KR101958235B1 - A pharmaceutical composition for prevention or treatment of obesity comprising cryptotanshinone or pharmaceutically accepted salts thereof as an effective component - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the prevention or treatment of obesity comprising cryptotanshinone (CT) or a pharmaceutically acceptable salt thereof, and more particularly to a pharmaceutical composition for preventing or treating obesity, Mesenchymal stem cells, pre-adipocytes or white adipose cells in brown adipose cells are treated by administering a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable salt thereof, Or beige adipose cells to prevent or treat obesity of the subject, and the like. When the CT of the present invention or a pharmaceutically acceptable salt thereof is administered to an individual, it is possible to effectively induce differentiation of the adipose tissue into brown fat and / or beige fat, and thus can be effectively used for obesity and related diseases, And is a very useful invention in the cosmetics industry.

Description

TECHNICAL FIELD The present invention relates to a pharmaceutical composition for preventing or treating obesity, which comprises cryptoxanthinone or a pharmaceutically acceptable salt thereof. The present invention relates to a pharmaceutical composition for prevention or treatment of obesity comprising cryptoxanthinone or a pharmaceutically acceptable salt thereof,

The present invention relates to a pharmaceutical composition for the prevention or treatment of obesity comprising cryptotanshinone (CT) or a pharmaceutically acceptable salt thereof, and more particularly to a pharmaceutical composition for preventing or treating obesity, Mesenchymal stem cells, pre-adipocytes or white adipose cells in brown adipose cells are treated by administering a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable salt thereof, Or beige adipose cells to prevent or treat obesity of the subject, and the like.

There are two types of adipose tissue in mammals that are functionally and metabolically active: white adipose tissue (WAT) and brown adipose tissue (BAT). WAT is specialized in large droplet form of triglycerides for the storage of chemical energy, and the expansion of WAT in terms of number and size causes obesity formation. In contrast, BAT contains a large number of small fat droplets and reacts upon exposure to cold to generate heat, which is named non-shivering heat generation [Non-Patent Document 1]. The heat-producing ability of brown adipocytes is due to the presence and expression of a large number of mitochondria and mitochondrial uncoupling protein 1 (UCP 1) [Non-Patent Document 2]. Thus, increased numbers of mitochondria or increased mitochondrial production are one of the key features of BAT and are associated with some of the nucleosomal and mitochondrial genes, such as Tfam, Nrf1 (nuclear transcription factors 1), cytochrome c oxidase subunits, Cox7a and Cox8b, Protein [Non-Patent Document 3]. BAT in humans has been reported to disappear after infancy, but recently there is evidence that metabolically active BAT is also present in adults. Furthermore, brown fat-like cells, also known as beige cells, have recently been found in WAT (Non-Patent Document 4). Recent studies have shown that brown fat saturation of WAT improves intracellular energy expenditure and reduces the risk of obesity-related metabolic syndrome [Non-Patent Literatures 5, 6, 7]. Thus, an exothermic reaction by BAT and brown fat saturation of WAT are considered as one of the new targets for the treatment or prevention of obesity and its associated metabolic complications.

It is known that PGC-1α (peroxisome proliferator-activated receptor γ coactivator 1α), which is a transcriptional activator of UCP1, plays an important role in increasing mitochondrial function and gene expression and promoting brown localization of WAT [Non-Patent Document 3]. Furthermore, some TGF-beta (transforming growth factor-beta), such as osteogenic proteins (Bmp4 and Bmp7), have similar characteristics to brown adipocytes in some cells such as mesenchymal stem cells C3H10T1 / 2 and preadipocytes 3T3-L1 (Non-Patent Documents 8 and 9).

Cryptotanshinone (CT), a natural compound present in plants of the Salvia miltiorrhiza plant, exhibits several pharmacological effects such as antioxidants, antiinflammatory agents, anticancer agents, antitumor agents, anti-angiogenic agents and anti- [Non-Patent Document 10]. CT inhibits tumor growth in vivo models [Non-Patent Document 11]. In vivo studies have confirmed that CT treatment can reduce the formation of body fat, cholesterol and triglycerides in mice, but the underlying molecular mechanism remains unclear [Non-Patent Document 10]. Furthermore, previous studies by the present inventors have reported that CT inhibits 3T3-L1 preadipocytes differentiation through regulation of the STAT3 pathway in early fat production (Non-Patent Document 31).

A problem to be solved by the present invention is to provide a pharmaceutical composition for effectively preventing and treating obesity of an individual by differentiating the fat tissue in the individual into brown fat and / or beige fat by treating the individual with CT or a pharmaceutically acceptable salt thereof And the like.

In order to solve the above problems, the present invention provides a pharmaceutical composition for preventing or treating obesity comprising CT or a pharmaceutically acceptable salt thereof as an active ingredient.

The present invention also provides a health functional food for preventing or ameliorating obesity comprising CT or a pharmaceutically acceptable salt thereof.

The present invention also provides a cosmetic composition for slimming comprising CT or a pharmaceutically acceptable salt thereof.

The cosmetic composition is preferably a formulation selected from the group consisting of lotion, lotion, cream, essence, foam and pack.

The present invention also provides a composition for inducing differentiation into mesenchymal stem cells, pre-adipocytes or brown adipose cells of white adipocytes or beige adipocytes comprising CT or a pharmaceutically acceptable salt thereof as an active ingredient.

The differentiation induction may be due to an increase in intracellular mitochondria number.

The differentiation induction may be due to the phosphorylation signal pathway of p38-MAPK and Smad1 / 5.

