KR102118771B1 - Compositions for inhibiting adipocyte differentiation and proliferation comprising 7'8′'-Dihydroxyflavone - Google Patents

Abstract

The present invention relates to a composition for inhibiting adipocyte differentiation and proliferation containing 7'8'-dihydroxyflavone, and the composition for adipocyte differentiation and proliferation inhibitor of the present invention is a conventional pharmaceutical Without the side effects that appeared in the composition, it is possible to specifically reduce only adipocytes, suppress the differentiation of adipocytes into adipocytes, and promote the expression of antioxidant enzymes, thereby accumulating free radicals in adipocytes and the activity of MAPK. Since it has a reducing effect, it can be usefully used as a pharmaceutical composition for preventing or treating obesity or as a health functional food for preventing or improving obesity.

Description

Composition for inhibiting adipocyte differentiation and proliferation comprising 7'8'-dihydroxyflavone}

The present invention relates to a composition for inhibiting adipocyte differentiation and proliferation comprising 7'8'-dihydroxyflavone, and more specifically, fat by reducing the expression level of adipocyte differentiation factor. It relates to a composition for inhibiting the differentiation and proliferation of adipocytes, which has the effect of inhibiting the differentiation of progenitor cells into adipocytes and reducing lipid, free radical accumulation and MAPK activity in adipocytes by promoting the expression of antioxidant enzymes.

In addition to the westernized diet, obesity in which fat tissue is excessively accumulated due to an inequality of calorie intake and consumption has increased significantly. Obesity, either independently or in close association with other diseases, causes serious health problems, especially increasing the prevalence and mortality of colorectal cancer, ovarian cancer, breast cancer, prostate cancer, etc. and coronary heart disease, diabetes, degenerative arthritis, and menstruation It causes a wide range of serious illnesses, including impurity and psychological disorders. Therefore, the prevention and treatment of obesity is considered to be a very effective means for the prevention of cancer and various adult diseases, and worldwide attention is gathering.

Obesity develops through the process of mitosis and differentiation of adipose progenitor cells, the number of adipocytes increases, and the accumulation of lipids in adipocytes. In this process, specific transcription factors are required. A representative transcription factor, peroxisome proliferator-activated receptors (γ, PPAR γ), is a nuclear hormone receptor superfamily and regulates expression by recognizing a promoter or enhancer of a target gene. C/EBP α and β (CCAAT/enhancer-binding protein-α,β, C/EBP-α,β) are important transcription factors that increase expression in fat cells during obesity. , When the expression levels of β and PPAR γ increase, the expression of adipocyte protein 2 (adipocyte protein 2), GAPDH, GLUT4, etc., is accelerated by obesity.

Furthermore, as the development of obesity, increased reactive oxygen species in the adipose tissue (Reactive Oxygen Species, ROS) causes dysregulation of adipocytokines and antioxidant enzymes secreted by fat cells, and ROS accumulated in the cytoplasm is fat It is known to further induce obesity by participating in dimer formation of C/EBP β, which is involved in cell differentiation.

ROS (Reactive Oxygen Species, ROS), the superoxide ion ^{_{(superoxide anion, O 2 -)}} , hydrogen peroxide (Hydrogen peroxide) and hydroxy radical there is the like (hydroxyl radical, OH) superoxide ions in the lipogenesis process It is generated in large quantities by the expressed NADPH oxidase. The superoxide ion is converted into hydrogen peroxide by the intracellular antioxidant enzyme Manganese Superoxide Dismutase (Mn-SOD), and the converted hydrogen peroxide is converted into water by the catalase enzyme to remove intracellular ROS. do.

In addition, HO-1 (heme oxygenase-1) is known as an enzyme that protects cells from oxidative stress. It has been reported that the increase in HO-1 expression suppresses the development of obesity. Therefore, controlling antioxidant enzymes and removing ROS can suppress obesity. ROS is also known to induce the activation of mitogen-activated protein kinase (MAPK), including extracellular signal regulated kinase (ERK), c-jun Nterminal kinase (JNK), and p38 MAP kinase. MAPK is involved in initiating differentiation and cell proliferation in adipocytes. As the number of adipocytes increased due to mitosis of adipocytes is involved in the development of obesity, inhibiting the activation of MAPK can also suppress the development of obesity.

In addition, ROS, which accumulates as obesity develops, induces oxidative stress by affecting other tissues, including muscle and liver tissues, through the blood in adipose tissue. Therefore, ROS removal and increased expression of antioxidant enzymes can inhibit the development of obesity by inhibiting the differentiation and cell proliferation of adipocytes, as well as protect against various diseases caused by oxidative stress.

Among the typical anti-obesity drugs developed to date are sibutramine, which has an appetite suppressant effect, and Xenical™, which inhibits fat absorption (Roche Pharmaceuticals, Switzerland). Sibutramine is a serotonin norepinephrine reuptake inhibitor (SNRI) that exhibits a sympathetic nerve-stimulating effect, and suppresses the satiety center to control the appetite. Sibutramine was approved in the U.S. on February 16, 2001 as Meridia, and was approved as a treatment for obesity to maintain weight loss and weight loss with low calorie intake.In Korea, in 2001, it was imported and sold by Ilsung New under the trade name Reductil. have. Xenical is a pancreatic lipase (lipase) inhibitor that prevents digestion and absorption of dietary fat, which is a major cause of obesity, and acts on the mucous membranes of the stomach and small intestine. Undigested fat is excreted in the stool in an unconverted state up to 30% of the total intake fat, and is currently used as a treatment for hyperobesity patients. However, side effects of these drugs cause heart disease, respiratory disease, blood pressure rise and insomnia, and have a problem in that the efficacy is low. Therefore, in the future, there is an urgent need to develop a high-value-added multi-functional product that has various side effects without preventing side effects, preventing and treating obesity, and reducing oxidative stress.

