KR20160091869A - Anti-obesitic composition comprising extract of Sigesbeckia orientalis L. - Google Patents

Abstract

The present invention relates to a composition for preventing, alleviating, or treating obesity, comprising a Siegesbeckia orientalis L. extract as an active ingredient. According to the present invention, a fraction thereof reduces the expression of main transcription factors (PPAR, C/EBP, and SREBP-1c) related to the differentiation of adipocytes inside the adipocytes, thereby having excellent effects with respect to the obesity. In addition, the composition of the present invention as a natural substance can be safely used without side effects, and can be used as a drug or a food.

Description

[Anti-obesitic composition comprising extract of Sigesbeckia orientalis L.}

The present invention relates to a composition for preventing or improving/treating obesity containing an extract of Sigesbeckia orientalis (Sigesbeckia spp.) as an active ingredient.

Obesity is a disease that occurs due to an ideal increase in adipose tissue due to an imbalance between energy accumulation and consumption. Obesity is recognized as a serious health problem in countries around the world, and it can act as a cause of many diseases such as insulinemia, arteriosclerosis, circulatory system disease, cancer, diabetes, etc., and its severity is increasing (Nature 404: 635). -643, 2000, Obes. Rev. 12: 1-13, 2011).

The Wnt protein is a cystein-rich-secreted ligand that binds to the receptor and activates the Wnt signaling pathway. During the development of vertebrates, Wnt signals act to regulate organ development, cell proliferation, morphology, motility and fate. When the Wnt protein binds to the cell membrane receptor, it is divided into a β-catenin-dependent pathway and an independent pathway depending on which signaling pathway is activated in the cell. The β-catenin-dependent Wnt pathway is activated by Wnt1, Wnt 2, Wnt 3a, Wnt 10a, etc. and is called the canonical Wnt pathway. The β-catenin-independent Wnt pathway is activated by Wnt 4, Wnt 5a, and Wnt 11, and is called the non-canonical Wnt pathway. In the presence of Wnt protein, the Canonical Wnt pathway binds to frizzled receptors on the cell membrane and low-density lipoprotein-receptor-related protein-5/6 (LRP), and the intracellular signal chain reaction is activated. Glycogensynthase kinase-3β) is inhibited. When GSK-3β is inhibited, the phosphorylation and decomposition of β-catenin is inhibited, and β-catenin accumulates in the cytoplasm. At a certain concentration, β-catenin enters the nucleus and binds to the Tcf/Lef transcription factor, and Cyclin D1 (CCND1), etc. It activates gene expression. Accordingly, activation of the Wnt/β-catenin signal inhibits CEBPs (CCAAT/enhancer binding proteins) and PPARγ (peroxisome proliferator-activated receptor gamma), which are major transcription factors for adipocyte differentiation. Prevent formation (Science 289: 950-953, 2000, Trends Endocrin. Met. 20: 16-24, 2009).

On the other hand, it is known that the balance control between the amount of β-catenin in the cell and PPARγ increased by Wnt is important for adipocyte differentiation, and the balance between these two proteins is dependent on protein degradation through phosphorylation of β-catenin by GSK-3β. (J. Biol. Chem. 279: 45020-45027, 2004, Biochem. J. 376: 607-613, 2003). In addition, it has been reported that Wnt plays a role in inhibiting adipocyte formation in the in vivo results of transgenic mice (J. Biol. Chem. 279: 45020-45027, 2004).

Among the representative anti-obesity drugs developed so far ^{, Reductil TM , which reduces appetite, and Xenical TM} , which inhibits fat absorption, are used clinically, but for heart disease and respiratory diseases. , Increased blood pressure and insomnia, and the persistence of its efficacy is low (Phytochem. 71: 1625-1641, 2010). Therefore, the present inventor conducted a study to develop an anti-obesity raw material using natural products with high safety while having few side effects, such as plant extracts, which are not chemically synthesized products.

J. Biol. Chem. 279: 45020-45027, 2004 Phytochem. 71: 1625-1641, 2010

Accordingly, the present inventors studied to search for a natural substance that has excellent anti-obesity activity and can be safely applied. As a result, Sigesbeckia orientalis (Hui Chum, Siegesbeckia spp.) extract or a fraction thereof has the effect of inhibiting adipogenesis. It was confirmed that the present invention was completed.

As a means for solving the above problems, the present invention provides a pharmaceutical composition for preventing or treating obesity, and a food composition for preventing or improving obesity, comprising an extract or a fraction thereof of Siges Beckia Orientalis as an active ingredient.

Sigesbeckia Orientalis extract or a fraction thereof according to the present invention inhibits adipogenesis and reduces the expression of key transcription factors (PPARγ, C/EBPα, and SREBP-1c) involved in fat differentiation, thereby having an excellent effect on obesity. have. In addition, since the present invention is a natural product, it can be safely used without side effects, and thus can be used as a medicine or food.

1 shows the result of measuring a fraction of Siges Beckia Orientalis extract using thin layer chromatography (TLC).
FIG. 2 shows the results of measuring the adipogenesis inhibitory activity by treatment with kylenol in 3T3-L1, which is an adipocyte precursor cell.
3 shows the mRNA expression level of the major transcription factors (PPARγ, C/EBPα, and SREBP-1c) of adipocyte differentiation (PPARγ, C/EBPα, and SREBP-1c) in 3T3-L1, which is adipocyte progenitor cells, relative to the control group ( Right) shows the measurement result.
Figure 4 shows the amount of mRNA expression (left) of adipogenesis-related genes (fatty acid synthesis (FAS) and acetyl-CoA carboxylase (ACC)) by kylenol treatment in 3T3-L1, which is an adipocyte progenitor cell, and relative mRNA expression level compared to the control group. (Right) shows the measurement results.
Figure 5 shows the mRNA expression level of inflammatory cytokines (adiponectin and leptin) expressed during adipocyte differentiation by kylenol treatment in adipocyte 3T3-L1 (left) and relative mRNA expression level compared to the control group. (Right) shows the measurement results.
6 is the mRNA expression level (left) of the major genes (LRP6, DVL2, β-catenin, and CCND1) of the Wnt/β-catenin signaling system by kylenol treatment in adipocytes 3T3-L1 and relative mRNA compared to the control group. The result of measuring the expression level (right) is shown.
Figure 7 is the mRNA of major transcription factors (PPARγ and C/EBPα) of adipocyte differentiation by treatment with Sigesbeckia Orientalis ethanol extract, methanol extract, hot water extract, ethyl acetate extract, and hexane extract in 3T3-L1 adipocytes. It shows the result of measuring the expression level.
FIG. 8 shows the results of measuring the mRNA expression levels of major transcription factors (PPARγ and C/EBPα) of adipocyte differentiation by treatment of a fraction of Sigesbeckia orientalis extract in 3T3-L1 adipocytes.
9 shows the results of measuring the adipogenesis inhibitory activity in 3T3-L1 adipocytes by treatment with Sigesbeckia Orientalis ultra-high pressure extract.
Figure 10 is the mRNA expression level of major transcription factors (PPARγ, C/EBPα, and SREBP-1c) of adipocyte differentiation by treatment with Sigesbeckia Orientalis ultra-high pressure extract in 3T3-L1 adipocytes (left) and compared to the control group The result of measuring the relative mRNA expression level (right) is shown.
FIG. 11 is a measurement of the mRNA expression level (left) and relative mRNA expression level (right) of the adipogenesis-related genes (FAS and ACC) by treatment with Sigesbeckia Orientalis ultra-high pressure extract in 3T3-L1, which is adipocyte progenitor cells. Show one result.
Figure 12 shows the mRNA expression levels of adiponectin and leptin, the inflammatory cytokines expressed during adipocyte differentiation by treatment with Sigesbeckia orientalis ultra-high pressure extract in adipocytes 3T3-L1 (left) and relative mRNA expression levels compared to the control (right ) Shows the measurement result.
13 shows the mRNA expression level of major genes (LRP5, DVL2, β-catenin, and CCND1) of the Wnt/β-catenin signaling system by treatment with Sigesbeckia Orientalis ultra-high pressure extract in adipocyte 3T3-L1 (left ) And the relative mRNA expression level (right) compared to the control group.
14 shows the effect of weight loss by administration of kylenol in obese mice induced by a high fat diet.
Fig. 15 shows the dietary amount of the ethanol extract administration group of Sigesbeckia Orientalis in obese mice induced by a high fat diet.
Figure 16 shows the effect of weight loss in obese mice induced by a high fat diet by administration of an ethanol extract of Sigesbeckia Orientalis.
17 shows the results of measuring total cholesterol, triglyceride, high-density cholesterol, and low-density cholesterol concentrations in blood by administration of an ethanol extract of Sigesbeckia Orientalis in obese mice induced by a high fat diet.
Figure 18 is the protein expression level of the major transcription factors (PPARγ and C/EBPα) of adipocyte differentiation (PPARγ and C/EBPα) in epididymal adipose tissue of obese mice induced by a high fat diet by administration of ethanol extract of Sigesbeckia Orientalis (left) and compared to the control group. The result of measuring the relative protein expression level (right) is shown.
Figure 19 shows the protein expression levels of major genes (β-catenin and p-Gsk3β) of the Wnt/β-catenin signaling system by administration of an ethanol extract of Sigesbeckia Orientalis in the epididymal adipose tissue of obese mice induced by a high fat diet ( It shows the results of measuring the relative protein expression level (right) compared to the left) and the control.