The differentiation induction may be caused by an increase in the expression level of a gene selected from the group consisting of Ebf2, Bmp7, Ucp1, Prdm16, Pgc-1 ?, Zic1, CD137, Tmem26, Sirt1, Tfam, Nrf1 and Cox7 ?.

The present invention also provides a method of inducing differentiation into brown adipocytes or beige adipocytes comprising treating isolated mesenchymal stem cells, pre-adipocytes or white adipocytes with CT or a pharmaceutically acceptable salt thereof.

When the CT of the present invention or a pharmaceutically acceptable salt thereof is administered to an individual, it is possible to effectively induce differentiation of the adipose tissue into brown fat and / or beige fat, and thus can be effectively used for obesity and related diseases, And is a very useful invention in the cosmetics industry.

Figure 1 shows the effect of CT on the differentiation of C3H10T1 / 2 mesenchymal stem cells into adipocytes.
FIG. 2 shows the expression pattern of BAT-specific markers by CT in C3H10T1 / 2 mesenchymal stem cells.
Figure 3 shows the effect of CT on mitochondrial generation.
FIG. 4 shows the effect of CT signaling pathways of p38 MAPK and Smad1 / 5 on C3H10T1 / 2 mesenchymal stem cells.
FIG. 5 shows that the large droplets of single triglyceride caused by CT in C3H10T1 / 2 mesenchymal stem cells change into small droplets and induce phosphorylation of lipid differentiation-related enzymes.

Hereinafter, the present invention will be described in detail.

The present inventors examined the effect of CT on brown fat saturation of C3H10T1 / 2 cells and observed various effects associated with brown fat saturation of CT with Bmp4. The present inventors have shown that CT effectively inhibits expression of white adipocyte-specific markers through the generation of Smad signals in C3H10T1 / 2 cells, while effectively upregulating key BAT-related markers, mitochondrial generation-related markers and some tan-specific markers Respectively.

A recent series of studies has demonstrated that adults have a significant amount of metabolically active brown adipocytes that have the ability to dissipate energy in the form of heat [Non-Patent Literature 4, 12, 13]. In addition, a number of studies using transgenic mice have revealed the appearance of brown adipose cells in the white fat reservoir (droplet pot). Evidence has shown that increased volume of BAT is associated with weight loss and energy consumption in obesity [Non-Patent Literatures 14 and 15]. Investigating factors that induce BAT-like traits in WAT may be a novel therapeutic strategy in the treatment of obesity and related metabolic complications.

Recently, it has been reported that bone morphogenetic protein (BMP) induces the production of brown fat in white adipocytes in both human and mouse cell models [Non-Patent Documents 8, 9, 16, 17]. Several studies have demonstrated that BMP4 and BMP7 induce brown fat saturation of human lipid stem cells [Non-Patent Document 16]. Another study has shown that BMP4 may induce brown fat production of C3H10T1 / 2 mesenchymal stem cells [Non-Patent Document 17]. In addition, recently, some plant-derived anti-fat-producing natural compounds such as curcumin [Non-Patent Document 24], resveratrol [Non-Patent Document 25], sulfolabane [Non-Patent Document 26], and butane [Non-Patent Document 27] Lt; RTI ID = 0.0 > 3T3-L1 < / RTI >

The inventors of the present invention have found that the effect of CT, a quinoid diterpene isolated from Danshen, on the differentiation of mesenchymal C3H10T1 / 2 stem cells into adipocytic lineage, and the effects of C3H10T1 / 2 and inguinal white To investigate the possible effects of brown fat-like features on lipid cells and their underlying molecular mechanisms. Also, in this specification, we compared the effect of CT used with BMP4 or Rosiglitazone as a positive control for the brownizing agent of white adipocytes.

In previous studies, the inventors have found that the treatment of CT during adipocyte differentiation of pre-adipocytes 3T3-L1 causes inhibition of the differentiation of these cells [Non-Patent Document 31]. Interestingly, the present inventors herein have found that pretreatment of CT in mesenchymal C3H10T1 / 2 stem cells induces restraint into adipocytic lineage prior to exposure to lipogenic hormone stimuli, which results in increased triglycerides Ride accumulation. From these results, it became necessary to confirm whether CT treatment induces these restricted cells into brown adipocyte lineage. However, Ebf2 (B-cell factor 2) acts to preserve pre-adipocyte fate in the precursor cell stage. This is because Ebf2-expressing cells from white adipose tissue differentiate into brown-like (or tan) adipocytes by expressing brown-fat-specific genes including Ucp1, Cidea and Pparα in animal body, It is considered to be an optional marker of the cell. The loss of Ebf2 in brown pre-adipocytes was reported to result in a decrease in the expression level of the brown pre-locus-specific gene, while the expression of ectopic Ebf2 in myoblasts leads to the expression of brown pre- Up [Non-Patent Documents 29, 30]. We have now found that in both C3H10T1 / 2 and inguinal white lipid cells, CT treatment induced upregulation of Ebf2 levels, while Ebf1 and Ebf3 levels were significantly reduced. Experimental results in this specification suggest that induction of Ebf2 levels may induce early brown fat cell line arrest due to CT treatment at the early stages of C3H10T1 / 2 and inguinal white lipid differentiation.

This indicates that all three primary members of the Ebf family (Ebf1, 2 and 3) are expressed in adipocytes, where Ebf1 binds specifically to PPARγ (peroxisome proliferator activated peptide γ) Respectively. Ebf1 also binds directly to the C / EBPa promoter and activates it, which gives positive feedback to C / EBPδ expression [Non-Patent Document 28]. Therefore, the CT-mediated inhibition of Ebf1 and Ebf3 can be maintained by antagonizing PPARγ to its anti-adipogenic activity [Non-patent document 31].