On the other hand, flavonoids are constituents of plant pigments and are secondary metabolites of plants as phenolic compounds. It has been found that thousands of kinds of flavonoids exist in plants by changing the positions of several residues around the basic structure, and in various forms, the functions and functions of the structures differ.

Among various types of flavonoids, 7'8'-Dihydroxyflavone has been reported for anti-inflammatory, vasodilatory, anti-hypertensive and antioxidant functions, and especially protects DNA, neurons and keratinocytes against oxidative stress. It is known to have excellent action. However, despite the fact that the antioxidant capacity of the substance in the development of obesity is closely related to the anti-obesity effect, 7'8'-dihydroxyflavones have not been studied or reported for direct antioxidant and anti-obesity activities against adipocytes. .

Thus, the present inventors have no side effects, and as a result of diligent efforts to select natural substances for prevention or treatment of obesity with excellent therapeutic effect, 7'8'-dihydroxyflavone (7'8'-Dihydroxyflavone) is a fat progenitor cell In addition to inhibiting the differentiation into adipocytes and the proliferation of adipocytes, it also promotes the expression of antioxidant enzymes, thereby reducing the accumulation of lipids and free radicals in adipocytes, thereby reducing fat differentiation factors and MAPK activity. It was confirmed that it can be utilized as a composition for inhibiting cell differentiation and proliferation, and the present invention was completed.

The present invention has been devised to solve the above problems, and the present invention provides a composition for inhibiting adipocyte differentiation and proliferation, comprising 7′8′-Dihydroxyflavone as an active ingredient. I want to.

In addition, the present invention prevents obesity or a pharmaceutical composition for preventing or treating obesity, including a composition for inhibiting fat cell differentiation and proliferation, including 7'8'-dihydroxyflavone as an active ingredient. Or to provide health functional food for improvement.

In order to solve the above-described problems of the present invention, the present invention includes a composition for inhibiting adipocyte differentiation and proliferation, comprising 7'8'-Dihydroxyflavone as an active ingredient.

According to one preferred embodiment of the present invention, the composition may include 7′8′-dihydroxyflavone at a concentration of 1 to 20 μM.

According to another preferred embodiment of the present invention, the composition may reduce the lipid accumulation or triglyceride (TG) content of adipocytes.

According to another preferred embodiment of the present invention, the composition can inhibit the expression of differentiation factors into adipocytes, and the differentiation factors into adipocytes are PPAR γ (peroxisome proliferator-activated receptors-γ), C/EBP α (CCAAT/enhancer-binding protein-α), C/EBP,β (CCAAT/enhancer-binding protein-β) and aP2 (adipocyte protein 2).

According to another preferred embodiment of the present invention, the composition can inhibit phosphorylation of extracellular signal regulated kinase (ERK) or p38 (p38 MAP kinase).

According to another preferred embodiment of the present invention, the composition can inhibit the reduction or accumulation of free radicals in adipocytes.

According to another preferred embodiment of the present invention, the composition can promote the expression of antioxidant enzymes.

According to another preferred embodiment of the present invention, the antioxidant enzyme may be one or more selected from the group consisting of catalase, manganese superoxide dismutase (Mn-SOD) and heme oxygenase-1 (HO-1).

In order to solve the second problem, the present invention includes a pharmaceutical composition for preventing or treating obesity or a health functional food for preventing or improving obesity, including the composition for inhibiting adipocyte differentiation and proliferation.

The composition comprising the 7'8'-dihydroxyflavone of the present invention as an active ingredient has no side effects caused by the conventional synthetic pharmaceutical composition, and suppresses the differentiation of adipocytes into adipocytes and proliferation of adipocytes, as well as the antioxidant enzymes. It promotes expression to reduce the accumulation of lipids and free radicals in adipocytes, thereby reducing the activity of fat differentiation factor and MAPK, which can effectively suppress adipocyte differentiation and proliferation, thus preventing or treating obesity. It can be useful as a composition or health functional food for preventing or improving obesity.