Hereinafter, the configuration of the present invention will be described in detail.

The present invention is used to prevent, ameliorate or treat obesity of the Sigesbeckia orientalis extract or a fraction thereof, or a compound represented by the following formula (1); A composition for preventing, improving, or treating obesity, including a Sigesbeckia orientalis extract or a fraction thereof, or a compound represented by the following formula (1); Or it provides a method for preventing, improving, or treating obesity, comprising applying a Sigesbeckia orientalis extract or a fraction thereof, or a compound represented by the following formula (1) to mammals, including humans.

[Formula 1]

In this specification,'Sigesbeckia Orientalis (Siegesbeckia orientalis L.)' is a plant of the genus Sigesbeckia of the family Asteraceae (Siegesbeckia spp.), it lives in Jeju Island and southern regions, and is also called Jeju Jindeukchal

In the present specification, the'Sygesbeckia orientalis extract' may be prepared using a conventional extraction method known in the art without limitation, and, for example, a Sigesbeckia orientalis plant or part of a plant (leaf or Root) can be obtained by extraction with one or more solvents selected from the group consisting of water, an organic solvent having 1 to 6 carbon atoms, and a subcritical or supercritical fluid.

In the present specification, "fraction" means a result obtained by a fractionation method for separating a specific component or a specific group from a mixture containing various constituents. A specific fraction in which the active material is concentrated can be prepared using techniques such as solvent fractionation, silica gel chromatography, and prep-HPLC.

In the present specification, the'anti-obesity composition' may be used interchangeably with the'composition for preventing, improving or treating obesity'. The term'prevention' refers to any action that inhibits or delays the onset of obesity, and the term'improvement or treatment' means alleviating, alleviating, or eliminating the symptoms of obesity, delaying, stopping or reversing the progression of obesity, etc. Includes all behavioral changes that are beneficial to obesity. 'Anti-obesity composition' or'a composition for preventing, improving or treating obesity' includes a pharmaceutical composition or a food composition.

The composition for preventing, improving, or treating obesity of the present invention may further contain one or more active ingredients exhibiting the same or similar functions in addition to the Sigesbeckia orientalis extract or a fraction thereof. For example, it may contain known ingredients that are effective for obesity. When the additional ingredients are included, the preventive, ameliorating or therapeutic effect of the composition of the present invention against obesity may be further enhanced. When the above ingredients are added, skin safety, ease of formulation, and stability of active ingredients can be considered in combination with use. In one embodiment of the present invention, the composition is a component for treating obesity known in the art, Myagropsis myagroides) extract, Staff purpura (Sargassum fulvellum) extract, Hoe life is Sargassum (Sargassum horneri) of the extract, acid citrus leaf extract, dumpling balls Sargassum extract, Quercus acuta leaf extract, carrot seed extract, nurukgyun of ginseng fermented, honggukgyun of ginseng It may further include one or more ingredients selected from the group consisting of fermented products, fermented koji bacteria of red ginseng or fermented red ginseng of red ginseng, namgasae extract and garlic species extract (Allium sativum L. stem extract). Additional ingredients may be included in an amount of 0.0001% by weight or more and 10% by weight or less based on the total weight of the composition. For example, 0.0001% by weight or more and 1% by weight or more, 0.0001% by weight or more and 0.1% by weight or less, 0.0001% by weight or more and 0.001% by weight or less, 0.001% by weight or more and 10% by weight or less, 0.001% by weight or more and 1 It may be less than or equal to 0.001% by weight and less than or equal to 0.1% by weight, less than or equal to 0.01% by weight and less than or equal to 10% by weight, and less than or equal to 0.01% by weight and less than 1% by weight. The content range may be adjusted according to requirements such as skin safety and ease of formulation of the compound of Formula 1.

The present invention provides a pharmaceutical composition for preventing or treating obesity, or a food composition for preventing or improving obesity, comprising the Siges Beckia Orientalis extract or a fraction thereof as an active ingredient.

In one embodiment, the Sigesveccia Orientalis extract can be obtained by extracting and purifying dried Sigesveccia Orientalis using purified water suitable for food processing, ethanol and subcritical water, or supercritical carbon dioxide, or an ultra-high pressure extraction device It can be obtained by extraction and purification using, or can be obtained by separating and purifying from oil obtained by directly compressing the Sigesbeckia Orientalis plant. For example, it is possible to obtain an extract by extracting Sigesbeckia orientalis under ultra-high pressure conditions of 100 MPa or more.

In one embodiment, the Sigesbeckia orientalis extract is obtained by extracting Sigesbeckia orientalis with one or more solvents selected from the group consisting of water, an organic solvent having 1 to 6 carbon atoms, a subcritical fluid, and a supercritical fluid. can do. For example, as the extraction solvent, various solvents such as ethanol, methanol, propanol, isopropanol, butanol, acetone, ether, benzene, chloroform, ethyl acetate, methylene chloride, hexane, cyclohexane, and petroleum ether may be used alone or in combination. I can. If necessary, it can be prepared by additionally including filtration and concentration steps according to methods known in the art.

In one embodiment, the organic solvent having 1 to 6 carbon atoms is an alcohol having 1 to 6 carbon atoms, acetone, ether, benzene, chloroform, ethyl acetate, It may be at least one selected from the group consisting of methylene chloride, hexane, cyclohexane, and petroleum ether.

In one embodiment, the Sigesbeckia orientalis extract or a fraction thereof may contain kylenol as an active ingredient.

In one embodiment, the Sigesbeckia Orientalis extract or a fraction thereof is Sigesbeckia Orientalis ethanol extract; Alternatively, it may be a hexane fraction obtained by fractionating the ethanol extract of Siges Beckia Orientalis with hexane. In addition, a hexane-ethyl acetate fraction obtained by fractionating the hexane fraction with a mixed solvent of hexane and ethyl acetate; An ethyl acetate fraction obtained by fractionating the hexane-ethyl acetate fraction with ethyl acetate; Alternatively, it may be an ethanol fraction obtained by fractionating the ethyl acetate fraction with ethanol. The hexane-ethyl acetate fraction comprises: a first hexane-ethyl acetate fraction obtained by fractionating with a mixed solvent in which hexane and ethyl acetate are mixed in a volume ratio of 9:1 to 8:2; Alternatively, the first hexane-ethyl acetate fraction may be a second hexane-ethyl acetate fraction obtained by fractionating with a mixed solvent in which hexane and ethyl acetate are mixed in a volume ratio of 7:3 to 6:4. Since the ethyl acetate fraction contains the most active ingredient kylenol, it has excellent anti-obesity effect.

The present invention also provides a pharmaceutical composition for preventing or treating obesity, or a food composition for preventing or improving obesity, comprising a compound represented by Formula 1 as an active ingredient:

[Formula 1]

The compound of Formula 1 may include all possible isomers, and may include, for example, the compound of Formula 2 below.

[Formula 2]

The compound of Formula 2 is referred to as kirenol.

Kirenol is Siegesbeckia Orientalis ( Siegesbeckia orientalis L.), Siegesbeckia pubescens Mak.), Siegesbeckia Glabrecens (Siegesbeckia glabrescens Shige's Becky inhabiting the material contained in Mak.) in Jeju and the southern provinces in Ah (Siegesbeckia spp.) It is an active ingredient of plants.

Kylenol or Sigesbeckia Orientalis extract has anti-inflammatory and analgesic effects (J. Ethnopharmacol. 137: 1089-1094, 2011), antibacterial effects (Pharmacogn. Mag. 8: 149-155, 2012), arthritis effect (Phytomedicine) 19: 882-889, 2012), but prior to the present invention, the effect of preventing and treating obesity was not known.

*Kylenol can be obtained by extracting and purifying dried Sigesbeckia Orientalis using purified water suitable for food processing, ethanol and subcritical water, or supercritical carbon dioxide, or by extraction and purification using an ultra-high pressure extraction device, or It can be obtained by separating and purifying from oil obtained by direct compression of the Sigesbeckia orientalis plant. Separation and purification of chilenol from the extract of Sigesbeckia Orientalis can be performed using column chromatography filled with various synthetic resins such as silica gel or activated alumina, and high-performance liquid chromatography (HPLC) alone or Although it can be used in parallel, the method of extraction and separation and purification of kylenol is not necessarily limited to the above method.

