KR20150061115A - Pharmaceutical composition comprising maysin for the prevention and treatment of obesity - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing and treating obesity including maysin and, more specifically, a pharmaceutical composition for preventing and treating obesity including maysin with good anti-obesity activity, which prevents the reduction of accumulating fat cells.

Description

[0001] The present invention relates to a pharmaceutical composition for preventing and treating obesity,

The present invention relates to a pharmaceutical composition for the prevention and treatment of obesity, which comprises meiosis, and more particularly to a pharmaceutical composition for prevention and treatment of obesity, which comprises meiosis, which has an excellent anti-obesity activity by reducing lipid accumulation of adipocytes, And to a pharmaceutical composition for preventing and treating such diseases.

Hyundai has been increasing in obesity due to lack of westernization and activity of dietary habits. Obesity is a major cause of hypertension, diabetes, and cardiovascular system obesity, a hormone deficiency in the brain called leptin, which is caused by overgrowth of adipocytes. These deficiencies of leptin hormone, except for genetic causes, are exacerbated by hyperglycemia, weakening of metabolism in vivo due to aging, and excessive accumulation of animal cholesterol in the body. As a cause of diseases and various cancers, There is much interest in the treatment of obesity worldwide.

Obesity is mostly consumed in the calories consumed and the remaining part is converted into adipocyte and accumulated in the subcutaneous tissue and abdominal cavity. Since there is a limit to the size of the adipocytes, it is necessary to increase the number of adipocytes in order to store the energy quickly when consuming the excess energy, and thus, a new adipocyte is produced in the human body. The new adipocytes differentiate from pro-adipocytes, which are formed by differentiation from stem cells. The process of differentiation of stem cells into adipocytes through adipocytes is called hormone, growth Factors such as urea, cytokine, and the like regulators are affected.

The main cause of obesity is a hormone deficiency in the brain called leptin, which is caused by overgrowth of adipocytes. This deficiency of leptin hormone, except for genetic causes, such as overeating, metabolism in vivo due to aging due to the accumulation of excess cholesterol in the body is reduced by the accumulation of animal cholesterol.

Saponins, essential amino acids, vitamin E, and vitamin C are the components that have lipid lowering effect. Recently, as the interest in the therapeutic effect of medicinal herbs has increased, the demand for natural products is increasing. Recently, research on anti-obesity agents using medicinal plants has been actively conducted. Among them, a method for producing a fat-soluble food product comprising a fresh neck, a corn mustache, a lycopene, a lycopodium, a squid, an amber, a ginger, a sesin, an arthaxis and a squid extract is disclosed in a registered patent 10-0903970 (public date: Mar. 18, 2009) .

Commercial anti-obesity agents include thiazolidinediones (TZDs), Xenical (Korean Roche) and sibutramine. The thiazolidinediones (TZDs) inhibit the production of sugars, and Xenical (Korean rosacea) and sibutramine inhibit lipid absorption and suppress the reabsorption of serotonin in synapses, .

However, these substances were originally used as antidepressants and caused serious side effects. In addition to these, anti-obesity agent called adipex is an appetite suppressant that contains pentamamine with sympathetic stimulant activity, and has a side effect by controlling the neurotransmitters associated with the hypothalamus.

Side effects of these drugs have been reported to include cardiovascular, central nervous, hepatic and renal disorders. Therefore, there is an urgent need to develop high value-added, multi-functional products that prevent side effects from long-term ingestion and prevent and treat obesity. Various herbal medicine extracts have been disclosed which minimize various side effects such as cardiovascular, central nervous, hepatic and renal disorders.

However, these herbal medicine extracts have a limited effect on reducing fat cells and inhibiting the production of triglycerides and thus being used as a composition for preventing and treating obesity and lipid-related metabolic diseases.

Disclosure of the Invention The present invention has been conceived to solve the above-mentioned problems, and it is an object of the present invention to provide a composition for prevention and treatment of obesity, which comprises Meicin without any side effects such as cardiovascular action, central action, hepatic disorder and kidney disorder induced by the conventional synthetic pharmaceutical composition And a method for manufacturing the same, and a health functional food using the same.

The present invention provides a pharmaceutical composition for preventing or treating obesity comprising mexin as an active ingredient.

According to a preferred embodiment of the present invention, the pharmaceutical composition may reduce lipid accumulation of adipocytes.