1 is a data confirming the degree of cytotoxicity for adipocytes (3T3-L1; A) and normal cells (HEK239 cells; B) of 7'8'-dihydroxyflavones.
Figure 2 is a data confirming the lipid accumulation in adipocytes by 7'8'-dihydroxyflavones.
Figure 3 is a data confirming the triglyceride content in adipocytes by 7'8'-dihydroxyflavones.
Figure 4 is a protein development of PPAR γ in adipocytes by 7'8'-dihydroxyflavones
It is the data that measured the change in cash flow.
5 is a data measuring the change in protein expression level of C/EBP α in adipocytes by 7′8′-dihydroxyflavones.
Figure 6 is a data measuring the change in protein expression of C/EBP β in adipocytes by 7'8'-dihydroxyflavones.
7 is a data measuring the change in protein expression of aP2 in adipocytes by 7'8'-dihydroxyflavones.
8 is data confirming the phosphorylation level of ERK in adipocytes by 7'8'-dihydroxyflavones.
9 is data confirming the phosphorylation level of p38 in adipocytes by 7'8'-dihydroxyflavones.
10 is data confirming the DPPH radical scavenging ability in adipocytes by 7'8'-dihydroxyflavones.
11 is data analyzing the level of free radicals accumulated in adipocytes by 7'8'-dihydroxyflavones.
12 is an image analysis data of the accumulation level of free radicals accumulated in the cytoplasm of adipocytes by 7'8'-dihydroxyflavones.
13 is data confirming the change in the expression level of catalase in adipocytes by 7'8'-dihydroxyflavones.
14 is data confirming the change in the expression level of Mn-SOD in adipocytes by 7'8'-dihydroxyflavones.
15 is data confirming the change in expression level of HO-1 in adipocytes by 7'8'-dihydroxyflavones.

Hereinafter, the present invention will be described in more detail.

As described above, drugs used as a preventive or therapeutic agent for obesity cause heart disease, respiratory disease, blood pressure rise and insomnia as side effects of drugs, and have a problem of low persistence of efficacy. Therefore, in the future, there is an urgent need to develop a high-value-added multi-functional product that has various side effects without preventing side effects, preventing and treating obesity, and reducing oxidative stress.

In the present invention, 7'8'-dihydroxyflavone (7'8'-Dihydroxyflavone)

By providing a composition for inhibiting the differentiation and proliferation of adipocytes containing as an active ingredient, the solution of the above-mentioned problems was sought. Through this, there is no side effect of the conventional pharmaceutical composition, specifically reducing only adipocytes, can inhibit the differentiation from adipocytes to adipocytes, promotes the expression of antioxidant enzymes, accumulates free radicals in adipocytes And it has the effect of providing a composition for inhibiting adipocyte differentiation and proliferation having an effect of reducing the activity of MAPK.

Therefore, the present invention includes a composition for inhibiting adipocyte differentiation and proliferation, comprising 7'8'-dihydroxyflavone as an active ingredient.

The 7'8'-dihydroxyflavones belong to flavonoids and have been reported for anti-inflammatory, vasodilatory, antihypertensive and antioxidant functions in general. The 7'8'-dihydroxyflavones are not particularly limited as long as they are usually manufactured and/or purchased.

In the present invention, in order to investigate the effect of 7'8'-dihydroxyflavones directly on the regulation of differentiation of adipocytes, in one embodiment of the present invention, cells of adipocytes of 7'8'-dihydroxyflavones Growth inhibition was measured. As a result, as shown in Fig. 1A, when treated with 7'8'-dihydroxyflavones of 1 to 20μM, when treated with 20'M concentration of 7'8'-dihydroxyflavones, about 11% of cell growth inhibition Appeared, and showed a slight cytotoxicity against mast cells.

However, as shown in Fig. 1B, as a result of treating the same concentration in HEK293 cells (Human embryonic kidney cells), which are normal cells, it was confirmed that there was little cytotoxicity to normal cells due to very little cell growth inhibition of about 4%. .

As a result, it was confirmed that 7'8'-dihydroxyflavones did not show cytotoxicity to normal cells, but adipocyte-specific toxicity, which indicates that 7'8'-dihydroxyflavones were specific to adipocytes. It can be said that it means that it can suppress proliferation.

In the present invention, the composition may include 1 to 20 μM of 7′8′-dihydroxyflavone. The solvent of the composition is not particularly limited as long as it does not interfere with the effect of inhibiting the adipocyte differentiation and proliferation of 7'8'-dihydroxyflavone, but more preferably water, ethanol, propyl alcohol, DMSO, and the like. When 7′8′-dihydroxyflavones with a concentration lower than 1 μM are included, the effect of suppressing adipocyte differentiation and proliferation may decrease, and when using 7′8′-dihydroxyflavones having a concentration of 20 μM or more, Cytotoxicity may occur due to 7'8'-dihydroxyflavones, and the efficacy of increasing concentration may not be noticeable.

In the present invention, the composition may reduce the lipid accumulation or triglyceride (TG) content of adipocytes.

In another embodiment of the present invention, to determine the effect of 7'8'-dihydroxyflavones on lipid accumulation and triglyceride content of adipocytes, adipocyte concentrations (0, 1, 10, 20 μM) were used. After treatment, lipids and triglycerides accumulated in adipocytes were observed.

As shown in FIG. 2, when the 7′8′-dihydroxyflavone was treated, it was confirmed that the amount of lipid accumulated in adipocytes was decreased in a concentration-dependent manner, and in particular, 7′8′-dihydroxy of 20μM When flavones were treated, it was confirmed that lipids in adipocytes were reduced by about 59% compared to the positive control group not treated with 7'8'-dihydroxyflavones.

In addition, as shown in FIG. 3, when the 7′8′-dihydroxyflavone was treated, it was confirmed that the amount of triglyceride accumulated in the adipocytes was concentration-dependent, and in particular, 7′8′- of 20μM When treated with dihydroxyflavones, it was confirmed that the amount of triglycerides in adipocytes decreased by about 54% compared to the positive control group without treatment with 7'8'-dihydroxyflavones.

