistical processing

[0092] All analytical values ​​are expressed as mean ± SEM. The collected results were analyzed using the GraphPad Prism 5.0 program (GraphPad software, San Diego, CA, USA). Student's t-test and one-way analysis of variance (ANOVA) were used to compare the differences between the test substance treatment group and the control group. Statistically significant was considered only when p < 0.05.

[0093] Experimental Example 1. Evaluation of the Effect on Cell Viability

[0094] Reduced cell proliferation due to test substance toxicity affects adipocyte differentiation and activity. To rule this out, we determined a concentration of test substance that did not exhibit cytotoxicity in 3T3-L1 cells. We treated the cell culture medium with various concentrations of the test substance and examined its effect on cell viability.

[0095] 3T3-L1 cells were treated with various concentrations (0, 1, 5, 10, 20, 40 μg / mL) of test substances in cell culture medium, and the cells were cultured for 72 hours. Then, an MTT assay was performed. The viability of 3T3-L1 cells was expressed as a percentage of the control group (0 μg / mL) that was not treated with the test substance.

[0096] As a result, compounds 1 to 3 and their combinations did not affect cell viability up to 10 μg / mL, and cell viability significantly decreased at a treatment concentration of 20 μg / mL. Based on this, the highest treatment concentration of the test substance was set to 10 μg / mL, and the following experiment was conducted.

[0097] Experimental Example 2. Evaluation of the Effect on Adipocyte Differentiation

[0098] To investigate the effects of test substances on the differentiation of preadipocytes into mature adipocytes, cells were cultured in differentiation-inducing medium containing various concentrations of the test substance. The cells were then stained with oil red O to measure fat accumulation, assessing the effects of the test substance on adipocyte differentiation.

[0099] As shown in Fig. 1 and Table 1, when differentiation was induced with the adipocyte differentiation-inducing medium (G1) compared to the undifferentiated control group (G0), fat accumulation was significantly increased. Compared to the control group (G1) not treated with the test substance, treatment with oregonine (ORE) significantly reduced fat accumulation starting from the treatment concentration of 5 μg / mL, and when treated at the highest treatment concentration of 10 μg / mL, fat accumulation was reduced by 17.5% compared to the control group (G1) not treated with the test substance.

[0100]

[0101] In the case of hersutanol and hersutanone, fat accumulation was significantly reduced starting from a treatment concentration of 5 μg / mL, and the reduction in fat accumulation was confirmed to be concentration-dependent. In addition, in the case of the combination of oregonine, hersutanol, and hersutanone, fat accumulation was confirmed to be significantly reduced in a concentration-dependent manner starting from a treatment concentration of 1 μg / mL.

[0102] Experimental Example 3. Evaluation of the Effect on Neutral Fat Accumulation in Adipocytes

[0103] Triglycerides, along with glucose, serve as an important energy source for cells in the body. However, excess triglycerides are absorbed and stored in adipocytes. Accumulated fat globules contribute to obesity and various chronic diseases. To investigate the effects of five test substances on intracellular triglyceride levels, the triglyceride content in adipocytes was measured using a triglyceride assay kit (Biomax) (Table 2).

[0104]

[0105] Compared to the undifferentiated control group (G0), when differentiation was induced with the adipocyte differentiation-inducing medium (G1), the neutral fat content in adipocytes significantly increased. Compared to the control group (G1) that was not treated with the test substance, treatment with oregonine significantly decreased the neutral fat content starting from the treatment concentration of 1 μg / mL, and when treated at the highest treatment concentration of 10 μg / mL, the neutral fat content decreased by 22.8% compared to the control group (G1) that was not treated with the test substance.

[0106] In the case of hersutanol and hersutanone, the neutral fat content was significantly reduced starting from a treatment concentration of 5 μg / mL, and the decrease in neutral fat content was confirmed to be concentration-dependent. In addition, in the case of the combination of oregonine, hersutanol, and hersutanone, the neutral fat content was confirmed to significantly decrease in a concentration-dependent manner starting from a treatment concentration of 1 μg / mL.

[0107] Experimental Example 4. Evaluation of the Effect on Fat Decomposition in Adipocytes

[0108] Neutral fat serves as an important energy source for cells in the body, but excessive accumulation of neutral fat contributes to obesity. Various hormones in the body stimulate the production or breakdown of neutral fat. Since neutral fat is broken down into fatty acids and glycerol, the glycerol content in the cell culture medium of 3T3-L1 adipocytes indirectly indicates the degree of neutral fat breakdown within the adipocytes.

[0109] To investigate the effect of test substances on lipolysis in adipocytes, the glycerol content in the cell culture medium was measured (Table 3).

[0110]

[0111] Compared to the undifferentiated control group (G0), when differentiation was induced with adipocyte differentiation-inducing medium (G1), the glycerol content in the cell culture medium significantly increased. Compared to the control group (G1) that was not treated with the test substance, treatment with oregonine significantly increased the glycerol content in the culture medium starting from a treatment concentration of 1 μg / mL.

[0112] In the case of hersutanol and hersutanone, the glycerol content significantly increased from a treatment concentration of 5 μg / mL, and in the case of the combination of oregonine, hersutanol, and hersutanone, the glycerol content significantly increased in a concentration-dependent manner from a treatment concentration of 1 μg / mL.

[0113] A composition comprising as an active ingredient at least one selected from compounds oregonine, hersutanol, hersutanone and a combination thereof isolated from the extract of the present invention can be usefully used as an anti-obesity composition by reducing fat accumulation and neutral fat content in fat cells and increasing lipolysis in fat cells.

[0114] While the embodiments of the present invention have been described above, the present invention is not limited to the above embodiments, but can be manufactured in various different forms. Those skilled in the art will understand that the present invention can be implemented in other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood to be illustrative in all respects and not restrictive.

Claims

1. A pharmaceutical composition for preventing or treating obesity, comprising a compound isolated from an extract of the alder tree as an active ingredient. A pharmaceutical composition characterized in that the compound is oregonine.

2. In paragraph 1, A pharmaceutical composition characterized in that the compound isolated from the above-mentioned extract of the orchard further comprises hersutanol and hersutanone.

3. In paragraph 1 or 2, A pharmaceutical composition, characterized in that the extraction solvent of the above-mentioned duckweed extract is alcohol.

4. In paragraph 3, A pharmaceutical composition, characterized in that the alcohol content is 50 to 70% (v / v).

5. In paragraph 1, A pharmaceutical composition characterized in that the above oregonine is obtained by solvent fractionation of a supercritical extract of an oregano tree.

6. In paragraph 5, A pharmaceutical composition, characterized in that the solvent is ethyl acetate.

7. In paragraph 2, The above hersutanol and hersutanone are pharmaceutical compositions characterized in that they are obtained by enzymatic decomposition of an extract of the ethanol of the alder tree, A pharmaceutical composition characterized in that the enzyme is a mixture of Pectinex Ultra pulp and Pectinex SP-L.

8. A health functional food composition for preventing or improving obesity, comprising a compound isolated from an extract of the alder tree as an active ingredient. A health functional food composition characterized in that the compound is oregonine.

9. In paragraph 8, A health functional food composition characterized in that the compound isolated from the above-mentioned extract of the duckweed tree additionally includes hersutanol and hersutanone.

10. A feed composition for preventing or improving obesity, comprising a compound isolated from an extract of the alder tree as an active ingredient. A feed composition characterized in that the compound is oregonine.

11. In paragraph 10, A feed composition characterized in that the compound isolated from the above-mentioned extract of the duckweed further comprises hersutanol and hersutanone.

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