In addition, the compound of Formula 1 or the compound of Formula 2 may be used separately or synthesized from a plant extract, or a commercially available compound may be used.

In one embodiment, the compound represented by Formula 1 or the compound represented by Formula 2 may be isolated from an extract of Sigesbeckia Orientalis.

The pharmaceutical composition for preventing or treating obesity of the present invention may contain a pharmaceutically acceptable salt of kylenol. As used herein, the term'pharmaceutically acceptable' refers to physiologically acceptable and when administered to humans, usually does not cause allergic reactions or similar reactions, and as the salt, a pharmaceutically acceptable free acid (free Acid addition salts formed by acid) are preferred.

The pharmaceutically acceptable salt of the chilenol may be an acid addition salt formed using an organic acid or an inorganic acid, and the organic acid may be, for example, formic acid, acetic acid, propionic acid, lactic acid, butyric acid, isobutyric acid, trifluoroacetic acid, malic acid, Maleic acid, malonic acid, fumaric acid, succinic acid, succinic acid monoamide, glutamic acid, tartaric acid, oxalic acid, citric acid, glycolic acid, glucuronic acid, ascorbic acid, benzoic acid, phthalic acid, salicylic acid, anthranilic acid, dichloroacetic acid, aminooxyacetic acid, benzenesulfonic acid , p-toluenesulfonic acid or methanesulfonic acid. Inorganic acids include, for example, hydrochloric acid, bromic acid, sulfuric acid, phosphoric acid, nitric acid, carbonic acid or boric acid. The acid addition salt may be preferably in the form of hydrochloride or acetate, and more preferably in the form of hydrochloride.

The above-mentioned acid addition salts are either a) directly mixing kylenol and an acid, b) dissolving and mixing one of them in a solvent or a water-containing solvent, or c) placing kylenol in an acid in a solvent or a submerged solvent. And it is prepared by a general salt preparation method of mixing them.

In addition to the above, additional salts are possible: gaba salt, gabapentin salt, pregabalin salt, nicotinic acid salt, adipate salt, hemimalonic acid salt, cysteine salt, acetylcysteine salt, methionine salt, arginine salt, lysine salt, ornithine salt or And aspartate.

In addition, the pharmaceutical composition for preventing or treating obesity of the present invention may further include a pharmaceutically acceptable carrier.

The pharmaceutically acceptable carrier may further include, for example, a carrier for oral administration or a carrier for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. In addition, the carrier for parenteral administration may include water, suitable oil, saline, aqueous glucose and glycol. In addition, it may further include a stabilizer and a preservative. Suitable stabilizers are antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives are benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. Other pharmaceutically acceptable carriers may be referred to as those described in the following literature (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).

The pharmaceutical composition of the present invention can be administered to mammals including humans by any method. For example, it can be administered orally or parenterally, and parenteral administration methods are not limited thereto, but intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal , Intranasal, intestinal, topical, sublingual or rectal administration.

The pharmaceutical composition of the present invention can be formulated as a formulation for oral administration or parenteral administration according to the route of administration as described above. When formulated, one or more buffers (e.g., saline or PBS), carbohydrates (e.g., glucose, mannose, sucrose, or dextran, etc.), antioxidants, bacteriostatic agents, chelating agents (e.g., EDTA) Or glutathione), fillers, extenders, binders, adjuvants (e.g., aluminum hydroxide), suspending agents, thickening agents, wetting agents, disintegrants or surfactants, diluents or excipients.

Solid preparations for oral administration include tablets, pills, powders, granules, liquids, gels, syrups, slurries, suspensions or capsules, and the like, and such solid preparations are at least one excipient in the pharmaceutical composition of the present invention, for example , Starch (including corn starch, wheat starch, rice starch, potato starch, etc.), calcium carbonate, sucrose, lactose, dextrose, sorbitol, mannitol, xylitol, erythritol maltitol, cellulose , Methyl cellulose, sodium carboxymethylcellulose, and hydroxypropylmethyl-cellulose or gelatin may be mixed to prepare a mixture. For example, tablets or dragees can be obtained by blending the active ingredient with a solid excipient, pulverizing it, adding a suitable auxiliary, and processing it into a granule mixture.

In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, liquid solutions, emulsions, or syrups, but may include various excipients, such as wetting agents, sweetening agents, fragrances, or preservatives, in addition to water or liquid paraffin, which are commonly used simple diluents. .

In addition, in some cases, cross-linked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate may be added as a disintegrant, and an anti-aggregating agent, a lubricant, a wetting agent, a fragrance, an emulsifying agent and a preservative, etc. may be additionally included. .

When administered parenterally, the pharmaceutical composition of the present invention may be formulated according to a method known in the art in the form of an injection, a transdermal administration, and a nasal inhalation agent together with a suitable parenteral carrier. In the case of the injection, it must be sterilized and protected from contamination by microorganisms such as bacteria and fungi. Examples of suitable carriers for injections include, but are not limited to, water, ethanol, polyols (e.g., glycerol, propylene glycol and liquid polyethylene glycol, etc.), mixtures thereof, and/or solvents or dispersion media containing vegetable oils. I can. More preferably, suitable carriers include isotonic solutions such as Hanks' solution, Ringer's solution, phosphate buffered saline (PBS) containing triethanol amine or sterile water for injection, 10% ethanol, 40% propylene glycol and 5% dextrose. Etc. can be used. In order to protect the injection from microbial contamination, various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid, thimerosal, and the like may be additionally included. In addition, the injection may further contain an isotonic agent such as sugar or sodium chloride in most cases.

In the case of transdermal administration, ointments, creams, lotions, gels, external solutions, pasta, liniment, and air rolls are included. In the above, "transdermal administration" means that the active ingredient in an effective amount contained in the pharmaceutical composition is delivered into the skin by topically administering the pharmaceutical composition to the skin.

In the case of inhalation dosages, the compounds used according to the invention can be used in pressurized packs or with suitable propellants, for example dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas It can be conveniently delivered from a nebulizer in the form of an aerosol spray. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve that delivers a metered amount. For example, gelatin capsules and cartridges for use in an inhaler or insufflator can be formulated to contain a powder mixture of the compound and a suitable powder base such as lactose or starch. Formulations for parenteral administration are described in Remington's Pharmaceutical Science, 15th Edition, 1975. Mack Publishing Company, Easton, Pennsylvania 18042, Chapter 87: Blaug, Seymour, a formula generally known for all pharmaceutical chemistry.

The pharmaceutical composition of the present invention may provide desirable obesity prevention, improvement or treatment effect when it contains an effective amount of kyrenol or Sigesbeckia orientalis extract or a fraction thereof. In the present specification, the term "effective amount" refers to an amount that exhibits a higher response compared to the negative control group, and preferably refers to an amount sufficient to prevent, ameliorate, or treat obesity. The pharmaceutical composition of the present invention may contain 0.01 to 99.99% of chilenol or a Sigesbeckia orientalis extract containing the same, and the balance may be occupied by a pharmaceutically acceptable carrier. The effective amount of the extract of Kyrenol or Sigesbeckia orientalis or fractions thereof contained in the pharmaceutical composition of the present invention will vary depending on the form in which the composition is commercialized.

The total effective amount of the pharmaceutical composition of the present invention may be administered to a patient in a single dose, and may be administered by a fractionated treatment protocol that is administered for a long time in multiple doses. . The pharmaceutical composition of the present invention may vary the content of the active ingredient according to the severity of the disease. For example, it is preferably administered in an amount of 0.001 to 100 mg, more preferably 0.01 to 10 mg per 1 kg of body weight per day based on kyrenol or a Sigesveccia orientalis extract containing the same. Can be administered separately. However, the dose of the Kylenol or Sigesbeckia Orientalis extract containing the same is not only the administration route and the number of treatments of the pharmaceutical composition, but also the age, weight, health condition, sex, severity of the disease, diet and excretion rate of the patient. Since the effective dosage for the patient is determined in consideration of the factors, those of ordinary skill in the art, considering these points, use the above Kylenol or Sigesveccia Orientalis extract containing the same for enhancing exercise performance. It will be possible to determine an appropriate effective dosage for a particular application. The pharmaceutical composition according to the present invention is not particularly limited in its formulation, route of administration, and method of administration as long as it exhibits the effects of the present invention.

The pharmaceutical composition for preventing or treating obesity of the present invention may be used alone or in combination with surgery, radiation therapy, hormone therapy, chemotherapy, or methods using a biological response modifier.

The pharmaceutical composition for preventing or treating obesity of the present invention may also be provided in the form of a formulation for external use comprising Kylenol or Sigesbeckia Orientalis extract or a fraction thereof as an active ingredient.