According to another preferred embodiment of the present invention, the pharmaceutical composition can inhibit the growth of adipocytes.

According to another preferred embodiment of the present invention, the pharmaceutical composition may reduce the triglyceride content in adipocytes.

According to another preferred embodiment of the present invention, the pharmaceutical composition may be contained at a concentration of 25 to 100 μg / ml of mexin.

According to another preferred embodiment of the present invention, the pharmaceutical composition comprises PPAR-γ (peroxisome proliferator-activated receptors-γ), C / EBP-α (CCAAT / enhancer- 2). ≪ / RTI >

According to another preferred embodiment of the present invention, the mechine may be derived from corn beard.

In addition, the present invention provides a health functional food for preventing or improving obesity including meisin.

According to a preferred embodiment of the present invention, the mechine may be derived from corn beard.

According to another preferred embodiment of the present invention, the health functional food may reduce lipid accumulation of adipocytes.

According to another preferred embodiment of the present invention, the health functional food may reduce the content of triglyceride in fat cells.

According to another preferred embodiment of the present invention, the health functional food may inhibit the growth of adipocytes.

According to another preferred embodiment of the present invention, the health functional food may be contained at a concentration of 25 to 100 μg / ml of mexin.

According to another preferred embodiment of the present invention, the health functional food comprises at least one selected from the group consisting of peroxisome proliferator-activated receptors (PPAR-gamma), C / EBP-alpha (CCAAT / enhancer- 2). ≪ / RTI >

According to another preferred embodiment of the present invention, when the pharmaceutical composition and / or the health functional food contains mei cine at a concentration of 25-100 μg / ml, the rate of inhibition of adipocyte growth is 15-25% .

According to another preferred embodiment of the present invention, when the pharmaceutical composition and / or the health functional food contains Meicin at a concentration of 50-100 μg / ml, the lipid accumulation reduction rate of the adipocyte is 20-50% Lt; / RTI >

According to another preferred embodiment of the present invention, when the pharmaceutical composition and / or the health functional food contains mexin at a concentration of 50 to 100 占 퐂 / ml, the decrease rate of triglyceride content in fat cells is 25 to 70 %. ≪ / RTI >

According to another preferred embodiment of the present invention, when the pharmaceutical composition and / or the health functional food contains Meicin at a concentration of 25-100 μg / ml, the decrease rate of PPAR-γ expression in adipocytes is 25 To 45%.

According to another preferred embodiment of the present invention, when the pharmaceutical composition contains mexin at a concentration of 75 to 100 μg / ml, the rate of decrease of aP2 expression in adipocytes may be 20 to 40%.

According to another preferred embodiment of the present invention, when the pharmaceutical composition and / or the health functional food is contained at a concentration of 50 to 75 μg / ml of mexin, the decrease rate of C / EBP- Can be 40 to 90%.

The present invention provides a method for inhibiting the growth of adipocytes, reducing lipid accumulation of adipocytes, decreasing triglyceride content, and reducing anti-obesity activity by preventing adverse effects such as cardiovascular action, central action, hepatic disorder and kidney disorder caused by conventional synthetic drug compositions A pharmaceutical composition for prevention and / or treatment of obesity including high mexin, or a health functional food for prevention and / or improvement of obesity.

Fig. 1 shows the measurement results of the inhibitory activity of macrocin against adipocyte growth, as confirmed in Example 1. Fig.
Fig. 2 shows the results of lipid-accumulating inhibitory activity of macecin in adipocytes as confirmed in Example 2. Fig.
FIG. 3 is a graph showing the results of analysis of triglyceride content in adipocytes according to the macine treatment identified in Example 3. FIG.
4 is the change in PPAR-gamma concentration in adipocytes as observed in Example 4. Fig.
5 is a change in aP2 concentration in adipocytes as observed in Example 4. Fig.
Fig. 6 shows the results of measurement of PPAR-gamma expression level in adipocytes as confirmed in Example 5. Fig.
FIG. 7 shows the results of measurement of C / EBP-alpha expression level in adipocytes as confirmed in Example 5. FIG.
8 shows the results of measurement of aP2 expression level in adipocytes as confirmed in Example 5. Fig.

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

As described above, the conventional synthetic medicines have various side effects such as cardiovascular action, central action, hepatic disorder and kidney disorder, and the herbal medicine extract prepared using the conventional hot water extraction method, And it is not effective to reduce lipid accumulation of fat cells and the content of triglyceride in adipocytes. Therefore, a new alternative is needed.