In the present invention, the composition can suppress the expression of differentiation factors into adipocytes, and the differentiation factors into adipocytes are PPAR γ (peroxisome proliferator-activated receptors-γ), C/EBP α (CCAAT/enhancer-binding) protein-α), C/EBP,β (CCAAT/enhancer-binding protein-β) and aP2 (adipocyte protein 2).

In addition, the composition can inhibit phosphorylation of extracellular signal regulated kinase (ERK) or p38 (p38 MAP kinase).

In another embodiment of the present invention, in order to investigate the mechanism of inhibition of adipocyte differentiation of 7'8'-dihydroxyflavones, protein expression levels of PPAR γ, C/EBP α, C/EBP β, and aP2 in adipocytes Was measured.

As shown in FIGS. 4 to 7, when the 7′8′-dihydroxyflavone was treated, the amount of PPAR γ, C/EBP α, C/EBP β, and aP2 expressed in adipocytes in a concentration-dependent manner was decreased. In particular, in the case of treatment with 7'8'-dihydroxyflavone of 20μM, PPAR γ, C/EBP α, C/EBP when compared with the positive control group not treated with 7'8'-dihydroxyflavone It was confirmed that β and aP2 expression levels decreased by 33%, 61%, 63%, and 50, respectively.

Also in another embodiment of the present invention, 7′8′-dihydroxyflavones

As a result of measuring the degree of phosphorylation change of ERK or p38 in one adipocyte, as shown in FIGS. 8 and 9, when treated with 7'8'-dihydroxyflavone, concentration of ERK and p38 in adipocytes was concentration-dependent. It was confirmed that the degree of phosphorylation decreases, especially when treated with 20'M of 7'8'-dihydroxyflavones, phosphorylation of ERK and p38 when compared to the positive control without treatment with 7'8'-dihydroxyflavones Was confirmed to decrease by 67% and 63%, respectively.

That is, the expression level of PPAR γ, C/EBP α, C/EBP β and aP2 in adipocytes is reduced by 7′8′-dihydroxyflavone of the present invention, and MAPK (mitogen-activated protein kinases such as ERK and p38) ) Activity was inhibited and it was confirmed that differentiation into adipocytes was inhibited.

In the present invention, the composition can inhibit the reduction or accumulation of free radicals in adipocytes.

In another embodiment of the present invention, in order to confirm the antioxidant activity and free radical scavenging activity of 7'8'-dihydroxyflavones, adipocytes are treated with 7'8'-dihydroxyflavones by concentration, and then DPPH free The radical scavenging capacity, the level of free radicals (ROS) in the cell and the amount of free radicals in the cell matrix were measured.

As shown in FIG. 10, it was confirmed that the free radical scavenging ability increased depending on the concentration of 7′8′-dihydroxyflavone, similar to ascorbic acid used as a positive control, and 300μM was 7′8′-dihydrate. It was confirmed that about 66.2% of DPPH free radicals were eliminated from oxyflavones.

As shown in FIG. 11, 7′8′-dihydroxyflavone was treated.

When it was confirmed that the amount of free radicals was decreased depending on the concentration, especially in the case of 20 μM of 7′8′-dihydroxyflavone, 7′8′-dihydroxyflavone was not treated with the positive control group. When compared, it was confirmed that the level of free radicals in adipocytes decreased by about 37%.

In addition, as shown in FIG. 12, in the group not treated with 7′8′-dihydroxyflavone, it was confirmed that active oxygen was present at the cytoplasmic site stained with rhodamine, but 7′8′-dihydroxyflavone In the treated group, it was confirmed that the amount of free radicals accumulated in the cytoplasmic location was significantly reduced.

In the present invention, the composition may promote the expression of antioxidant enzymes, and the antioxidant enzymes are catalase, Mn-SOD (manganese superoxide dismutase) and HO-1 (heme oxygenase-1) or more. Can be.

In another embodiment of the present invention, in order to measure the change in antioxidant enzyme activity by 7'8'-dihydroxyflavones in adipocytes, the amount of catalase, Mn-SOD and HO-1 present in adipocytes Was measured.

13 to 15, when the 7′8′-dihydroxyflavone was treated, it was confirmed that the expression amount of catalase, Mn-SOD and HO-1 increased in a concentration-dependent manner, in particular, 7 of 20 μM. When ′8′-dihydroxyflavone was treated, the expression of catalase, Mn-SOD and HO-1 in adipocytes was 59%, respectively, compared to the positive control group not treated with 7′8′-dihydroxyflavone. , 35% and 30% increase.

Due to this, 7'8'-dihydroxyflavones eliminate free radicals, thereby reducing the amount of free radicals accumulated in adipocytes, and increasing the amount of antioxidant enzymes acting on adipocytes to eliminate free radicals, thereby It was confirmed that it suppressed the development of obesity promoted by oxygen.

Therefore, the composition for inhibiting adipocyte differentiation and proliferation containing 7'8'-dihydroxyflavone as an active ingredient of the present invention has no side effects that have been shown in conventional pharmaceutical compositions, and promotes the expression of antioxidant enzymes to promote adipocytes. It reduces the accumulation of lipids and free radicals in this, thereby reducing the differentiation factor and MAPK activity, and thus can inhibit the differentiation and proliferation of adipocytes, and thus contains 7'8'-dihydroxyflavone as an active ingredient. It may be usefully used as a pharmaceutical composition for preventing or treating obesity using a composition for inhibiting adipocyte differentiation and proliferation, or as a health functional food for preventing or improving obesity.