When the pharmaceutical composition for preventing or treating obesity of the present invention is used as an external preparation for skin, additionally fatty substances, organic solvents, solubilizers, thickening and gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents , Fragrance, surfactant, water, ionic emulsifier, nonionic emulsifier, filler, sequestering agent, chelating agent, preservative, vitamin, blocker, wetting agent, essential oil, dye, pigment, hydrophilic activator, lipophilic activator or It may contain adjuvants commonly used in the field of dermatology such as any other ingredients commonly used in skin external preparations such as lipid vesicles. In addition, the above ingredients may be introduced in an amount generally used in the field of dermatology.

When the pharmaceutical composition for preventing or treating obesity of the present invention is provided as an external preparation for skin, it may be a formulation such as an ointment, a patch, a gel, a cream, or a spray, but is not limited thereto.

The food composition for preventing or improving obesity of the present invention includes all forms such as functional food, nutritional supplement, health food, food additives and feed, and human or Animals, including livestock, are targeted for eating. Food compositions of this type can be prepared in various forms according to conventional methods known in the art.

Food compositions of this type can be prepared in various forms according to conventional methods known in the art. General foods include, but are not limited to, beverages (including alcoholic beverages), fruits and processed foods thereof (e.g., canned fruit, canned food, jam, marmalade, etc.), fish, meat and processed foods thereof (e.g. ham, sausage) Corn beef), bread and noodles (e.g. udon, buckwheat noodles, ramen, spagate, macaroni, etc.), fruit juice, various drinks, cookies, sweets, dairy products (e.g. butter, cheese, etc.), edible vegetable oil, margarine , Vegetable protein, retort food, frozen food, various seasonings (eg, soybean paste, soy sauce, sauce, etc.), and the like can be prepared by adding the kyrenol or the Sigesbeckia Orientalis extract containing the same. In addition, as a nutritional supplement, it is not limited thereto, but may be prepared by adding the kylenol or the Sigesbeckia Orientalis extract containing the same to capsules, tablets, pills, and the like. In addition, the health functional food is not limited thereto, but for example, the kylenol or the Sigesbeccia Orientalis extract itself containing the same is prepared in the form of tea, juice, and drink, and liquefied so that it can be consumed (health drink). , Can be ingested by granulating, encapsulating and powdering. In addition, in order to use the kyrenol or the Sigesbeckia orientalis extract containing the same in the form of a food additive, it may be prepared and used in the form of a powder or a concentrate. In addition, it can be prepared in the form of a composition by mixing the kyrenol or the Siges Beckia Orientalis extract containing the same with a known active ingredient known to have an effect of enhancing exercise performance.

When the food composition for preventing or improving obesity of the present invention is used as a health drink composition, the health drink composition may contain various flavors or natural carbohydrates as an additional component, such as a conventional beverage. The natural carbohydrates described above include monosaccharides such as glucose and fructose; Disaccharides such as maltose and sucrose; Polysaccharides such as dextrin and cyclodextrin; It may be a sugar alcohol such as xylitol, sorbitol, and erythritol. Sweeteners include natural sweeteners such as taumatin and stevia extract; Synthetic sweeteners such as saccharin and aspartame can be used. The proportion of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 mL of the composition of the present invention.

Kylenol or Sigesbeckia Orientalis extract containing the same may be contained as an active ingredient in a food composition for preventing or improving obesity, the amount of which is not particularly limited to an amount effective to obtain an effect of preventing or improving obesity, but It is preferably 0.01 to 100% by weight based on the total weight of the composition. The food composition of the present invention may be prepared by mixing with chirenol or a Sigesbeckia orientalis extract containing the same and other active ingredients known to have an effect on obesity.

In addition to the above, the health food of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, colorants, pectic acid, salts of pectic acid, alginic acid, salts of alginic acid, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, It may contain glycerin, alcohol, or a carbonating agent. In addition, the health food of the present invention may contain flesh for the manufacture of natural fruit juice, fruit juice beverage, or vegetable beverage. These ingredients may be used independently or in combination. Although the proportion of these additives is not very important, it is generally selected from 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

Hereinafter, the present invention will be described in detail by examples. However, the following examples are provided for illustrative purposes only to aid understanding of the present invention, and the scope of the present invention is not limited thereto. The experimental results of the following examples were expressed as mean ± standard deviation, and statistical analysis was evaluated as having significance when the p value was 0.05 or less using t test or ANOVA analysis method (Scheff test).

Reference Example 1: Information on kirenol substances

Name: Kirenol; Kirel; (1R,3S,4aS,4bS,7S,10aS)-1,2,3,4,4a,4b,5,6,7,9,10,10a-Dodecahydro-3-hydroxy-7-[(R) -1,2-dihydroxyethyl]-1,4a,7-trimethylphenanthrene-1-methanol

CAS No.: 52659-56-0

[Example 1] Preparation of Sigesbeckia Orientalis Extract

[Example 1-1] Preparation of Ethanol Extract of Sigesbeckia Orientalis

The dried Siges Veckia Orientalis was pulverized with a mixer, and then 100 g of the ground Siges Veckia Orientalis sample was added to 1 L of ethanol and extracted at room temperature for 48 hours. The extracted sample was filtered through Whatman No. 2 filter paper, and the filtered extract was concentrated with a vacuum rotary concentrator to remove the solvent component, and then an ethanol extract of Sigesbeckia Orientalis was obtained.

[Example 1-2] Preparation of methanol extract of Sigesbeckia Orientalis

The dried leaves and stems of Siges Beckia Orientalis were pulverized with a mixer, and then 100 g of the pulverized Siges Beckia Orientalis sample was added to 1 L of methanol and extracted while stirring at 50° C. for 60 minutes. The extracted sample was filtered through Whatman No. 2 filter paper, and the filtered extract was concentrated with a vacuum rotary concentrator to remove the solvent component, thereby obtaining a methanol extract of Sigesbeckia Orientalis.

[Example 1-3] Preparation of Sigesbeckia Orientalis Hot Water Extract

The dried leaves and stems of Siges Beckia Orientalis were pulverized with a mixer, and then 100 g of the ground Siges Beckia Orientalis sample was added to 1 L of water and extracted with stirring at 100° C. for 4 hours. The extracted sample was filtered through Whatman No. 2 filter paper, and the filtered extract was concentrated with a vacuum rotary concentrator to remove the solvent component, thereby obtaining a Sigesbeckia Orientalis hot water extract.

[Example 1-4] Preparation of Sigesbeckia Orientalis ethyl acetate extract

The dried leaves and stems of Siges Beckia Orientalis were pulverized with a mixer, and then 100 g of the ground Siges Beckia Orientalis sample was added to 1 L of ethyl acetate and extracted with stirring at 50° C. for 60 minutes. The extracted sample was filtered through Whatman No. 2 filter paper, and the filtered extract was concentrated with a vacuum rotary concentrator to remove the solvent component, thereby obtaining a Sigesbeckia Orientalis ethylacetate extract.

[Example 1-5] Preparation of hexane extract of Sigesbeckia Orientalis

The dried leaves and stems of Siges Beckia Orientalis were pulverized with a mixer, and then 100 g of the ground Siges Beckia Orientalis sample was added to 1 L of hexane and extracted while stirring at 50° C. for 60 minutes. The extracted sample was filtered through Whatman No. 2 filter paper, and the filtered extract was concentrated with a vacuum rotary concentrator to remove the solvent component, thereby obtaining a Sigesbeckia Orientalis hexane extract.

[Example 1-6] Preparation of Sigesbeckia Orientalis Ultra High Pressure Extract

After pulverizing the dried leaves and stems of Siges Beckia Orientalis with a mixer, put 1 g of the crushed Siges Veckia Orientalis sample and 76 mL of 18% ethanol into a polyethylene pack, sealed, and then ultra-high pressure extraction device (Frescal MFP-7000; Mitsubishi Heavy Industries, Tokyo, Japan) was used to extract. In the ultra-high pressure extraction conditions, the extraction pressure was 320 MPa and the extraction time was 5 min. The extracted sample was filtered through Whatman No. 2 filter paper, and the filtered extract was concentrated with a vacuum rotary concentrator to remove the solvent component, thereby obtaining a Sigesbeckia Orientalis ultra-high pressure extract.

[Example 2] Isolation and structure determination of kylenol

[Example 2-1] Isolation of kylenol

Using a solvent system in which the concentrated Sigesbeckia Orientalis ethanol extract obtained in Example 1-1 was loaded on a column filled with silica gel and ethyl acetate and methanol were mixed in a ratio of 10:0.5 (v/v). Aliquoted. According to the preparative sequence, it was divided into a total of 7 fractions, and each fraction was concentrated and dried. Fraction 6 of the 7 fractions (fraction 6) was fractionated with 10% ethyl acetate as a developing solvent using RP-18 reverse phase column chromatography (Lichroprep RP-18 25-40um, Merck&Co., Whitehouse Station, NJ, USA). . According to the preparative sequence, a total of two fractions were divided and each fraction was concentrated to dryness. Of the two fractions, fraction 2 (fraction 6-2) was concentrated to dryness, and again fractionated with 20% ethyl acetate as a developing solvent using Rp-18 reverse phase column chromatography. According to the preparative sequence, it was divided into three fractions, and each fraction was concentrated to dryness. Finally, fraction 2 of the three fractions (fraction 6-2-2) was concentrated to dryness to separate a pure single active substance.