Accordingly, the present invention provides a pharmaceutical composition for prevention and treatment of obesity including meiosis, and a health functional food for prevention and improvement of obesity, thereby solving the above-mentioned problems. Thus, without adverse effects such as cardiovascular action, central action, hepatic disorder and kidney disorder, which were the side effects of the conventional pharmaceutical composition, it was found that the lipid accumulation in the adipocyte, And a health functional food for obesity prevention and improvement. The present invention also provides a health functional food for obesity prevention and improvement.

The present invention provides a pharmaceutical composition for the prevention and treatment of obesity comprising mexin.

The maysin is a yellow plant-flavonoid flavonoid substance found in corn, teosinte, and centipedegrass, and has antioxidant and anticancer activities. However, in the present invention, meishin derived from corn beard has confirmed the preventive and therapeutic effect on obesity.

The meicin of the present invention is not particularly limited as long as it can be usually prepared and / or purchased, but it may preferably be one derived from corn hairs.

And found a novel physiological activity of mexin derived from the corn beard as an obesity prevention and / or therapeutic activity.

Specifically, as shown in Example 1, the meicin inhibited the growth of adipocytes and confirmed the activity of preventing and treating obesity.

Moreover, as shown in Example 2, the meicin showed lipid accumulation of adipocytes and activity of preventing and treating obesity.

Also, as shown in Example 3, the Mechin reduced the content of triglyceride in adipocytes and confirmed the activity of preventing and treating obesity.

Further, the pharmaceutical composition for prevention and treatment of obesity according to the present invention can be used as a prophylactic or therapeutic agent for obesity, such as PPAR-γ (peroxisome proliferator-activated receptors-γ), C / EBP-α (CCAAT / enhancer- Lt; RTI ID = 0.0 > mRNA < / RTI >

Specifically, as shown in Examples 4 to 5 and Figs. 5 to 9, it was confirmed that the maccin of the present invention exhibited anti-obesity activity by decreasing the expression of PPAR-y, C / EBP-a and aP2.

In addition, the pharmaceutical composition for prevention and treatment of obesity is not particularly limited as long as it contains meicin, but it may preferably be contained at a concentration of 25 to 100 占 퐂 / ml, more preferably 50 to 100 占 퐂 / Ml. ≪ / RTI >

If it contains less than 25 μg / ml of meicin, it may not exhibit sufficient anti-obesity activity, and if it contains an amount of meicin in excess of 100 μg / ml, Problems can arise.

More specifically, the pharmaceutical composition for the prevention and treatment of obesity of the present invention is characterized in that, when the myc synthase is contained at a concentration of 25 to 100 μg / ml, To 15% to 25%, and when the macronutrient is contained at a concentration of 25 to 75 μg / ml, the fat cell growth can be suppressed to 15 to 23%.

Furthermore, the pharmaceutical composition for the prevention and treatment of obesity of the present invention is characterized in that lipid accumulation of adipocytes is 20 to 50% higher than that of adipocyte-free adipocyte, when it contains mexin at a concentration of 50 to 100 占 퐂 / , And preferably the lipid accumulation of adipocytes can be reduced to 22-45% when the macrocin is included at a concentration of 50-100 [mu] g / ml.

In addition, the pharmaceutical composition for the prevention and treatment of obesity of the present invention is characterized in that when the mycin is contained at a concentration of 50 to 100 占 퐂 / ml, the content of triglyceride in the adipocyte is 25 to 70 %, And preferably, when the mycelium is contained at a concentration of 50 to 100 占 퐂 / ml, the content of triglyceride in the adipocyte can be reduced by 40 to 65%, more preferably, Ug / ml, the content of triglyceride in fat cells can be reduced to 60 to 65%.

In addition, the pharmaceutical composition for the prevention and treatment of obesity of the present invention can inhibit the expression of PPAR-gamma in adipocytes by 25-50% compared with adipocytes not containing meicin, when the macrocin is contained at a concentration of 50-100 占 퐂 / 45%, and when the macronutrient is contained at a concentration of 50-100 占 퐂 / ml, the amount of PPAR-gamma expression in adipocytes can be reduced to 25-40%.