Several clues, such as the generation of Bmp signals, have been reported to activate Ebf2, which is particularly critical for early BAT development [Non-Patent Document 32]. Interestingly, our experimental results have shown that CT treatment significantly upregulates Bmp7 transcription levels than transcription levels of other Bmp family members such as Bmp2, Bmp4 and Bmp5. These data strongly support that CT treatment may have the potential to induce a brown fat-like phenotype in bound C3H10T1 / 2 cells.

The heat-producing properties of brown adipocytes are governed by the activity of UCP1 [Non-Patent Document 2]. Interestingly, we found that CT treatment significantly upregulated UCP1 mRNA levels in C3H10T1 / 2 mesenchymal stem cells, suggesting that CT could induce brown fat production of C3H10T1 / 2 mesenchymal stem cells I suggest. A similar effect was also observed in the Bmp4-mediated browning of C3H10T1 / 2 cells by increased expression of UCP1 [Non-Patent Document 17]. Our data also showed that CT significantly induced mRNA levels of Prdm16, an early regulator of brown fat production. Expression of Prdm16 in white adipocytes leads to a complete program of brown fat differentiation including PGC-1 alpha, UCP1, Deiodinase-2, Cidea and genes involved in mitochondrial generation [Non-patent Documents 18, 19]. Consistently, we observed increased expression of UCP1, PGC-1 alpha, Dio2, and Cidea. In addition, the present inventors have also found that CT treatment upregulates mRNA levels of two classic brown fat markers Eval and Zicl. Our data show that CT treatment significantly induces mRNA levels of highly expressed PPARα in white adipocytes treated with β ₃ -adrenergic agonist in brown fat-like adipocytes.

Differentiation and physiological action of brown adipocytes is associated with enhanced mitochondrial production. The transcription factor activator PGC-1 alpha is the master modulator of mitochondrial action [Non-patent document 20]. This protein stimulates mitochondrial production and activates some genes, such as Nrf1, that regulate the expression of Tfam. Although Tfam is encoded in the nuclear genome, it enters the mitochondria and additionally stimulates replication and transcription of the mitochondrial genome [Non-Patent Document 21].

The present inventors observed that CT significantly induced mRNA levels of PGC-1 alpha, and Tfam and Nrf1 were also significantly induced. These data suggest that CT has effectively induced mitochondrial generation through up-regulation of these mitochondrial generation-related genes. In addition, the cytochrome oxidase subunit VIII a (Cox7a) was significantly induced by CT treatment, indicating an increase in the number of mitochondria.

Rodents, on the other hand, have two distinct types of brown fat, classic brown and beige. Classic brown fats originate from the Myf5 ⁺ line and are located between the scapular bones of the mouse, while yellow brown brown fats originate from the Myf5 ^- line and are present in white adipose tissue. Most of the morphological and functional characteristics of the tan adipocytes are similar to classic brown adipose cells, but they have a unique pattern of gene expression [Non-patent document 19]. As CT induces the expression of classic brown adipocyte markers including Zic1, Cidea and Dio2, the present inventors further found that expression of a gene specific for yellowish brown adipocytes such as CD137, Tmem26, Tbx1, Fgf21, Hspb7 and arginase 1 [Non-Patent Document 22]. The experimental results of the present inventors showed that CD137 and Tmem26 were up-regulated by CT treatment while Tbx1, Fgf21, Hspb7 and arginase 1 were not induced by CT treatment, suggesting that CT is partially induced in C3H10T1 / 2, . Furthermore, CT decreased the expression of highly expressed Resistin and Psat-1 in white adipocytes, whereas Serpina3k, another white adipocyte marker, was hardly affected by CT treatment. These experimental results suggest that CT may inhibit the production of white fats, supported by previous studies by the present inventors that CT inhibited 3T3-L1 adipogenesis.

In addition, since Smad signaling pathway plays an important role in the generation of brown fat [Non-patent Documents 8 and 23], we observed the role of CT in C3H10T1 / 2 cells during Smad signaling. BMP4 appears to activate brown fat production through Smad signaling in various in vitro and in vivo models. The results of the present invention demonstrate that CT in BMP4 pretreated cells highly activates the Smad1 / 5 phosphorylation reaction compared to only BMP4 pretreated cells, suggesting that CT stimulates Smad signaling through activation of Smad1 / 5 phosphorylation Suggesting activation.

Overall, the experimental results of the present invention demonstrate that CT effectively induces brown fat production in C3H10T1 / 2 mesenchymal stem cells with induction of mitochondrial production by Smad signaling. CT also partially activates some of the yellowish brown selective genes and inhibits the white fat cell specific gene Resistin. The effect of CT on the production of brown adipose fat in vivo has not been studied, and further studies will be required. In conclusion, the discovery of the present invention will provide a pathway for new therapies for the treatment or prevention of obesity.

Accordingly, the present invention provides a pharmaceutical composition for preventing or treating obesity comprising CT or a pharmaceutically acceptable salt thereof as an active ingredient.

The term " obesity " refers to a clinical condition in which the amount of fat in an individual exceeds a normal range. The obesity also includes metabolic syndrome due to various metabolic abnormalities associated with obesity.

The pharmaceutical composition containing the active ingredient of the present invention may be formulated into tablets, capsules, suspensions, emulsions, oral preparations such as syrups and aerosols, injections of sterilized injection solutions And may be administered by various routes including oral administration or intravenous, intraperitoneal, subcutaneous, rectal, topical administration, and the like.

Such pharmaceutical compositions may further comprise carriers, excipients or diluents, and examples of suitable carriers, excipients or diluents that may be included include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, But are not limited to, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, amorphous cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, And the like. In addition, the pharmaceutical composition of the present invention may further include a filler, an anti-coagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent, an antiseptic, and the like.