[Example 2-2] Structure determination of kylenol

In order to determine the structure of the single active material isolated in Example 2-1, ¹ H-NMR spectrum and ¹³ C-NMR spectrum were measured at 500 MHz and 125 MHz (solvent: MeOH), respectively. The ¹ H ¹ H COSY spectrum and ¹ H ¹³ C HSQC spectra in order to obtain the ¹ H-NMR measurement of correlation between the spectrum and the ¹³ C-NMR correlation of the ¹ H ¹ H on the basis of the result of the spectrum related to the ¹ H ¹³ C relationships It was measured, and the result was measured by discriminating each carbon signal by the wavelength emitted through the carbon resonance.

In addition, FAB-MS measured for mass spectrometry of the isolated single substance was measured. This compound was found to have a molecular weight of 338.48 as [M] was observed at m/z _{338.48 in FAB-MS, and had a molecular formula of C 20} H ₃₄ O ₄ .

The above ¹ H-NMR, ¹³ C-NMR, and FAB-MS results were identified by comparative analysis and previously published research reports (Wang JP et al., Pharmacogn. Mag., 8:149-155, 2012). As a result, the single substance isolated above was identified as a kirenol compound represented by the following formula (2).

[Formula 2]

[Example 3] Sigesbeckia Orientalis Fraction Preparation

The concentrated Sigesbeckia Orientalis ethanol extract obtained in Example 1-1 was completely dissolved in ethanol, then silica gel was added thereto, concentrated to dryness, and coated on the silica gel surface. Fraction 1 was eluted with 100% hexane on silica gel coated with Sigesbeckia Orientalis ethanol extract, and concentrated to dryness. After obtaining the first fraction, the second fraction was eluted and concentrated to dryness using a solvent in which hexane and ethyl acetate were mixed in a ratio of 9:1 (v/v) to the remaining extract. After obtaining the second fraction, fraction 3 was eluted and concentrated to dryness using a solvent in which hexane and ethyl acetate were mixed in a ratio of 7:3 (v/v) to the remaining extract. After obtaining fraction 3, fraction 4 was eluted with 100% ethyl acetate to the remaining extract and concentrated to dryness. Finally, after obtaining the fourth fraction, fraction 5 was eluted with 100% ethanol for the remaining extract and concentrated to dryness. As a result of measuring each of a total of five fractions using thin layer chromatography (TLC), it was found that kylenol was concentrated in the fourth fraction as shown in FIG. 1.

[Example 4] Anti-obesity effect of kylenol

[Example 4-1] Effect of kylenol on inhibiting fat production

3T3-L1 adipocytes (American Type Culture Collection (ATCC), Manassas, VA, USA) were cultured in DMEM (Dulbecco's Modified Eagle's Media) medium containing 10% calf serum, and then cultured in a 24-well plate incubator (24 -well microtiter plate) to 1,00,000 cells / well (well). After growing to 90%, leave it for 2 more days, and when the adipocyte density reaches 100%, dexamethasone, IBMX (3-isobutyl-1-methylxanthine), and kylenol dissolved in a culture medium containing insulin are added 10, 20, respectively. In addition, adipocyte differentiation was induced by treating the adipocytes at a concentration of 40 μM. Then, every 2 days, the cell culture was treated with 10% fetal calf serum and 0.1% insulin in a culture solution containing 10, 20, and 40 μM of kylenol, respectively, and compared with the control group not treated with kylenol. On the 10th day of induction of differentiation, adipocytes that had undergone differentiation were treated with Oil Red O staining solution (Sigma, St. Louis, MO, USA) and stained, and the stained lipid droplets were 100% isopropanol. After extraction with (isopropanol), the absorbance was measured at 500 nm using a spectrophotometer. The fat accumulation rate was expressed as a percentage (%) compared to the group not treated with the sample during induction of adipocyte differentiation.

As a result, as shown in Fig. 2, it was found that kylenol effectively inhibited adipogenesis in 3T3-L1 adipocytes.

[Example 4-2] Effect of kylenol on reducing major transcription factors on adipocyte differentiation

RT-PCR was performed to determine the mRNA expression level of a major transcription factor in adipocyte differentiation in 3T3-L1 adipocytes using kylenol. Total RNA was harvested and reverse transcribed from 3T3-L1 adipocytes differentiated in the same manner as in Example 4-1 using a TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and then reverse transcription-polymerase chain (RT-PCR). reaction) analysis was performed as follows. First, for cDNA synthesis, the RNA was reverse transcribed with a reverse transcriptase. RT-PCR was performed with the following primers:

β-actin:

SEQ ID NO: 1: 5'-AGCCATGTACGTAGCCATCC-3' (Forward primer)

SEQ ID NO: 2: 5'-CTCTCAGCTGTGGTGGTGAA-3' (Reverse primer)

PPARr:

SEQ ID NO: 3: 5'-CCTGTTGACCCAGAGCATGG-3' (Forward primer)

SEQ ID NO: 4: 5'-CGAGTGGTCTTCCATCACGC-3' (Reverse primer)

C/EBPα:

SEQ ID NO: 5'-CTGCCCCTCAGTCCCTGTC-3' (Forward primer)

SEQ ID NO: 6: 5'-GTTCCTTCAGCAACAGCGG-3' (Reverse primer)

SREBP-1c:

SEQ ID NO: 7: 5'-GCGCTACCGGTCTTCTATCA-3' (Forward primer)

SEQ ID NO: 8: 5'-TGCTGCCAAAAGACAAGGG-3' (Reverse primer)

*In addition, based on the amount of mRNA expression measured by RT-PCR, the relative amount of mRNA expression by kylenol treatment compared to the control was calculated.

As a result, as shown in FIG. 3, it was confirmed that mRNA expression of major transcription factors (PPARγ, C/EBPα, and SREBP-1c) of adipocyte differentiation was reduced by treatment with kylenol. This means that the signaling of adipogenesis is suppressed by reducing the major transcription factor for adipocyte differentiation in 3T3-L1 adipocytes by treatment with kylenol.

[Example 4-3] Effect of kylenol on reducing adipogenesis-related genes

RT-PCR was performed to determine the mRNA expression level of adipogenesis-related genes in 3T3-L1 adipocytes using kylenol. Total RNA was harvested and reverse transcribed from 3T3-L1 adipocytes differentiated in the same manner as in Example 4-1 using a TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and then reverse transcription-polymerase chain (RT-PCR). reaction) analysis was performed as follows. First, for cDNA synthesis, the RNA was reverse transcribed with a reverse transcriptase. RT-PCR was performed with the following primers:

β-actin:

SEQ ID NO: 1: 5'-AGCCATGTACGTAGCCATCC-3' (Forward primer)

SEQ ID NO: 2: 5'-CTCTCAGCTGTGGTGGTGAA-3' (Reverse primer)

FAS:

SEQ ID NO: 9: 5'-GGTTCGGAATGCTATCCAGG-3' (Forward primer)

SEQ ID NO: 10: 5'-CTGCGGAAACTTCAGGAAAT-3' (Reverse primer)

ACC:

SEQ ID NO: 11: 5'-TGACCGTGGGCACAAAGTT-3' (Forword primer)

SEQ ID NO: 12: 5'-AGGAGGAACCGCATTTATCGA-3' (Reverse primer)

In addition, based on the mRNA expression level measured by RT-PCR, the relative mRNA expression level by the kylenol treatment compared to the control group was calculated.

As a result, as shown in Fig. 4, it was confirmed that mRNA expression of adipogenesis-related genes (FAS and ACC) was reduced by treatment with kylenol. Therefore, it was confirmed that the signaling of adipogenesis was suppressed by reducing the adipogenesis-related genes in 3T3-L1 adipocytes by treatment with kylenol.