In addition, the pharmaceutical composition for the prevention and treatment of obesity according to the present invention can inhibit the expression of aP2 in adipocytes by 20 to 40% when compared with adipocyte-free adipocytes, when the macrocin is contained at a concentration of 75 to 100 占 퐂 / , And preferably the amount of aP2 expression in adipocytes can be reduced to 20 to 35% when the macrocin is contained at a concentration of 75 to 100 占 퐂 / ml.

Further, when the pharmaceutical composition for preventing and treating obesity of the present invention contains mexin at a concentration of 25 to 75 占 퐂 / ml, the expression level of C / EBP-α in adipocytes The amount of C / EBP-α expression in the adipocyte can be reduced to 40 to 90% when the myc synthase is contained at a concentration of 50 to 75 μg / ml, More preferably 40 to 85%.

The pharmaceutical compositions for preventing and treating obesity according to the present invention may be formulated into oral compositions such as powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols, external preparations, May be formulated in the form of sterile injectable solutions.

More specifically, when formulating the composition, it can be prepared using a diluent or an excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, and the like.

Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose, sucrose, lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used.

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

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

The pharmaceutical composition for preventing and treating obesity according to the present invention may vary depending on the age, sex and body weight of a patient, but is generally in an amount of 0.01 to 50 mg / kg, preferably 0.1 to 10 mg / kg May be administered once or several times per day. Also, the dosage of the composition containing meicin may be increased or decreased depending on the route of administration, degree of disease, sex, weight, age, and the like. Accordingly, the dosage is not limited in any way to the scope of the present invention.

Further, the present invention provides a health functional food for preventing or improving obesity including meisin.

The maysin is a yellow plant-flavonoid flavonoid substance found in corn, teosinte, and centipedegrass, and has antioxidant and anticancer activities. However, in the present invention, meishin derived from corn beard has confirmed the preventive and therapeutic effect on obesity.

The meicin of the present invention is not particularly limited as long as it can be usually prepared and / or purchased, but it may preferably be one derived from corn hairs.

The present inventors have found an activity of preventing and / or improving obesity, which is a new physiological activity of mexin derived from corn beard.

Specifically, as shown in Example 1, the meicin inhibited the growth of adipocytes and confirmed the activity of preventing and treating obesity.

Moreover, as shown in Example 2, the meicin showed lipid accumulation of adipocytes and activity of preventing and treating obesity.

Furthermore, as shown in Example 3, the meicin showed an activity of preventing and treating obesity by decreasing the content of triglyceride in adipocytes.

Further, the health-functional food for preventing and improving obesity according to the present invention can be produced by using PPAR-γ (peroxisome proliferator-activated receptors-γ), C / EBP-α (CCAAT / enhancer-binding protein-α) and aP2 (adipocyte protein 2) Lt; RTI ID = 0.0 > mRNA < / RTI >

Specifically, as shown in Examples 4 to 5 and Figs. 5 to 9, it was confirmed that the maccin of the present invention exhibited anti-obesity activity by decreasing the expression of PPAR-y, C / EBP-a and aP2.

In addition, the health functional food for prevention and improvement of obesity is not particularly limited as long as it contains meicin, but it may preferably be contained at a concentration of 25 to 100 占 퐂 / ml, more preferably 50 to 100 占 퐂 / Ml. ≪ / RTI >

If it contains less than 25 μg / ml of meicin, sufficient anti-obesity activity may not be seen, and if it contains an amount of meicin in excess of 100 μg / ml, Problems may arise.

More specifically, the health functional food for prevention and improvement of obesity according to the present invention is characterized in that when the mexin is contained at a concentration of 25 to 100 占 퐂 / ml, To 15% to 25%, and when the macronutrient is contained at a concentration of 25 to 75 μg / ml, the fat cell growth can be suppressed to 15 to 23%.

In addition, the health functional food for prevention and improvement of obesity according to the present invention is characterized in that lipid accumulation of adipocyte is 20 to 50% higher than that of adipocyte not containing meicin, when it contains meicin at a concentration of 50 to 100 占 퐂 / , And preferably the lipid accumulation of adipocytes can be reduced to 22-45% when the macrocin is included at a concentration of 50-100 [mu] g / ml.

In addition, when the health functional food for preventing and improving obesity of the present invention contains mexin at a concentration of 50 to 100 占 퐂 / ml, the content of triglyceride in fat cells is 25 to 70 %, And preferably, when the mycelium is contained at a concentration of 50 to 100 占 퐂 / ml, the content of triglyceride in the adipocyte can be reduced by 40 to 65%, more preferably, Ug / ml, the content of triglyceride in fat cells can be reduced to 60 to 65%.