In a preferred embodiment, the solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient, for example starch, calcium carbonate, Sucrose, lactose, gelatin and the like are mixed and formulated. In addition to simple excipients, lubricants such as magnesium stearate, talc, and the like may also be used.

Examples of the oral liquid preparation include suspensions, solutions, emulsions, syrups and the like. In addition to water and liquid paraffin which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, Perfumes, preservatives, and the like.

As a preferable specific example, the preparation for parenteral administration includes sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-drying agents, suppositories, and the like. Examples of the non-aqueous solvent and suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Injectables may include conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, preservatives, and the like.

The active ingredient of the present invention is administered in a pharmaceutically effective amount. In the present invention, " pharmaceutically effective amount " means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level will depend on the type of disease, severity, The sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of the treatment, factors including co-administered drugs, and other factors well known in the medical arts. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiply. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without side effects, which can be easily determined by those skilled in the art.

As a preferable example, the effective amount of the active ingredient in the pharmaceutical composition of the present invention may vary depending on the age, sex, and body weight of the patient, and generally 1 to 5,000 mg, preferably 100 to 3,000 mg per kg of body weight per day Or every other day or one to three times a day. However, the dosage may not be limited in any way because it may be increased or decreased depending on route of administration, severity of disease, sex, weight, age, and the like.

The pharmaceutical composition of the present invention can be administered to a subject through various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

In the present invention, " administration " means providing a predetermined substance to a patient by any suitable method, and the administration route of the pharmaceutical composition of the present invention is either oral or non-oral May be administered orally. The composition of the present invention may also be administered using any device capable of delivering an effective ingredient to a target cell.

In the present invention, the term " object " includes, but is not limited to, human, monkey, cow, horse, sheep, pig, chicken, turkey, quail, cat, dog, mouse, rat, rabbit or guinea pig , Preferably a mammal, more preferably a human.

The present invention also provides a health functional food for preventing or ameliorating obesity comprising CT or a pharmaceutically acceptable salt thereof.

The health functional food of the present invention can be variously used for foods and beverages effective for preventing or improving various symptoms related to obesity. Examples of foods containing the active ingredient of the present invention include various foods, beverages, gums, tea, vitamin complex, health supplement foods and the like, and they can be used in the form of powder, granule, tablet, capsule or beverage .

The active ingredient of the present invention may generally be added in an amount of 0.01 to 15% by weight of the total food, and the health beverage composition may be added in a proportion of 0.02 to 10 g, preferably 0.3 to 1 g, based on 100 ml.

The health functional food of the present invention may contain, as an additional ingredient, a food-acceptable food-aid additive such as natural carbohydrates and various flavors, in addition to containing the above-mentioned compound as an essential ingredient in the indicated ratio.

Examples of the natural carbohydrate include sugar sugars such as glucose, monosaccharides such as fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol.

Examples of the flavoring agents include natural flavoring agents such as tautatin, rebaudioside A and stiglycerin such as glycyrrhizin, and synthetic flavoring agents such as saccharin and aspartame. The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the health functional food of the present invention. In addition to the above, the health functional food of the present invention may contain various kinds of nutrients, vitamins, minerals, flavors such as synthetic flavors and natural flavors, colorants and heavy stabilizers, pectic acid and its salts, alginic acid and its salts, Thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. In addition, the health functional food of the present invention may contain flesh for producing natural fruit juice, fruit juice drink, vegetable drink and the like. These components may be used independently or in combination. The ratio of such additives is generally selected in the range of 0.01 to about 20 parts by weight per 100 parts by weight of the active ingredient of the present invention.

The present invention also provides a cosmetic composition for slimming comprising CT or a pharmaceutically acceptable salt thereof.

The term " slimming " means a phenomenon in which the volume of the whole or a specific part of the body is reduced, for example, the elimination or reduction of fat in the whole or a specific part of the body.

The active ingredient of the cosmetic composition of the present invention may be contained in an amount of 0.01 to 50% by weight, preferably 0.1 to 20% by weight, more preferably 1 to 10% by weight based on the total weight of the composition. Appropriate formulation stability can be ensured within such a content range, and a desired slimming effect can be expected.

The composition of the present invention may further contain, in addition to the effective ingredient of the present invention, a known natural extract or other ingredients for the antioxidative, anti-aging, moisturizing and skin regeneration promoting effects within the range not inhibiting the active activity of the present invention .

In addition, the composition may also contain components commonly added to cosmetic compositions, such as fatty substances, organic solvents, solubilizers, thickeners, gelling agents, softeners, antioxidants, suspending agents, stabilizers, foaming agents, A cosmetic or dermatological composition such as a perfume, a perfume, a surfactant, water, an ionic or nonionic type emulsifier, a filler, a sequestering and chelating agent, a preservative, a vitamin, a barrier, a wetting agent, an essential oil, a dye, a pigment, a hydrophilic or lipophilic active agent May be formulated into a particular formulation, including adjuvants or carriers commonly used in the field of dermatology.

The cosmetic composition of the present invention may be prepared in any form conventionally produced in the art and may be formulated into various forms such as solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, Containing cleansing, oil and spray, but are not limited thereto. More specifically, the present invention relates to a lotion, a lotion, a flexible lotion, a nutritional lotion, a cream, a nutritional cream, a massage cream, an essence, an eye cream, a powder foundation, an emulsion foundation, a wax foundation, a cleansing cream, a foam, a cleansing foam, , Spray, powder, fact, lip gloss, lipstick, shadow, shampoo and rinse.