In addition, since the 7'8'-dihydroxyflavone of the present invention can effectively suppress the differentiation of adipocytes into adipocytes, it can be used as a medium composition for suppressing the differentiation of adipocytes.

The present invention provides a pharmaceutical composition for preventing or treating obesity, including a composition for inhibiting adipocyte differentiation and proliferation, comprising the 7'8'-dihydroxyflavone as an active ingredient.

The pharmaceutical composition for preventing or treating obesity comprising the composition for inhibiting adipocyte differentiation and proliferation according to the present invention may further include other natural substances or compounds having anti-obesity efficacy, and the pharmaceutical composition of the present invention is a rat , It can be administered to mammals such as mice, livestock, and humans through various routes including oral, transdermal, subcutaneous, intravenous or muscle. In addition, the pharmaceutical composition for preventing or treating obesity comprising the composition for inhibiting adipocyte differentiation and proliferation according to the present invention can be formulated into various dosage forms. In the case of formulation, it may be formulated using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, and capsules, and these solid preparations contain at least one excipient (eg, starch, sucrose) in 7'8'-dihydroxyflavones. , Lactose and gelatin). Lubricating agents may also be used in addition to simple excipients. Liquid preparations for oral administration include suspending agents, intravenous solutions, emulsions, and syrups. In addition to water and liquid paraffin, which are commonly used diluents, various excipients, such as wetting agents, sweeteners, sweeteners, fragrances and preservatives, etc. Can be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween61, cacao butter, laurin butter, and glycerogelatin may be used.

The pharmaceutical composition for preventing or treating obesity comprising the composition for inhibiting adipocyte differentiation and proliferation according to the present invention may vary depending on the patient's age, sex, and weight, but is generally in an amount of 0.01 to 50 mg/kg, preferably Can be administered by dividing the amount of 0.1 to 10 mg/kg once to several times a day. In addition, the dosage of the composition containing 7'8'-dihydroxyflavones may be increased or decreased depending on the route of administration, the degree of disease, sex, weight, and age. Therefore, the dosage does not limit the scope of the present invention in any way.

In addition, the present invention provides a health functional food composition for preventing or improving obesity comprising a composition for inhibiting adipocyte differentiation and proliferation, comprising the 7'8'-dihydroxyflavone as an active ingredient. The type of health functional food containing the health functional food composition for preventing or improving obesity, which includes the composition for inhibiting fat cell differentiation and proliferation of the present invention, is not particularly limited, for example, meat, sausage, bread, chocolate, candy, It may be snacks, confectionery, pizza, ramen, other noodles, gum products, dairy products including ice cream, various soups, beverages, teas, drinks, alcoholic beverages, and vitamin complexes.

The health food is used in combination with other foods or food additives other than the 7'8'-dihydroxyflavone, and can be suitably used according to a conventional method. For example, the beverage for preventing obesity containing the 7'8'-dihydroxyflavone as an active ingredient, in addition to the 7'8'-dihydroxyflavone being included as an active ingredient, calcium, thorny peel concentrate, liquid fructose, Purified water and the like can be added and mixed, filled into a bottle for drinks, sterilized, and then cooled to room temperature to produce a beverage. In addition, the 7'8'-dihydroxyflavones as an active ingredient for preventing obesity is a health supplement for 7'8'-dihydroxyflavones (vitamins B1, B2, B5, B6, E and acetic acid esters) , Nicotinic acid amide), oligosaccharides, 50% ethanol, purified water are added and mixed to form granules, dried in a vacuum dryer, and uniformly granulated by passing through 12 to 14 mesh to extrude and purified by an appropriate amount. Alternatively, it can be prepared as a hard capsule product by powdering or filling a hard capsule.

The effective dose of the 7'8'-dihydroxyflavone contained in the healthy food can be used in accordance with the effective dose of the pharmaceutical composition, and the amount of the active ingredient is suitable for the purpose of use such as prevention or therapeutic treatment. Can be decided. It may be below the above range for long-term intake for health and hygiene purposes or for health control purposes.

Hereinafter, the present invention will be described in detail with reference to preferred embodiments so that those skilled in the art to which the present invention pertains can easily practice. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein.

Example 1. 7′8′- Dihydroxyflavone Toxicity measurement for adipocytes

MTT assay (methylthiazolyldiphenyl tetrazolium bromide assay) was performed to measure the cytotoxicity of 7'8'-dihydroxyflavones to 3T3-L1 (Mouse embryonic fibroblast-adipose like cell line) adipocytes. In addition, it was also specified whether 7'8'-dihydroxyflavones are toxic to normal cells other than adipocytes.