In addition, when the health functional food for obesity prevention and improvement of the present invention contains mexin at a concentration of 50 to 100 占 퐂 / ml, PPAR-gamma expression in adipocytes is reduced to 25 ~ 45%, and when the macronutrient is contained at a concentration of 50-100 占 퐂 / ml, the amount of PPAR-gamma expression in adipocytes can be reduced to 25-40%.

In addition, when the health functional food for preventing and improving obesity of the present invention contains mexin at a concentration of 75-100 μg / ml, the expression of aP2 in adipocytes is 20-40% higher than that of adipocytes not containing meicin. , And preferably the amount of aP2 expression in adipocytes can be reduced to 20 to 35% when the macrocin is contained at a concentration of 75 to 100 占 퐂 / ml.

In addition, when the health functional food for prevention and improvement of obesity of the present invention contains mexin at a concentration of 25 to 75 占 퐂 / ml, the adipocyte C / EBP-α expression level The amount of C / EBP-α expression in the adipocyte can be reduced to 40 to 90% when the myc synthase is contained at a concentration of 50 to 75 μg / ml, More preferably 40 to 85%.

There is no particular limitation on the kind of the health functional food of the present invention. Examples of the foods to which the meicin can be added include dairy products including meats, sausages, breads, chocolates, candies, snacks, confections, pizza, ramen noodles, gums, ice cream, soups, drinks, tea, Alcoholic beverages, and vitamin complexes, and can be used in the form of pills, powders, granules, infusions, tablets, capsules or beverages, and includes health functional foods in a conventional sense.

In this case, the addition amount of meicin in the food or beverage is generally in the range of 1 to 200 μg / mg, preferably 2 to 100 μg / mg, for the whole food in the case of the health functional food composition of the present invention And may be added at a ratio of 100 to 20,000 占 퐂, preferably 200 to 10,000 占 퐂 based on 100 ml in the case of a health beverage composition.

The health beverage composition of the present invention is not particularly limited to liquid ingredients other than those containing the above-mentioned macroscene as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; Polysaccharides such as dextrin, cyclodextrins; And sugar alcohols such as xylitol, sorbitol, and erythritol. As natural flavors other than those described above, natural flavors (such as tau martin, stevia extract (e.g., rebaudioside A, glycyrrhizin)) and synthetic flavors (saccharin, aspartame, etc.) have.

In addition to the above, the health functional food of the present invention may contain various kinds of nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and heavies (cheese, chocolate etc.), pectic acid and its salts, And salts thereof, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like.

In addition, the health functional foods of the present invention may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks. These components may be used independently or in combination.

Hereinafter, the present invention will be described in detail with reference to examples. However, these examples are intended to further illustrate the present invention, and the scope of the present invention is not limited to these examples.

[ Example ]

Example  1. Measurement of adipocyte growth inhibitory activity

MTT assay (methylthiazolyldiphenyl tetrazolium bromide assay) was performed to measure the inhibitory activity of macrophages on 3T3-L1 (mouse embryonic fibroblast-adipose like cell line) adipocytes.

The 3T3-L1 adipocytes were cultured in 96-well plates (96-well plate) in ⁴ × 10 4 cells / ㎖ to dispense with 5% CO ₂ for 24 hours, 37 ℃ incubator (incubator). After the culture, the culture medium was removed, and the medium was replaced with Dulbecco's Modified Eagle's Medium (DMEM medium; Thermo Scientific, USA) containing 1% penicillin streptomycin and cultured for 2 hours. After that, DMEM medium and Meishin were used (concentration of 0 ㎍ / ㎖, 25 ㎍ / ㎖, 50 ㎍ / ㎖, 75 ㎍ / ㎖ or 100 [mu] g / ml) for 24 hours.

Then, a solution of MTT (3- [4,5-dimethyl-thiazole] -2,5-diphenyl-tetrazolium bromide) dissolved in 5 mg / ml of DBS in a solvent DPBS (Dulbecco's Phosphate-Buffered Saline, And incubated for 4 hours in a 5% CO ₂ incubator at 37 ° C. After the culture, the culture solution was removed, and 200 μl / well of dimethyl sulfoxide (DMSO) was added to each well, followed by well stirring using a pipette. The absorbance was then measured at 570 nm with an ELISA reader. The measurement results are shown in FIG. 1 below.