When the formulation of the present invention is a paste, cream or gel, an animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as the carrier component .

When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as the carrier component. In particular, in the case of a spray, a mixture of chlorofluorohydrocarbons, Propane / butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, a solubilizing agent or an emulsifying agent is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid esters of sorbitan.

In the case where the formulation of the present invention is a suspension, a carrier such as water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Cellulose, aluminum metahydroxide, bentonite, agar or tracant, etc. may be used.

When the formulation of the present invention is an interfacial active agent-containing cleansing, the carrier component may include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters.

The present invention also provides a composition for inducing differentiation into mesenchymal stem cells, pre-adipocytes or brown adipose cells of white adipocytes or beige adipocytes comprising CT or a pharmaceutically acceptable salt thereof as an active ingredient.

The differentiation induction may be due to an increase in intracellular mitochondria number.

The differentiation induction may be due to the phosphorylation signal pathway of p38-MAPK and Smad1 / 5.

The differentiation induction may be caused by an increase in the expression level of a gene selected from the group consisting of Ebf2, Bmp7, Ucp1, Prdm16, Pgc-1 ?, Zic1, CD137, Tmem26, Sirt1, Tfam, Nrf1 and Cox7 ?.

The composition for inducing differentiation can be applied, for example, as a reagent composition for research. In general, the composition for inducing differentiation of the present invention can be administered to a cell or an experimental animal under laboratory conditions. It is preferable that the specific administration method and safety rule are performed according to the internal regulations of each laboratory of the experimenter.

The present invention also provides a method of inducing differentiation into brown adipocytes or beige adipocytes comprising treating isolated mesenchymal stem cells, pre-adipocytes or white adipocytes with CT or a pharmaceutically acceptable salt thereof.

The mesenchymal stem cells, whole adipocytes or white adipocytes are those which have been separated from the subject by natural or artificial methods. The cells isolated from the individual can be cultured for several days to several weeks under appropriate culture conditions, and may be stored in a freezer as the case may be.

Methods for administering the active ingredient of the present invention to the isolated cells are obvious to those of ordinary skill in the art, and preferably those described in the following Examples of the present invention may be followed.

Hereinafter, the present invention will be described in more detail with reference to specific examples. The following examples illustrate one preferred embodiment of the present invention, and it is apparent that the scope of the present invention is not to be construed as limited by the matters described in the following examples.

[ Example ]

1. Materials and Methods

1.1. chemical substance

(Purity ≥ 98%, HPLC), dexamethasone, isobutyl-1-methylxanthine (IBMX), insulin and dibutyryladenosine 3 ', 5'-cyclic monophosphate (cAMP, %, HPLC) was purchased from Sigma-Aldrich (St. Louis, MO). Dulbecco's modified Eagle's medium (DMEM), newborn calf serum (NBCS) and recombinant human BMP4 were purchased from Gibco (Grand Island, NY, USA). Fetal serum was purchased from Atlas Biologicals (Fort Collins, Colo., USA). The penicillin-streptomycin solution was purchased from Hyclone Laboratories, Inc. (Saugogan, NY, USA). Antibodies to Mitotracker red probe, P-Smad1 / 5, Smad1, P-AMPK, AMPK and HRP-linked anti-rabbit IgG were purchased from Cell signaling Technology (Danvers, Mass., USA) . Antibodies to β-actin were purchased from Abcam (Cambridge, Mass., USA). The BCA protein assay kit was purchased from Thermo Scientific (Rockford, Ill.). Protein loading buffer was purchased from Bio-Rad Laboratories, Inc. (Hercules, CA, USA).

1.2. Cell culture, differentiation and treatment

C3H10T1 / 2 mouse pluripotent stem cells to Korea Cell Line Bank (Korean Cell Line Bank) obtained from the (KCLB-10226), and that in a humidified incubator of one, 37 ℃ containing 5% CO ₂ - glucose DMEM, 10% (Volume / volume) heat-inactivated newborn calf serum and 1% (volume / volume) antibiotic / antifungal agent (100 U / mL penicillin, 100 U / mL streptomycin). To induce commitment, C3H10T1 / 2 cells were inoculated at low density to obtain confluence of 20-30% the following day. The next day, the cells were treated with or without 50 ng / mL human recombinant BMP4 until the cells were completely confluent. The medium was supplemented every 2 to 3 days. 48 hours after confluence inducing differentiation by changing the medium to DMEM containing 10% FBS, 0.5 mM IBMX, 1 μM dexamethasone and 10 μg / ml insulin (MDI), in the presence or absence of 8 μM CT designated at day 0 Respectively. The medium was then supplemented with DMEM containing 10% FBS and 10 [mu] g / ml insulin with or without 8 [mu] M CT every other day for up to 8 days. Differentiated cells were exposed to 500 μM dibutyryl cAMP for 4 h to stimulate the heat-generating program.

1.3. Mito Tracker  By red dyeing Maitocondria  Analysis of content

To label the mitochondria, the cells were stained with a 50 nM mitotracker red probe (manufactured by Cell Signaling) for 30 min at 37 ° C during treatment. After the incubation, fluorescence microscopy was performed using an Axiovert-25 microscope (manufactured by Carl Zeiss, Germany) for 579 nm excitation and 599 nm emission wavelength.