For general cells, HEK293 cells (Human embryonic kidney cells; CRL-1573, ATCC, USA) were used, and HEK293 cells and 3T3-L1 adipocytes (CL-173, ATCC, USA) were 96-well plates. ) To 4×10 ⁴ cells/ml and incubated in 5% CO ₂ , 37℃ incubator for 24 hours. After incubation, the culture solution was removed and DMEM medium (Thermo Fisher Scientific, USA) and 7′8′-dihydroxyflavone (D5446, Sigma-Aldrich, USA) for each concentration (0, 1, 10, 20 μM) for 24 hours Treatment. Subsequently, a solution of MTT (3-[4,5-dimethyl-thiazol]-2,5-diphenyl-tetrazolium bromide) dissolved in 5 mg/ml in solvent DPBS (Dulbecco's Phosphate-Buffered Saline, 1X) was added to each well. 120 µl was treated and incubated in a 5% CO2, 37°C incubator for 4 hours. After the culture, the culture solution was removed, and then dimethyl sulfoxide (DMSO) was added to each well at 200 µl/well and mixed well using a pipette. Thereafter, the absorbance was measured at 570 nm using an ELISA reader (Molecular Devices, USA).

As a result, as shown in Figure 1, when treated with 7'8'-dihydroxyflavones 1 to 20μM, when treated with 7'8'-dihydroxyflavones at a concentration of 20μM about 11% cytotoxicity Could confirm.

Example 2. 7′8′- Dihydroxyflavones Measurement of lipid accumulation inhibitory activity of adipocytes

The effect of 7'8'-dihydroxyflavones on lipid accumulation in adipocytes

To confirm, in the same manner as in Example 1, 7′8′-dihydroxyflavone was treated by concentration (1, 10, 20 μM) while inducing differentiation (0.5 μM IBMX (3-Isobutyl-1-methylxanthine). , 1 μM Dexamenthason, 10 μg/mL Insulin-containing differentiation medium was used to remove the culture medium of the cells) and washed with phosphate buffered saline (PBS). The adipocytes that did not induce differentiation into adipocytes were used as a negative control, and adipocyte differentiation was induced, but adipocytes not treated with 7'8'-dihydroxyflavone were used as a positive control.

500 μl of 10% formaldehyde was added to each well in each group, fixed at 4° C. for 1 hour, then the formaldehyde was removed, washed three times with phosphate buffered saline and dried in a CO 2 incubator. Thereafter, when the plate was dried, 500 μl of Oil red O dye was added and stained for 30 minutes in a dark state at room temperature, and then washed three times with phosphate buffered saline. The stained cells were observed under a microscope, and after observation, a dye stained in adipocytes was extracted with 300 μl of iso-propanol per well, and an optical density (OD) value was measured at 500 nm with an ELISA reader. The Oil Red O dye is a solution in which 500 mg of Oil Red O dye is dissolved in 100 ml of iso-propanol at a ratio of 6:4 and filtered with a 0.45 μm filter.

As a result, when the 7′8′-dihydroxyflavone was treated, it was confirmed that the amount of lipid accumulated in the adipocytes in a concentration-dependent manner was reduced. In particular, the 20′M 7′8′-dihydroxyflavone was treated. In the case, it was confirmed that the lipid in adipocytes decreased by about 59% compared to the positive control group not treated with 7'8'-dihydroxyflavone.

Example 3. 7′8′- Dihydroxyflavones Analysis of triglyceride content in fat cells

Dispense 3T3-L1 adipocytes into 6-well plates at a density of 7 × 10 ⁴ cells/ml and differentiate adipocytes for 8 days while simultaneously adding 7′8′-dihydroxyflavones by concentration. (0, 1, 10, 20 μM). After 8 days, each well was washed with phosphate-buffered saline, and then cells were obtained using a cell scraper (SPL), and then triglyceride in the cells was extracted using an ultrasonic grinder (SONIFIER 450, BRANSON). Then, the triglyceride kit (TG-S, Asan Pharmaceutical) was used to measure the content of triglyceride extracted above.

As a result, as shown in FIG. 3, when the 7′8′-dihydroxyflavone was treated, it was confirmed that the amount of triglyceride accumulated in the adipocytes in a concentration-dependent manner decreased, and in particular, 7′8′ of 20μM -When treated with dihydroxyflavones, it was confirmed that the amount of triglycerides in adipocytes decreased by about 54% compared to the positive control without 7'8'-dihydroxyflavones.

Example 4. 7′8′- Dihydroxyflavones Investigation of the mechanism by which adipocyte differentiation is inhibited

To investigate the mechanism of inhibition of adipocyte differentiation of 7′8′-dihydroxyflavones, peroxisome proliferator-activated receptors-γ (PPAR γ), C/EBP α (CCAAT/enhancer-binding protein-α) in adipocytes, Protein expression levels of C/EBP,β (CCAAT/enhancer-binding protein-β) and aP2 (adipocyte protein 2) were measured by Western blot method. In addition, the change in phosphorylation of ERK or p38 in adipocytes by 7'8'-dihydroxyflavones was also measured.

7'8'-dihydroxyflavones were treated by concentration (0, 1, 10, 20 μM) while differentiating 3T3-L1 cells into 6 well plates for 8 days. After 8 days, the cells were obtained by washing with phosphate buffered saline and centrifuging at 1,400 rpm for 5 minutes. Protein was obtained by adding 50 μl of lysis buffer to the precipitated cells, reacting on ice for 60 minutes, and centrifuging at 14,000 rpm for 30 minutes. After quantifying the protein using the Bradford method, it was denatured at 95°C for 5 minutes, and then electrophoresed using a SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) gel.