As shown in Fig. 1, when treated with a concentration of 25 μg / ml, 50 μg / ml, 75 μg / ml or 100 μg / ml of mexin, the adipocytes treated with 100 μg / Cell growth inhibition was confirmed.

Example  2. Measurement of lipid accumulation inhibitory activity of adipocytes

The cells were cultured in the same manner as in Example 1 except that the cells were treated with different concentrations (0 μg / ml, 25 μg / ml, 50 μg / ml, 75 μg / ml or 100 μg / And washed with PBS (phosphate buffered saline). Then, 500 쨉 l of 10% formaldehyde was added to each well to fix the cells, followed by incubation at 4 째 C for 1 hour. After that, formaldehyde was removed, washed three times with phosphate-buffered saline, and then dried in a CO ₂ incubator.

After the plates were dried, 500 μl of Oil Red Dye was added, and the mixture was stained for 30 minutes at room temperature in a dark state, and washed three times with phosphate-buffered saline. The stained cells were observed under a microscope. After observation, the stained dye in the adipocyte was extracted with 300 μl of 2-propanol per well and the optical density value (OD value) was measured at 500 nm with an ELISA reader. The measurement results are shown in Fig.

The oil red ointment agent is a solution obtained by mixing 500 mg of Oil Red O dyeing agent in 100 ml of 2-propanol with distilled water in a ratio of 6: 4, followed by filtration through a 0.45 μm filter.

As shown in FIG. 2, when the fat cells differentiated from the control group treated with meiocin were compared with the fat cells differentiated from the fat cells without treatment with meiocin, the adipocyte differentiation was about 50% when 100 μg / As shown in Fig.

Example  3. Analysis of triglyceride content in adipocytes by Meicin treatment

3T3-L1 adipocytes were dispensed in 6-well plates at a density of 7 × 10 ⁴ cells / ml and differentiated into adipocytes for 8 days, while Mechin was added to 0 μg / ml, 25 μg / ml, 50 μg / Ml, 75 [mu] g / ml or 100 [mu] g / ml. After the Meishin treatment, the cells were washed with phosphate-buffered saline, collected using a scraper, extracted with intracellular triglycerides using an ultrasonic sonicator, and analyzed with a Triglyceride Kit And the content thereof was measured. The measured triglyceride content is shown in FIG.

As shown in Fig. 3, when the control group in which Mechicine was treated with 25 ㎍ / ㎖, 50 ㎍ / ㎖, 75 ㎍ / ㎖ or 100 ㎍ / ㎖ and differentiated into adipocytes and adipocytes was compared, It was confirmed that the triglyceride of fat cells treated with 100 ㎍ / ㎖ of gins reduced about 70%.

Example  4. Adipocyte mRNA Mechanism of expression

4-1. Adipocyte RNA  detach

3T3-L1 cells were treated with different concentrations (0 μg / ml, 25 μg / ml, 50 μg / ml, 75 μg / ml or 100 μg / ml) while differentiating them into 60-mm dishes. Then, the cells were washed with phosphate-buffered saline and treated with 1 ml of Trizol to transfer the cells to an eppendorf tube.

After that, 200 μl of chloroform per 1 ml of trisole was added to the solution, and the mixture was centrifuged (13,000 rpm, 10 minutes, 4 ° C) and the supernatant RNA was recovered. 400 μl of Binding Buffer was added to the recovered RNA and mixed by hand for about 1 minute. After removing the chloroform using a dedicated RNA column and Washing Buffer 1 (700 μl) and 2 (700 μl), the RNA-only column was removed and the new EP tube (Eppendorf tube) was transferred RNA was eluted with an Elution Buffer to be pulled down under the column to recover only the RNA.

4-2. Adipocyte cDNA  synthesis

The RNA recovered in Example 4-1 was quantitatively determined with a microplate reader (Bio-Tek, USA) to prepare RNA, and DEPC water (diethyl pyrocarbonate water) was prepared.

The quantified RNA and DEPC water were added to the tubes for PCR and 1 μl of reverse transcriptase Oligo (dT) 15 was added. Then, the reaction was carried out at 75 ° C for 5 minutes using a PCR machine and the cDNA synthesis kit (Intronbio, Korea) was mixed well. Thereafter, the mixture is spun down to precipitate, and then reacted at 42 ° C for 1 hour. After 5 minutes at 75 ° C, 50 μl of DEPC water was added to the obtained cDNA. Finally, the PCR product was used for RT-PCR.