[Example 4-4] Effect of kylenol on reducing inflammatory cytokines expressed during adipocyte differentiation

RT-PCR was performed to determine the mRNA expression level of inflammatory cytokines expressed during adipocyte differentiation in 3T3-L1 adipocytes using kylenol. Total RNA was harvested and reverse transcribed from 3T3-L1 adipocytes differentiated in the same manner as in Example 4-1 using a TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and then reverse transcription-polymerase chain (RT-PCR). reaction) analysis was performed as follows. First, for cDNA synthesis, the RNA was reverse transcribed with a reverse transcriptase. RT-PCR was performed with the following specific primers:

β-actin:

SEQ ID NO: 1: 5'-AGCCATGTACGTAGCCATCC-3' (Forward primer)

SEQ ID NO: 2: 5'-CTCTCAGCTGTGGTGGTGAA-3' (Reverse primer)

Adiponectin:

SEQ ID NO: 13: 5'-TGTGCAGGTTGGATGGCAGG-3' (Forward primer)

SEQ ID NO: 14: 5'-CTCCAGCCCCACACTGAACG-3' (Reverse primer)

Leptin:

SEQ ID NO: 15: 5'-CCAAAACCCTCATCAAGACC-3' (Forword primer)

SEQ ID NO: 16: 5'-CTCAAAGCCACCACCTCTGT-3' (Reverse primer)

In addition, based on the mRNA expression level measured by RT-PCR, the relative mRNA expression level by the kylenol treatment compared to the control group was calculated.

As a result, as shown in FIG. 5, it was confirmed that mRNA expression of inflammatory cytokines (adiponectin and leptin) expressed during adipocyte differentiation by treatment with kylenol was reduced. Therefore, it was confirmed that the signaling of adipogenesis was suppressed by reducing the inflammatory cytokines expressed during adipocyte differentiation in 3T3-L1 adipocytes by treatment with kylenol.

[Example 4-5] Effect of kylenol on promoting Wnt/β-catenin signaling system activity

RT-PCR was performed to determine the mRNA expression level of major genes of the Wnt/β-catenin signaling system in 3T3-L1 adipocytes using kylenol. Total RNA was harvested and reverse transcribed from 3T3-L1 adipocytes differentiated in the same manner as in Example 4-1 using a TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and then reverse transcription-polymerase chain (RT-PCR). reaction) analysis was performed as follows. First, for cDNA synthesis, the RNA was reverse transcribed with a reverse transcriptase. RT-PCR was performed with the following specific primers:

β-actin:

SEQ ID NO: 1: 5'-AGCCATGTACGTAGCCATCC-3' (Forward primer)

SEQ ID NO: 2: 5'-CTCTCAGCTGTGGTGGTGAA-3' (Reverse primer)

LRP5:

SEQ ID NO: 17: 5'-AAGGGTGCTGTGTACTGGAC-3' (Forward primer)

SEQ ID NO: 18: 5'-AGAAGAGAACCTTACGGGACG-3' (Reverse primer)

LRP6:

SEQ ID NO: 19: 5'-ACCTCAATGCGATTTGTTCC-3' (Forward primer)

SEQ ID NO: 20: 5'-GGTGTCAAAGAAGCCTCTGC-3' (Reverse primer)

β-catenin:

SEQ ID NO: 21: 5'-GCCAAGTGGGTGGTATAGAG-3' (Forward primer)

SEQ ID NO: 22: 5'-CTGGGTATCCTGATGTGC-3' (Reverse primer)

DVL2:

SEQ ID NO: 23: 5'-GCTTCCACATGGCCATGGGC-3' (Forward primer)

SEQ ID NO: 24: 5'-TGGCACTGCTGGTGAGAGTCACAG-3' (Reverse primer)

CCND1:

SEQ ID NO: 25: 5'-AAAATCGTGGCCACCTGGAT-3' (Forward primer)

SEQ ID NO: 26: 5'-CATCCGCCTCTGGCATTTTG-3' (Reverse primer)

In addition, based on the mRNA expression level measured by RT-PCR, the relative mRNA expression level by the kylenol treatment compared to the control group was calculated.

As a result, as shown in Fig. 6, it was confirmed that mRNA expression of LRP6, DVL2, β-catenin, and CCND1, which are major genes of the Wnt/β-catenin signaling system, was increased by treatment with kylenol. Therefore, it was confirmed that the Wnt/β-catenin signaling system activity was promoted in 3T3-L1 adipocytes by treatment with kylenol.

[Example 5] Anti-obesity effect of Sigesbeckia orientalis extract

[Example 5-1] The mRNA reduction effect of major transcription factors on adipocyte differentiation of Sigesbeckia orientalis ethanol extract, methanol extract, hot water extract, ethyl acetate extract, and hexane extract

The ethanol extract, methanol extract, hot water extract, ethyl acetate extract, and hexane extract of Sigesbeckia Orientalis prepared in Examples 1-1 to 1-5 were treated at a concentration of 10 ppm, respectively, to obtain 3T3-L1 adipocytes. In order to determine the mRNA expression level of the major transcription factor in adipocyte differentiation, RT-PCR was performed after differentiation in the same manner as in Example 4-2.

As a result, mRNA expression of PPARγ and C/EBPα, key transcription factors in the adipogenesis process, by treatment with ethanol extract, methanol extract, hot water extract, ethyl acetate extract, and hexane extract of Sigesbeckia Orientalis as shown in FIG. 7 It was confirmed that this decreased. Therefore, it was confirmed that the signal transduction of adipogenesis was suppressed by reducing the major transcription factor for adipocyte differentiation in 3T3-L1 adipocytes by treatment with Sigesbeckia orientalis extract.

[Example 5-2] The effect of reducing major transcription factors on adipocyte differentiation of Sigesbeckia orientalis fraction

In the same manner as in Example 4-2, fraction 1, fraction 2, fraction 3, fraction 4, and fraction 5 of Sigesbeckia Orientalis prepared in Example 3 were each treated at a concentration of 10 ppm. I did. As a result, as shown in FIG. 8, the major transcription factors PPARγ and C/EBPα were most reduced in adipocyte differentiation in fraction 4, which contained the most kyrenol among the five Sigesbeckia orientalis fractions, and then Fraction 5 reduced the major transcription factor in adipocyte differentiation. Fraction 1, Fraction 2, and Fraction 3 did not show the effect of reducing major transcription factors on adipocyte differentiation. These results showed that among the active substances contained in the Sigesveccia Orientalis extract, kyrenol had the best effect of inhibiting adipogenesis.

[Example 5-3] Measurement of Adipogenesis Inhibitory Activity of Sigesbeckia Orientalis Ultra High Pressure Extract

In the same manner as in Example 4-1, the Sigesbeckia Orientalis ultra-high pressure extract of Example 1-6 was treated at concentrations of 10, 20, and 40 ppm, respectively. As a result, as shown in Fig. 9, it was found that adipogenesis was effectively suppressed in adipocytes by treatment with the Sigesbeckia Orientalis ultra-high pressure extract.

[Example 5-4] Effect of Sigesbeckia Orientalis Ultra-High Pressure Extract on Adipocyte Differentiation on the Reduced mRNA Expression of Major Transcription Factors

In the same manner as in Example 4-2, the Sigesbeckia Orientalis ultra-high pressure extract of Example 1-6 was treated at concentrations of 10, 20, and 40 ppm, respectively. As a result, as shown in FIG. 10, signaling of adipogenesis by reducing the major transcription factors PPARγ, C/EBPα, and SREBP-1c in adipocyte differentiation in adipocytes by treatment with Sigesbeckia Orientalis ultra-high pressure extract It was confirmed that it was suppressed.

[Example 5-5] Effect of Sigesbeckia Orientalis Ultra-high Pressure Extract on Reducing mRNA Expression of Adipogenesis-related Genes

In the same manner as in Example 4-3, the Sigesbeckia Orientalis ultra-high pressure extract of Example 1-6 was treated at concentrations of 10, 20, and 40 ppm, respectively. As a result, as shown in Fig. 11, it was confirmed that the signaling of adipogenesis was suppressed by reducing the adipogenesis-related genes FAS and ACC in adipocytes by treatment with the Sigesbeckia Orientalis ultra-high pressure extract.

[Example 5-6] Effect of Sigesbeckia Orientalis Ultrahigh Pressure Extract on Reducing mRNA Expression of Inflammatory Cytokines Expressed in Adipocyte Differentiation

In the same manner as in Example 4-4, the Sigesbeckia Orientalis ultra-high pressure extract of Example 1-6 was treated at concentrations of 10, 20, and 40 ppm, respectively. As a result, it was confirmed that the signaling of adipogenesis was suppressed by reducing the inflammatory cytokines adiponectin and leptin, which are expressed during adipocyte differentiation in adipocytes by treatment with Sigesbeckia Orientalis ultra-high pressure extract as shown in FIG. 12.

[Example 5-7] Wnt/β-catenin signaling system activation promotion effect

In the same manner as in Example 4-5, the Sigesbeckia Orientalis ultra-high pressure extract of Example 1-6 was treated at concentrations of 10, 20, and 40 ppm, respectively. As a result, as shown in Fig. 13, it was confirmed that the mRNA expression of LRP5, DVL2, β-catenin, and CCND1, which are major genes of the Wnt/β-catenin signaling system, was increased. Therefore, it was confirmed that the Wnt/β-catenin signaling system activity was promoted in 3T3-L1 adipocytes by treatment with Sigesbeckia orientalis extract.