After electrophoresis and transfer to a nitrocellulose membrane, primary PPAR γ antibody (1:500; #2430, Cell Signaling Technology, USA), anti-C/EBP α antibody (1:500; # 2295, Cell Signaling Technology, USA), anti-C/EBP β antibody (1:500; #3087, Cell Signaling Technology, USA), anti-aP2 antibody (1:1000; #2120, Cell Signaling Technology, USA), Anti-phospho ERK antibody (1:500; #4377, Cell Signaling Technology, Danvers, USA), anti-phospho p38 antibody (1:500; #9126, Cell Signaling Technology, USA), anti-beta actin antibody (1: 1000; #4967, Cell Signaling Technology, USA) was diluted in 5% skim milk at each ratio, treated on a nitrocellulose membrane, and reacted at 4° C. for 24 hours. Then, washed three times every 10 minutes with TBST (TBS, 0.1% Tween 20) and HRP (horseradish peroxidase)-conjugated secondary antibody (1:2000; #7076, Cell Signaling Technology, USA) was added to 5% skim milk powder. After diluting 1:1000, it was reacted at room temperature for 2 hours. After washing three times using TBST in the same manner as above, the ECL (Enchanced Chemiluminescence) substrate solution was reacted on the membrane for 1 minute and then developed on an X-ray film. The obtained results were quantified using an image J program.

As a result, as shown in FIGS. 4 to 7, when treated with 7′8′-dihydroxyflavones, the amount of PPAR γ, C/EBPα, C/EBP β and aP2 expressed in adipocytes in a concentration-dependent manner was reduced. PPAR γ, C/EBP α, C compared to the positive control group not treated with 7′8′-dihydroxyflavone, especially when treated with 20'M of 7'8'-dihydroxyflavone. It was confirmed that /EBP β and aP2 expression levels decreased by 33%, 61%, 63%, and 50, respectively.

In addition, as shown in FIGS. 8 and 9, it was confirmed that when the 7′8′-dihydroxyflavone was treated, the concentration of ERK and p38 phosphorylation in adipocytes was decreased in a concentration-dependent manner, in particular, 7′ of 20μM. When 8′-dihydroxyflavones were treated, it was confirmed that phosphorylation of ERK and p38 decreased by 67% and 63%, respectively, when compared to the positive control group not treated with 7′8′-dihydroxyflavone.

That is, the expression level of PPAR γ, C/EBP α, C/EBP β and aP2 in adipocytes is reduced by 7′8′-dihydroxyflavone of the present invention, and MAPK (mitogen-activated protein kinases such as ERK and p38) ) Activity was inhibited and it was confirmed that differentiation into adipocytes was inhibited.

Example 5. 7′8′- Dihydroxyflavones Antioxidant effect in adipocytes

Reactive Oxygen Species (ROS) accumulate in mast cells during the development process of obesity, and accumulated ROS causes adipocytokine dysregulation in mast cells and decreases the expression of antioxidant enzymes in adipose tissue. While promoting the obesity gradually. Therefore, substances that have antioxidant effects are candidates for suppressing the development of obesity.

5-1. Confirmation of DPPH free radical scavenging ability

DPPH radical scavenging activity was measured to measure the antioxidant effect of 7'8'-dihydroxyflavones. Add 0.2 ml of 7′8′-dihydroxyflavone solution prepared at different concentrations (0 to 300 μM) and 1.8 ml of 0.1 mM DPPH (1,1-diphenyl-2-picrylhydrazyl) solution, mix well, and mix the light. After reacting for 10 minutes in the blocked state, absorbance was measured at 518 nm.

DPPH radical scavenging capacity was expressed as% by [1-(absorbance of sample/absorbance of control group]×100] by comparing the absorbance of the sample addition group with respect to the control group. As a positive control group, ascorbic acid (Ascrobic acid) was used.

As a result, as shown in FIG. 10, it was confirmed that the free radical scavenging ability increased depending on the concentration of 7'8'-dihydroxyflavone, similar to ascorbic acid used as a positive control, and 300μM was 7'8'. -It was confirmed that about 66.2% of DPPH free radicals were eliminated in dihydroxyflavones.

5-2. Measurement of ROS (Reactive Oxygen species) level in adipocytes

DCF-DA (cell permeable acetylated forms of 2'7'-dichlorofluorescein) was used to confirm that free radicals (ROS) accumulated in adipocytes were eliminated by 7'8'-dihydroxyflavones during adipocyte differentiation. It was confirmed by using.

7'8''-dihydroxyflavones were treated by concentration (0, 1, 10, 20 μM) while differentiating 3T3-L1 cells into 6 well plates for 8 days. After 8 days, the cells were obtained by washing with phosphate buffered saline and centrifuging at 1,400 rpm for 5 minutes. The precipitated cells were treated with DCF-DA of 10 and cultured at 37 for 45 min. After dispensing 100 μl into 96-well plates blocked with light, a fluorescence photometer was used to measure the fluorescence at excitation 492 nm and emission 530 nm. Did.

As shown in FIG. 11, when the 7′8′-dihydroxyflavone was treated, it was confirmed that the amount of free radicals was decreased depending on the concentration, and in particular, the 20′M 7′8′-dihydroxyflavone was treated. In the case, it was confirmed that the level of free radicals in adipocytes decreased by about 37% compared to the positive control group not treated with 7'8'-dihydroxyflavone.