1.4. qRT PCR  analysis

Total RNA was extracted using an RNA extraction kit (Qiagen, Valencia, Calif., USA) according to the manufacturer's instructions and the concentration was determined using a Scandrop spectrometer from Analytik jena AG (Zena, Germany). 1 μg of the RNA was subjected to reverse transcriptase PCR to synthesize cDNA with a Maxime RT PreMix kit (manufactured by Intron Biotechnology Co., Seoul, Korea), and the reaction was performed in a Veriti 96-well thermal cycler (Applied Biosystems, Singapore) Respectively. Quantitative real-time PCR was performed on a CFX96 TM real-time PCR detection system (Bio-Rad, Singapore) using the iQTM SYBR Green Supermix kit (Bio-Rad, Singapore). The sequences for the primers are listed in Table 1. The expression level was normalized to Gapdh.

[Table 1]

1.5. Whole cell extract preparation and Western Blot  analysis

Before harvesting, untreated cells and treated cells were washed twice with ice-cold 1X phosphate buffered saline (PBS) and lysed in RIPA lysis buffer supplemented with proteolytic enzyme inhibitor cocktail, 2mM PMSF and 1mM sodium orthovanadate (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The lysates were scraped off, harvested, incubated on ice for 15 minutes, and centrifuged at 14,000 rpm for 15 minutes at 4 ° C to collect the supernatant. Protein concentrations were determined using a BCA protein assay kit (Pierce, Rockford, IL). Equal amounts of protein were loaded onto each sample and separated on a 4-20% sodium dodecyl sulfate polyacrylamide gradient gel (Mini-PROTEAN Precast Gel, Bio-Rad). After separation of the electrophoresis, the protein was then transported onto a polyvinylidene fluoride (PVDF) membrane at 13V using a semi-dry transfer cell (Bio-Rad) for about 1.5 hours. The membranes were then blocked for 1 hour at room temperature on a shaker using 5% dry skim milk in 1 x Tris buffered saline (TBS) containing 0.1% Tween 20 and incubated with primary antibody overnight at 4 < 0 & . After incubation with the primary antibody, the membrane was washed three times at room temperature for about 5 minutes. The nitrocellulose membrane was then incubated for 1.5 hours at room temperature under stirring on a horseradish peroxidase-conjugated secondary antibody shaker, and then washed three times each for about 5 minutes. All washing steps were performed in 1 x TBS containing 0.1% Tween 20. Immunoreactive protein signals were detected using chemiluminescent ECL analysis and measured using molecular imaging software (Bio-Rad). A small adjustment of brightness and contrast was performed for better visualization of the data, but the same change was applied to the entire image panel as a whole and there was no loss of information in the image. Anti-beta-actin was used as a load control for each protein expression.

2. result

2.1. By CT Versatility C3H10T1 /2 Stem cell  Induction of restraint into adipocyte system

To investigate the role of CT during C3H10T1 / 2 cell differentiation, we treated CT and Bmp4 during confinement and browning of C3H10T1 / 2 pluripotent stem cells (Figure 1A). Consistent with our previous findings, non-patent document 31, co-treatment of CT with adipogenic hormone stimulation (MDI) inhibited the differentiation of 3T3-L1 adipocytes (FIG. 1A). Interestingly, however, CT co-treatment during MDI-stimulated arrest of pluripotent C3H10T1 / 2 stem cells affected the binding of multiple pluripotent C3H10T1 / 2 stem cells to the adipocyte lineage, As evidenced by increased triglyceride accumulation as measured by oil red O) staining (Figure IB). Next, we compared the effect of CT with the known inductor Bmp4 of C3H10T1 / 2 stem cell confinement, and our data showed that CT treatment significantly induced fat accumulation compared to MDI treatment, but not Bmp4 (Fig. 1C). Further, this data shows that CT treatment significantly increased the levels of RNA and protein in B-cell factor 2 (Ebf2), known as derivatives of early brown fat adipocyte lineage restriction, in both C3H10T1 / 2 and inguinal WAT cells (Fig. 1D). This data shows that CT treatment significantly increased the RNA level of bone morphogenetic protein 7 (Bmp7), which is known to be a derivative of mesenchymal C3H10T1 / 2 stem cells to pre-adipocytes (Ref), while Bmp2, Bmp4 and Bmp5 Lt; RTI ID = 0.0 > 1E). ≪ / RTI > The data suggest that CT may contribute to fat-producing phylogeny in MDI-induced C3H10T1 / 2 mesenchymal cells rather than terminal differentiation.

2.2. By CT Versatility C3H10T1 /2 In stem cells  Increased expression of BAT and tan specific genes and WAT  Specific gene reduction

Since Bmp4 is known to induce BAT-specific markers in white adipocytes, in order to investigate the CT ability to induce brown fat-like features in C3H10T1 / 2 adipocytes and compare the brown effect of CT to Bmp4, The inventors classified the cells into several treatment groups: MDI unstimulated, MDI stimulated and supplemented with MDI stimulated cells by CT or Bmp4 or a combination thereof (FIG. 2).

Consistent with recent studies, pretreatment of Bmp4 in cells prior to exposure to MDI resulted in significant inhibition of BAT-specific markers Ucp1, Pgc-1α, Prdm16, deiodination agent-2 (Dio2) Cidaa, PPARa, epithelial V-like antigen 1 (Eba1) and Zic1. Surprisingly, we observed that CT treatment significantly induced mRNA levels of Pgc-1 alpha (up to about 4-fold) and co-treatment of CT with Bmp4 induced Pgc-1 alpha mRNA levels (8-fold). Consistently, Prdm16, an important early regulator of brown cell fat production, was significantly induced both by Bmp4-dependent and independently by CT. In particular, Ucp1, a major brown fat heat generation marker, is up-regulated by CT, and co-treatment of CT with Bmp4 results in significant induction. Additionally, we also observed upregulation at the mRNA level of some other important brown fat-selective genes such as Dio2, Sidera, Pparα, Eval and Zicl by CT treatment in the presence or absence of Bmp4 (Figure 2A) . As CT induced the major brown fat-selective markers Ucp1 and Pgc-lα, we further evaluated the effect of CT on yellowish fat cell markers. Interestingly, CT induced two major yellowish brown selective genes, Cd137 and Tmem26, where Tbx1, Fgf21, Hspb7, and other tan genes, including arginine 1, were not induced by CT treatment. Combination treatment of CT with Bmp4 showed elevated levels of Cd137 and Tmem26 expression relative to CT alone or Bmp4 alone treatment (Fig. 2B). On the other hand, CT treatment significantly reduced the RNA levels of the major white lipid tissue-selective genes Psat1 and ristigin, while the level of Serpina was not significantly changed (Fig. 2C).