4-3. cDNA Of adipocytes mRNA  Mechanism of expression

1 μl of the cDNA synthesized in Example 4-2 and 2 μl of the primer shown in Table 1 were placed in a Master Mix Solution (iNtRON, i-StatTaq, Korea), 17 μl of DEPC water was added thereto, . The specific PCR method is shown in Table 1.

The PCR product was then electrophoresed on 2% agarose gel, stained with ethidium bromide, and checked on a UV lamp. After the PCR, the concentration of peroxisome proliferator-activated receptors-γ (PPAR-γ) and aP2 (adipocyte protein 2) was calculated using an image station (Kodak, USA) 5.

As can be seen in FIG. 4, when comparing the control group in which the adipocyte and adipocyte were differentiated after treatment with Mechicine at a concentration of 25 μg / ml, 50 μg / ml, 75 μg / ml or 100 μg / And PPAR-gamma (PPAR-gamma) decreased to 43% in adipocytes treated with 100 μg / ml of ginsenoside.

As can be seen in FIG. 5, when comparing the control group in which the adipocyte and adipocyte were differentiated after treatment with mexin at a concentration of 25 μg / ml, 50 μg / ml, 75 μg / ml or 100 μg / It was confirmed that the amount of aP2 decreased to 40% in adipocytes treated with 100 μg / ml of ginsenoside.

As a result, it was confirmed that the macronutrient of the present invention decreased PPAR-γ and aP2 concentrations.

Example  5. Mechanism of protein expression in adipocytes

To investigate the mechanism of anti-obesity in mexin, 3T3-L1 adipocytes were treated with PPAR-γ, C / EBP-α and adipocyte protein-α (CCAAT / enhancer- 2) was investigated by western blot method.

Specifically, 3T3-L1 adipocytes were seeded in a 6-well plate at a concentration of 7 × 10 ⁴ cells / ml, followed by differentiation at 37 ° C for 8 days. 25 μg / ml, 50 μg / ml, 75 μg / ml or 100 μg / ml. After 8 days, the cells were washed with phosphate buffered saline (PBS buffer) and centrifuged at 1,400 rpm for 5 minutes to obtain cells.

The obtained cells were added with 50 占 퐇 of lysis buffer, reacted on ice for 30 minutes, and centrifuged at 12,000 rpm for 10 minutes to obtain proteins. Proteins were then quantitated by Bradford assay (Bradford assay), denatured at 95 ° C for 5 minutes in a sample buffer, and then subjected to SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) And then electrophoresed. After electrophoresis, the cells were transferred to a nitrocellulose membrane, and the primary antibody (1: 1000, v / v) was diluted in 5% skim milk and placed in a nitrocellulose membrane and reacted at 4 ° C for 24 hours . Afterwards, HRP (horeseradish peroxidase) -conjugated secondary antibody was washed 1: 2000 (v / v) in 5% non-fat milk milk with TBST (mixture of Tris-Buffered Saline and Tween 20, 0.1% Tween 20) And 5 μl of the mixture was added thereto, followed by reaction at 25 ° C for 2 hours.

After washing three times with TBST, ECL (enchanced chemiluminescence) substrate solution was blocked and reacted for 1 minute and developed on X-ray film. The results obtained were digitized using the Image J program and are shown in Figures 6 to 8.

As can be seen in FIG. 6, when comparing the control group in which the adipocyte and adipocyte differentiated with mexin at a concentration of 25 μg / ml, 50 μg / ml, 75 μg / ml or 100 μg / PPAR-γ was reduced to 7% when treated at a concentration of 75 μg / ml, and lower than that of the non-differentiated control (UC) when treated with 100 μg / ml of mexin I could confirm.

As can be seen in FIG. 7, when comparing the control group in which the adipocyte and adipocyte were treated with 25 μg / ml, 50 μg / ml, 75 μg / ml or 100 μg / C / EBP-alpha was reduced by 17% when treated with 75 ㎍ / ㎖, and similar to control group (UC) when treated with 100 ㎍ / ㎖ of mexin.

As can be seen in FIG. 8, when comparing the control group in which the adipocyte and adipocyte differentiated with mexin at a concentration of 25 μg / ml, 50 μg / ml, 75 μg / ml or 100 μg / 75 ㎍ / ㎖, the concentration of aP2 decreased to 7%, and when it was treated with 100 ㎍ / ㎖ of mexin, it appeared to be similar to that of the non - differentiated control (UC).