5-3. Measurement of changes in free radical accumulation in adipocyte matrix

Cell permeable acetylated forms of 2'7'-dichlorofluorescein (DCF-DA) and rhodamine to confirm that ROS accumulated in the adipocyte's matrix during adipocyte differentiation is reduced by 7'8'-dihydroxyflavones Fluorescence microscopy images were analyzed using (Rhodamine).

20, 30 μM of 7′8′-dihydroxyflavones were treated simultaneously while 3T3-L1 cells were differentiated into 8 well plates for 8 days. After 8 days, after washing with phosphate buffered saline, the cells were fixed with 1% paraformaldehyde, treated with rhodamine, and then cultured at 37°C for 3 hours, followed by 10 μM of DCF-DA And incubated at 37°C for 45 minutes. After the stained cells were covered and covered with cover glass, images were analyzed using a fluorescence microscope (Magnification, ×100).

As a result, as shown in FIG. 12, in the group not treated with 7′8′-dihydroxyflavone, it was confirmed that active oxygen was present in the cytoplasmic site stained with rhodamine, but 7′8′-dihydrate In the group treated with oxyflavone, it was confirmed that the amount of free radicals accumulated at the cytoplasmic position was significantly reduced.

Example 6. 7′8′- Dihydroxyflavones Antioxidant Enzyme Activity in Fat Cells

In order to measure the change in antioxidant enzyme activity by 7'8'-dihydroxyflavones in adipocytes, the expression levels of catalase, Mn-SOD and HO-1 present in adipocytes were measured by Western blot method.

Primary antibodies for measuring the expression levels of catalase, Mn-SOD and HO-1 include anti-catalase antibodies (1:1000, #12980, Cell Signaling Technology, USA), anti-Mn-SOD antibodies (1:1000, #06-984, Merck KGaA, Germany), anti-HO-1 antibody (1:1000, #D60G11, Cell Signaling Technology, USA) was used, and the Western blot method was performed in the same manner as in Example 4.

As a result, as shown in FIGS. 13 to 15, when the 7′8′-dihydroxyflavone was treated, it was confirmed that the expression levels of catalase, Mn-SOD, and HO-1 increased in a concentration-dependent manner. When treated with 20μM of 7'8'-dihydroxyflavone, the expression level of catalase, Mn-SOD and HO-1 in adipocytes compared to the positive control without 7'8'-dihydroxyflavone It was confirmed that the increase was 59%, 35%, and 30%, respectively.

For this reason, 7'8'-dihydroxyflavones eliminate free radicals, thereby reducing the amount of free radicals accumulated in adipocytes, and increasing the amount of antioxidant enzymes acting on adipocytes to eliminate free radicals, thereby It was confirmed that it suppressed the development of obesity promoted by oxygen.

Therefore, the composition for inhibiting the differentiation and proliferation of adipocytes containing 7'8'-dihydroxyflavone as an active ingredient of the present invention reduces the accumulation of lipids and triglycerides in adipocytes and reduces the number of adipocytes. It is possible to suppress the differentiation mechanism into adipocytes, thereby suppressing the differentiation of adipocytes. In addition, by accelerating the expression of antioxidant enzymes can reduce the accumulation of free radicals in the adipocytes and MAPK activity, using a composition for inhibiting adipocyte differentiation and proliferation containing 7'8'-dihydroxyflavone as an active ingredient It may be useful as a pharmaceutical composition for preventing or treating obesity or as a health functional food for preventing or improving obesity.

As described above, since a specific part of the present invention has been described in detail, for those skilled in the art, this specific technique is only a preferred embodiment, and the scope of the present invention is not limited thereby. It will be obvious. Therefore, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (11)

7'8'-dihydroxyflavone (7'8'-Dihydroxyflavone) as an active ingredient, and 10 to 20 μM of 7'8'-dihydroxyflavone (7'8'-Dihydroxyflavone), ERK (extracellular signal regulated kinase) or p38 (p38 MAP kinase) characterized in that inhibiting the phosphorylation, in vitro (in vitro) adipocyte differentiation and proliferation inhibition composition.
delete According to claim 1, wherein the composition is characterized in that to reduce the lipid accumulation or triglyceride (TG) content of adipocytes, a composition for inhibiting adipocyte differentiation and proliferation in vitro.
delete According to claim 1, wherein the differentiation factors into adipocytes are PPAR γ (peroxisome proliferator-activated receptors-γ), C/EBP α (CCAAT/enhancer-binding protein-α), C/EBP, β (CCAAT/enhancer- A composition for inhibiting adipocyte differentiation and proliferation in vitro, characterized in that at least one selected from the group consisting of binding protein-β) and aP2 (adipocyte protein 2).
delete According to claim 1, The composition is characterized in that to inhibit the reduction or accumulation of free radicals in adipocytes, a composition for inhibiting adipocyte differentiation and proliferation in vitro.
According to claim 1, The composition is characterized in that to promote the expression of antioxidant enzymes, in vitro (in vitro) adipocyte differentiation and proliferation inhibition composition.
The method of claim 8, wherein the antioxidant enzyme is catalase, Mn-SOD (manganese superoxide dismutase) and HO-1 (heme oxygenase-1), characterized in that at least one selected from the group consisting of, in vitro (in vitro) A composition for inhibiting adipocyte differentiation and proliferation.
delete delete

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