2.3. Promoting the production of mitochondria by CT

Mitochondrial formation and action is critical to the development of brown fat. Since CT induces the browning of C3H10T1 / 2 white adipocytes in both BMP4-dependent and independent ways, we further assessed the effect of CT on the expression of mitochondrial mass and mitochondrial generation related genes. We found that CT treatment markedly upregulated the expression of Pgc-1 alpha, the major regulator of mitochondrial production and action in both Bmp4 pretreated or untreated differentiated C3H10T1 / 2 cells. Nuclei respiratory factor 1 (Nrf1), mitochondrial transcription factor A (Tfam), cytochrome oxidase subunit VIIIa (Cox7a) were also significantly induced by CT treatment in Bmp4 pretreated cells while cytochrome c The oxidase subunit VIIIb (Cox8b) expression was maintained unchanged (Figure 3).

2.4. By CT C3H10T1 / 2 and < RTI ID = 0.0 > 3T3-L1 & MAPK  And Smad1 / 5 Specific activation of signal generation

Smad is the main signaling pathway activated by BMP during the arrest of white pre-adipocytes or the browning of white adipocytes. Regardless of whether CT activated Smad signaling during C3H10T1 / 2 differentiation, we pretreated 30-40% confluence cells with 8 μM CT for the indicated time period. The data of the present invention demonstrate that the p38-MAPK phosphorylation reaction is activated within the first 5 minutes of CT treatment, which is maintained for up to 10 minutes and thereafter decreased. Immediately afterwards, a phosphorylation reaction of Smad1 / 5 appeared, which lasted up to 60 minutes (Fig. 4A). Furthermore, although CT treatment could reverse the inhibitory effect of SB20358 on p38 MAPK phosphorylation, the inhibitory effect of PD98059 on ERK1 / 2 phosphorylation could be reversed, suggesting that CT specifically activates p38 MAPK phosphorylation, but ERK1 / 2 phosphorylation is not activated (Fig. 4B).