Example 6 Fat Weight loss effect due to the play Kastrup administration in obese mice leading to

Four-week-old C57BL/6 mice were adapted for 1 week and fed a high-fat diet (Research Diets D12492, 60% kcal from fat, New Brunswick, NJ, USA) for 6 weeks to induce obesity. As an experimental group, each of 5 animals was randomly divided into 2 groups and used for the experiment. As an experimental group, the kylenol prepared in Example 2-2 was suspended in 0.25% carboxymetylcellulose and orally administered once a day for 6 weeks at an administration concentration of 20 mg/kg body weight, and a control group Was orally administered only the same amount of 0.25% carboxymethyl cellulose. The dietary amount of the mice to which the sample was administered and the weight of the individual were measured every week. There was no difference between the experimental group and the control group as a result of measuring the dietary amount of the experimental group and the control group for 6 weeks after administration of the sample.

As a result of measuring the weight gain rate between the experimental group and the control group after 6 weeks, as shown in FIG. 14, the weight gain rate of the kylenol experimental group was significantly lower than that of the control group (*p<0.05). These results confirmed that kylenol was very effective in weight loss.

[Example 7] Shige in the obese mice led to a fat Oh Becky's Weight Loss Effect by Administration of Orientalis Ethanol Extract

Four-week-old C57BL/6 male mice were acclimated for 1 week and fed a high-fat diet (Research Diets D12492, 60% kcal from fat) for 6 weeks to induce a normal diet (Research Diets D12450B, 10% kcal from fat) and obesity. Then, each of 5 animals was randomly divided into 4 groups and used in the experiment. As the experimental group, the ethanol extract of Sigesbeckia Orientalis prepared in Example 1-1 was used as a group administered with the ethanol extract of Sigesbeckia Orientalis on a high-fat diet and the ethanol extract of Sigesbeckia orientalis on a high-fat diet. 0.25% carboxymethyl It was suspended in cellulose and administered orally at a constant time for 6 weeks, once a day at an administration concentration of 250 mg/day/kg body weight of Sigesbeckia Orientalis ethanol extract. As a control group, only the same amount of 0.25% carboxymethylcellulose consumed by the experimental group was administered orally in the normal diet group and the high fat diet group. The dietary amount of the mice to which the sample was administered and the weight of the individual were measured every week.

As a result of measuring the dietary amount of the experimental group and the control group for 6 weeks after administration of the sample, as shown in FIG. 15, there was no statistically significant difference in the dietary amount between the experimental group and the control group. In addition, as a result of measuring the change in body weight of the experimental group and the control group for 6 weeks after administration of the sample, a significant difference between the normal diet control group and the high fat diet control group was shown by the high fat diet as shown in FIG. 16 (##p<0.01). In addition, there was no statistically significant difference between the normal diet control group and the normal diet Sigesbeckia Orientalis ethanol extract experimental group, but the high fat diet control group and the high fat diet were statistically significant between the Sigesbeckia Orientalis ethanol extract experimental group. There was one difference (**p<0.01). Compared with the high-fat diet control group, the body weight of the experimental group of Sigesbeckia Orientalis ethanol extract on the high-fat diet decreased by 12.1%. These results confirmed that the ethanol extract of Sigesbeckia Orientalis has a very excellent weight loss effect.

[Example 8] Shige in the obese mice led to a fat Oh Becky's Changes in blood triglyceride, total cholesterol, high-density cholesterol, and low-density cholesterol concentrations by administration of orientalis ethanol extract

Blood was collected from the hearts of the normal diet mice and high fat diet mice of Example 7. The collected blood was centrifuged at 4,000 rpm for 15 minutes to separate the serum, and the separated serum was stored at -70°C. Serum triglyceride, total cholesterol, low-density cholesterol, and high-density cholesterol concentrations were analyzed using an automated biochemical analyzer (Mindray, Nanshan, Shenzhen, China). As a result, as shown in FIG. 17, the serum triglyceride, total cholesterol, and low-density cholesterol contents showed a significant difference between the normal diet control group and the high fat diet control group by the high fat diet (##p<0.01). In addition, there was no statistically significant difference between the normal diet control group and the normal diet Sigesbeckia Orientalis ethanol extract experimental group, but the high fat diet control group and the high fat diet were statistically significant between the Sigesbeckia Orientalis ethanol extract experimental group. There was one difference (*p<0.05, **p<0.01). Compared with the high-fat diet control group, the serum triglyceride, total cholesterol, and low-density cholesterol content of the high-fat diet Sigesbeckia Orientalis ethanol extract experimental group decreased by 45.1%, 27.6%, and 33.8%, respectively. There was no statistically significant difference in serum high-density cholesterol content between the control group and the experimental group. Therefore, it was confirmed that the ethanol extract of Sigesbeckia Orientalis has an excellent effect of improving blood lipids.

[Example 9] Effect of reducing transcription factors related to adipocyte differentiation in adipose tissue of an animal model

After extracting epididymal fat from the normal diet mice and high fat diet mice of Example 7, protein expression of PPARγ, C/EBPα and SREBP-1c was confirmed through western blot, and α -It was shown that the protein loading amount was constant with tubulin. In addition, based on the protein expression amount measured by Western blot, the relative protein expression amount by treatment with the ethanol extract of Sigesbeckia Orientalis compared to the control was calculated.

As a result, as shown in FIG. 18, the protein expression level of the key transcription factor in the fat differentiation process was significantly increased in the high-fat diet control group compared to the normal diet control group (##p<0.01). In addition, protein expression levels of PPARγ, C/EBPα, and SREBP-1c, which are key transcription factors in adipocyte differentiation process, were statistically significantly reduced in the high-fat diet-treated Sigesbeckia Orientalis extract experimental group compared to the high-fat diet control group. (**p<0.01). Therefore, it was confirmed that the Sigesveccia orientalis extract reduced the key transcription factor in the adipocyte differentiation process, thereby inhibiting the signaling of adipogenesis in adipose tissue.

[ Example 10] Effect of promoting Wnt /β- catenin signaling system activity in adipose tissue of an animal model

After extracting epididymal fat from the normal diet mice and high fat diet mice of Example 7, protein expression of β-catenin and p-Gsk3β was confirmed through western blot, and α-tu It was shown that the protein loading amount was constant by boolean. In addition, based on the protein expression amount measured by Western blot, the relative protein expression amount by treatment with the ethanol extract of Sigesbeckia Orientalis compared to the control was calculated.

As a result, as shown in FIG. 19, the protein expression level of the major genes of the Wnt/β-catenin signaling system was significantly decreased in the high-fat diet control group compared to the normal diet control group (##p<0.01). In addition, the protein expression levels of β-catenin and p-Gsk3β, which are major genes of the Wnt/β-catenin signaling system, were statistically significantly reduced in the experimental group of Sigesbeckia orientalis extract from the high-fat diet compared to the control group. (**p<0.01). Therefore, it was confirmed that the Sigesbeckia orientalis extract promoted the Wnt/β-catenin signaling system activity in adipose tissue.

Hereinafter, examples of the manufacture of foods and pharmaceuticals for preventing, treating or improving obesity containing chilenol according to the present invention or a Sigesbeckia orientalis extract containing the same as an active ingredient will be described, but the present invention is intended to limit it. It is not intended to be explained in detail. Foods and pharmaceuticals for preventing, improving or treating obesity of Preparation Examples 1 to 2 according to the following composition and composition ratio, having Kylenol having excellent effect on preventing, treating or improving obesity, or Sigesbeckia Orientalis extract containing the same The composition was prepared according to a conventional method.

[Production Example 1] Preparation of food

[Production Example 1-1] Preparation of health food

Sigesveccia orientalis extract of Example 1, Sigesveccia orientalis fraction of Example 4 or kylenol 1000 mg, vitamin A acetate 70 ug, vitamin E 1.0 mg, vitamin B1 0.13 mg, vitamin B2 0.15 mg , Vitamin B6 0.5 mg, vitamin B12 0.2 ug, vitamin C 10 mg, biotin 10 ug, nicotinic acid amide 1.7 mg, folic acid 50 ug, pantothenic acid calcium 0.5 mg, ferrous sulfate 1.75 mg, zinc oxide 0.82 mg, magnesium carbonate 25.3 mg , Potassium dibasic 15 mg, calcium dibasic 55 mg, potassium citrate 90 mg, calcium carbonate 100 mg, and magnesium chloride 24.8 mg may be mixed and prepared, and the mixing ratio may be arbitrarily modified. After mixing the above ingredients according to a health food manufacturing method, granules are prepared, and can be used for preparing a health food composition according to a conventional method.