<110> SOON CHUN HYANG UNIVERSITY INDUSTRY ACADEMY COOPERATION FOUNDATION <120> A PHARMACEUTICAL COMPOSITION FOR PREVENTION OR TREATMENT OF          OBESITY COMPRISING CRYPTOTANSHINONE OR PHARMACEUTICALLY ACCEPTED          SALTS THEREOF AS AN EFFECTIVE COMPONENT <130> YP160043 <160> 64 <170> KoPatentin 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Mouse Bmp2 forward primer <400> 1 gggacccgct gtcttctagt 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Mouse Bmp2 reverse primer <400> 2 tcaactcaaa ttcgctgagg 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Mouse Bmp4 forward primer <400> 3 gacttcgagg cgacacttct 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Mouse Bmp4 reverse primer <400> 4 gccggtaaag atccctcatg 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Mouse Bmp5 forward primer <400> 5 ttacttaggg gtattgtggg 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Mouse Bmp5 reverse primer <400> 6 ccgtctctca tggttccgta 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Mouse Bmp7 forward primer <400> 7 acggacaggg cttctcctac 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Mouse Bmp7 reverse primer <400> 8 atggtggtat cgagggtgga 20 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Mouse Cidea forward primer <400> 9 tgctcttctg tatcgcccag t 21 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Mouse Cidea reverse primer <400> 10 gccgtgttaa ggaatctgct g 21 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Mouse Cox1 forward primer <400> 11 tctactattc ggagcctgag 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Mouse Cox1 reverse primer <400> 12 ctactgatgc tcctgcatgg 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Mouse Cox7alpha1 forward primer <400> 13 cagcgtcatg gtcagtctgt 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Mouse Cox7alpha1 reverse primer <400> 14 agaaaaccgt gtggcagaga 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Mouse Cox8b forward primer <400> 15 gaaccatgaa gccaacgact 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Mouse Cox8b reverse primer <400> 16 cgcaagttca cagtcgttcc 20 <210> 17 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Mouse Dio2 forward primer <400> 17 cagtgtggtg cacgtctcca atc 23 <210> 18 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Mouse Dio2 reverse primer <400> 18 tgaacccccg ttgaccacca g 21 <210> 19 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Mouse Ebf1 forward primer <400> 19 aaggatggac gacgggaggc g 21 <210> 20 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Mouse Ebf1 reverse primer <400> 20 gggatgtgcc gaggaggtgt ct 22 <210> 21 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Mouse Ebf2 forward primer <400> 21 gctgcgggaa ccggaacgag a 21 <210> 22 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Mouse Ebf2 reverse primer <400> 22 acacgacctg gaaccgcctc a 21 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Mouse Ebf3 forward primer <400> 23 cgaaaggacc gcttttgtgg 20 <210> 24 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Mouse Ebf3 reverse primer <400> 24 agtgaatgcc gttgttggtt t 21 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Mouse Gapdh forward primer <400> 25 gacatgccgc ctggagaaac 20 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Mouse Gapdh reverse primer <400> 26 agcccaggat gccctttagt 20 <210> 27 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Mouse Hspb7 forward primer <400> 27 gagcatgttt tcagacgact ttg 23 <210> 28 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Mouse Hspb7 reverse primer <400> 28 ccgagggtct tcatgtttcc tt 22 <210> 29 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Mouse Aginase 1 forward primer <400> 29 tggcttgcga gacgtagac 19 <210> 30 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Mouse Aginase 1 reverse primer <400> 30 gctcaggtga atcggccttt t 21 <210> 31 <211> 22 <212> DNA <213> Artificial Sequence <220> Mouse CD137 forward primer <400> 31 cgtgcagaac tcctgtgata ac 22 <210> 32 <211> 21 <212> DNA <213> Artificial Sequence <220> Mouse CD137 reverse primer <400> 32 gtccacctat gctggagaag g 21 <210> 33 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Mouse Cox2 forward primer <400> 33 gactgggcca tggagtgg 18 <210> 34 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Mouse Cox2 reverse primer <400> 34 cacctctcca ccaatgacc 19 <210> 35 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Mouse Eva1 forward primer <400> 35 ccacttctcc tgagtttaca gc 22 <210> 36 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Mouse Eva1 reverse primer <400> 36 gcattttaac cgaacatctg tcc 23 <210> 37 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Mouse Fgf21 forward primer <400> 37 agatcaggga ggatggaaca 20 <210> 38 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Mouse Fgf21 reverse primer <400> 38 tcaaagtgag gcgatccata 20 <210> 39 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Mouse wTbx1 forward primer <400> 39 ggcaggcaga cgaatgttc 19 <210> 40 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Mouse wTbx1 reverse primer <400> 40 ttgtcatcta cgggcacaaa g 21 <210> 41 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Mouse mTmem26 forward primer <400> 41 accctgtcat cccacagag 19 <210> 42 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Mouse mTmem26 reverse primer <400> 42 tgtttggtgg agtcctaagg tc 22 <210> 43 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Mouse Nrf1 forward primer <400> 43 caacagggaa gaaacggaaa 20 <210> 44 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Mouse Nrf1 reverse primer <400> 44 gcaccacatt ctccaaaggt 20 <210> 45 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Mouse P0 forward primer <400> 45 gcactttcgc tttctggagg gtgt 24 <210> 46 <211> 24 <212> DNA <213> Artificial Sequence <220> Mouse P0 reverse primer <400> 46 tgacttggtt gctttggcgg gatt 24 <210> 47 <211> 19 <212> DNA <213> Artificial Sequence <220> Mouse Pgc1a forward primer <400> 47 acagctttct gggtggatt 19 <210> 48 <211> 20 <212> DNA <213> Artificial Sequence <220> Mouse Pgc1a reverse primer <400> 48 tgaggaccgc tagcaagttt 20 <210> 49 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Mouse Ppara forward primer <400> 49 gcgtacggca atggctttat 20 <210> 50 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Mouse Ppara reverse primer <400> 50 gaacggcttc aggttctt 18 <210> 51 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Mouse Prdm16 forward primer <400> 51 cagcacggtg aagccattc 19 <210> 52 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Mouse Prdm16 reverse primer <400> 52 gcgtgcatcc gcttgtg 17 <210> 53 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Mouse Psat1 forward primer <400> 53 taccgccttg tcaagaaacc 20 <210> 54 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Mouse Psat1 reverse primer <400> 54 agtggagcgc cagaatagaa 20 <210> 55 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Mouse Resistin forward primer <400> 55 tgccagtgtg caaggataga ct 22 <210> 56 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Mouse Resistin reverse primer <400> 56 cgctcacttc cccgacat 18 <210> 57 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Mouse Serpina3k forward primer <400> 57 ggctgaaggc aaagtcagtg t 21 <210> 58 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Mouse Serpina3k reverse primer <400> 58 tggaatctgt cctgctgtcc t 21 <210> 59 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Mouse Tfam forward primer <400> 59 gtccataggc accgtattgc 20 <210> 60 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Mouse Tfam reverse primer <400> 60 cccatgctgg aaaaacactt 20 <210> 61 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Mouse Ucp1 forward primer <400> 61 ggcattcaga ggcaaatcag ct 22 <210> 62 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Mouse Ucp1 reverse primer <400> 62 caatgaacac tgccacacct c 21 <210> 63 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Mouse Zic1 forward primer <400> 63 ctgttgtggg agacacgatg 20 <210> 64 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Mouse Zic1 reverse primer <400> 64 ctgttgtggg agacacgatg 20

Claims (9)

delete delete delete delete A reagent composition for inducing differentiation of mesenchymal stem cells into brown adipocytes or beige adipocytes comprising cryptotanshinone or a pharmaceutically acceptable salt thereof as an active ingredient. 6. The reagent composition according to claim 5, wherein the induction of differentiation is caused by an increase in intracellular mitochondria number. 6. The reagent composition according to claim 5, wherein the differentiation induction is by a phosphorylation signal pathway of p38-MAPK and Smad1 / 5. 6. The method according to claim 5, wherein the differentiation induction is caused by an increase in the expression level of a gene selected from the group consisting of Ebf2, Bmp7, Ucp1, Prdm16, Pgc-1 ?, Zic1, CD137, Tmem26, Sirt1, Tfam, Nrf1 and Cox7? &Lt; / RTI &gt; A method of inducing differentiation into brown adipocytes or beige adipocytes comprising treating isolated mesenchymal stem cells with cryptotanshinone or a pharmaceutically acceptable salt thereof.

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