[Production Example 1-2] Preparation of health drink

Purified water was added to the Sigesveccia orientalis extract of Example 1, the Sigesveccia orientalis fraction of Example 4, or Kylenol 1000 mg, citric acid 1000 mg, oligosaccharide 100 g, plum concentrate 2 g, and taurine 1 g. Total 900 ml After mixing the above ingredients according to the usual health drink manufacturing method, stirring and heating at 85 for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed and sterilized, and then stored in the refrigerator. It can be used to prepare the following health beverage compositions.

[Production Example 1-3] Chewing gum

20% by weight of gum base, 76.9% by weight of sugar, 1% by weight of flavor, and 2% by weight of water, and Sigesbeckia Orientalis extract of Example 1, Sigesveccia Orientalis fraction of Example 4 or 0.1% by weight of Kylenol % Was blended to prepare a chewing gum by a conventional method.

[Production Example 1-4] Candy

60% by weight of sugar, 39.8% by weight of starch syrup, and 0.1% by weight of flavor, and the Sigesveccia Orientalis extract of Example 1, the Sigesveccia Orientalis fraction of Example 4, or 0.1% by weight of kylenol are conventionally formulated. Candy was prepared by the method.

[Production Example 1-5] Biscuit

Power Level 1 25.59% by weight, Gravity Level 1 22.22% by weight, sugar 4.80% by weight, salt 0.73% by weight, glucose 0.78% by weight, palm shortening 11.78% by weight, ammonium 1.54% by weight, sodium bicarbonate 0.17% by weight, sodium bisulfite 0.16% by weight , Rice flour 1.45% by weight, vitamin B 0.0001% by weight, milk flavor 0.04% by weight, water 20.6998% by weight, whole milk powder 1.16% by weight, substitute powdered milk 0.29% by weight, monobasic calcium phosphate 0.03% by weight, spray salt 0.29% by weight and spray A biscuit was prepared by a conventional method by mixing 7.27% by weight of milk, the Sigesveccia orientalis extract of Example 1, the Sigesveccia orientalis fraction of Example 4, or the weight% of kylenol.

[Production Example 2-Pharmaceuticals]

[Production Example 2-1] Powder

After mixing the Sigesveccia orientalis extract of Example 1, the Sigesveccia orientalis fraction of Example 4 or 50 mg of kylenol, and 2 g of crystalline cellulose, it was filled in an airtight cloth according to a conventional powder preparation method. A powder was prepared.

[Production Example 2-2] Tablet

After mixing the Sigesveccia orientalis extract of Example 1, the Sigesveccia orientalis fraction of Example 4 or 50 mg of kylenol, 400 mg of crystalline cellulose, and 5 mg of magnesium stearate, according to a conventional tablet manufacturing method. Tablets were prepared by tableting.

[Production Example 2-3] Capsule

After mixing the Sigesveccia orientalis extract of Example 1, the Sigesveccia orientalis fraction of Example 4 or kylenol 30 mg, whey protein 100 mg, crystalline cellulose 400 mg, and magnesium stearate 6 mg, Capsules were prepared by filling in gelatin capsules according to the capsule preparation method.

[Production Example 2-4] Injection

According to a conventional injection preparation method, the active ingredient was dissolved in distilled water for injection and the pH was adjusted to about 7.5. Nol 100 mg, distilled water for injection, and a pH adjuster were mixed, filled in a 2 ml ampoule, and sterilized to prepare an injection.

<110> University-Industry Foundation, Yonsei University <120> Anti-obesitic composition comprising kirenol or extract of Sigesbeckia orientalis L. <130> P14U16C0905 <150> 2013-0084953 <151> 2013-07-18 <160> 26 <170> KopatentIn 2.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> beta-actin forward primer <400> 1 agccatgtac gtagccatcc 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> beta-actin reverse primer <400> 2 ctctcagctg tggtggtgaa 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PPARr forward primer <400> 3 cctgttgacc cagagcatgg 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PPARr reverse primer <400> 4 cgagtggtct tccatcacgc 20 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> CEBP alpha forward primer <400> 5 ctgcccctca gtccctgtc 19 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> CEBP alpha reverse primer <400> 6 gttccttcag caacagcgg 19 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SREBP 1c forward primer <400> 7 gcgctaccgg tcttctatca 20 <210> 8 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> SREBP 1c reverse primer <400> 8 tgctgccaaa agacaaggg 19 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FAS forward primer <400> 9 ggttcggaat gctatccagg 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FAS reverse primer <400> 10 ctgcggaaac ttcaggaaat 20 <210> 11 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> ACC forward primer <400> 11 tgaccgtggg cacaaagtt 19 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> ACC reverse primer <400> 12 aggaggaacc gcatttatcg a 21 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> adiponectin forward primer <400> 13 tgtgcaggtt ggatggcagg 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> adiponectin reverse primer <400> 14 ctccagcccc acactgaacg 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> leptin forward primer <400> 15 ccaaaaccct catcaagacc 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> leptin reverse primer <400> 16 ctcaaagcca ccacctctgt 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LRP5 forward primer <400> 17 aagggtgctg tgtactggac 20 <210> 18 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> LRP5 reverse primer <400> 18 agaagagaac cttacgggac g 21 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LRP6 forward primer <400> 19 acctcaatgc gatttgttcc 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LRP6 reverse primer <400> 20 ggtgtcaaag aagcctctgc 20 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> beta catenin forward primer <400> 21 gccaagtggg tggtatagag 20 <210> 22 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> beta catenin reverse primer <400> 22 ctgggtatcc tgatgtgc 18 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> DVL2 forward primer <400> 23 gcttccacat ggccatgggc 20 <210> 24 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> DVL2 reverse primer <400> 24 tggcactgct ggtgagagtc acag 24 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CCND1 forward primer <400> 25 aaaatcgtgg ccacctggat 20 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CCND1 reverse primer <400> 26 catccgcctc tggcattttg 20

Claims (12)

A pharmaceutical composition for preventing or treating obesity comprising an extract of Shigella beccia orientalis or a fraction thereof as an active ingredient. The method according to claim 1,
The Shigesbeckia orientalis extract is obtained by extracting Shigusbeckia orientalis with at least one solvent selected from the group consisting of water, an organic solvent having 1 to 6 carbon atoms, a subcritical fluid and a supercritical fluid, Or &lt; / RTI &gt; The method according to claim 1,
A pharmaceutical composition for preventing or treating obesity, which comprises extracting Shigusbeckia orientalis under an ultra-high pressure condition of 100 MPa or more, and obtaining the Shigusubekia orientalis extract. The first hexane-ethyl acetate fraction obtained by fractionating the hexane fraction obtained by fractionating the extract of Shigeysubekia orientalis ethanol with hexane by the mixed solvent of the first hexane and ethyl acetate was fractionated with a mixed solvent of the second hexane and ethyl acetate A pharmaceutical composition for preventing or treating obesity comprising an ethyl acetate fraction of a second hexane-ethyl acetate fraction as an active ingredient. 5. The method of claim 4,
The mixed solvent of the first hexane-ethyl acetate fraction is a mixture of hexane and ethyl acetate in a volume ratio of 8 to 10: 1,
Wherein the mixed solvent of the second hexane-ethyl acetate fraction is hexane and ethyl acetate mixed in a volume ratio of 2: 4: 1. 6. The method according to any one of claims 1 to 5,
A pharmaceutical composition for the prevention or treatment of obesity wherein the Shigesbeckia orientalis extract or a fraction thereof contains a pyrenyl group represented by the following formula
(2)

A food composition for preventing or improving obesity comprising an extract of Shigesbeckia orientalis or a fraction thereof as an active ingredient. 8. The method of claim 7,
The Shigesbeckia orientalis extract is obtained by extracting Shigusbeckia orientalis with at least one solvent selected from the group consisting of water, an organic solvent having 1 to 6 carbon atoms, a subcritical fluid and a supercritical fluid, Or improving food composition. 8. The method of claim 7,
Wherein the Shigesbeckia orientalis extract is obtained by extracting Shigusbeckia orientalis under an ultra-high pressure condition of 100 MPa or more. The first hexane-ethyl acetate fraction obtained by fractionating the hexane fraction obtained by fractionating the extract of Shigeysubekia orientalis ethanol with hexane by the mixed solvent of the first hexane and ethyl acetate was fractionated with a mixed solvent of the second hexane and ethyl acetate A composition for preventing or improving obesity comprising an ethyl acetate fraction of a second hexane-ethyl acetate fraction as an active ingredient. 11. The method of claim 10,
The mixed solvent of the first hexane-ethyl acetate fraction is a mixture of hexane and ethyl acetate in a volume ratio of 8 to 10: 1,
Wherein the mixed solvent of the second hexane-ethyl acetate fraction is hexane and ethyl acetate mixed in a volume ratio of 2: 4: 1. 12. The method according to any one of claims 7 to 11,
Wherein the Shigesbeckia orientalis extract or a fraction thereof contains a pyrenyl group of the following formula (2):
(2)

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