As a result, the present invention provides a method of decreasing the expression of PPAR-γ (peroxisome proliferator-activated receptors-γ), C / EBP-α (CCAAT / enhancer-binding protein-α) and aP2 (adipocyte protein 2) Lt; / RTI > activity.

<110> CATHOLIC UNIVERSITY INDUSTRY ACADEMIC COOPERATION FOUNDATION <120> Pharmaceutical composition comprising maysin for the prevention          and treatment of obesity <130> 1040229 <160> 6 <170> Kopatentin 2.0 <210> 1 <211> 23 <212> DNA <213> Mouse embryonic fibroblast <400> 1 aggtatcctg accctgaagt acc 23 <210> 2 <211> 23 <212> DNA <213> Mouse embryonic fibroblast <400> 2 gttgccaata gtgatgacct ggc 23 <210> 3 <211> 20 <212> DNA <213> Mouse embryonic fibroblast <400> 3 gtctgtgggg ataaagcatc 20 <210> 4 <211> 20 <212> DNA <213> Mouse embryonic fibroblast <400> 4 ctgatggcat tgtgagacat 20 <210> 5 <211> 21 <212> DNA <213> Mouse embryonic fibroblast <400> 5 cctggaagac agctcctcct c 21 <210> 6 <211> 23 <212> DNA <213> Mouse embryonic fibroblast <400> 6 atccaggcct cttcctttgg ctc 23

Claims (15)

A pharmaceutical composition for the prevention or treatment of obesity comprising mexin as an active ingredient. The pharmaceutical composition for preventing or treating obesity according to claim 1, wherein the pharmaceutical composition inhibits the growth of adipocytes, decreases lipid accumulation of adipocytes and decreases triglyceride content in adipocytes. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is one selected from the group consisting of peroxisome proliferator-activated receptors (γ), C / EBP-α (CCAAT / enhancer-binding protein-α) and aP2 (adipocyte protein 2) A method for preventing or treating obesity comprising administering to a subject in need thereof an effective amount of at least one mRNA. The pharmaceutical composition for preventing or treating obesity according to claim 1, wherein the pharmaceutical composition comprises mexin at a concentration of 25 to 100 占 퐂 / ml. The pharmaceutical composition for preventing or treating obesity according to claim 4, wherein the pharmaceutical composition contains 15-25% of inhibiting growth of adipocyte when the macroscopic concentration is 25-100 μg / ml. [Claim 5] The pharmaceutical composition for preventing or treating obesity according to claim 4, wherein the pharmaceutical composition has a lipid accumulation reduction rate of 20 to 50% when the macrocycle is contained at a concentration of 50 to 100 占 퐂 / ml. . [Claim 5] The pharmaceutical composition for preventing or treating obesity according to claim 4, wherein the pharmaceutical composition comprises 25 ~ 70% of a decrease in triglyceride content in adipocytes when the macrocycle is contained at a concentration of 50-100 [mu] g / Composition [Claim 5] The pharmaceutical composition according to claim 4, wherein the pharmaceutical composition contains 25 to 45% of the reduction rate of expression of PPAR-gamma in adipocytes when the macroscopic concentration is 25 to 100 占 퐂 / ml. A pharmaceutical composition. The pharmaceutical composition for preventing or treating obesity according to claim 4, wherein the pharmaceutical composition has a decrease rate of aP2 expression in adipocyte by 20 to 40% when it contains mexin at a concentration of 75 to 100 占 퐂 / ml Composition. [Claim 5] The method according to claim 4, wherein the pharmaceutical composition has a decrease rate of C / EBP-alpha expression in adipocytes is 40 to 90% when the pharmaceutical composition is contained at a concentration of 50 to 75 mu g / ml of mexin. A pharmaceutical composition for therapeutic use. The pharmaceutical composition for preventing or treating obesity according to claim 1, wherein the meicin is derived from a corn beard. Health functional food for prevention or improvement of obesity including mexin. 13. The method of claim 12, wherein the health functional food is selected from the group consisting of peroxisome proliferator-activated receptors (gamma), C / EBP- alpha (CCAAT / enhancer-binding protein- alpha) and aP2 (adipocyte protein 2) A health functional food for preventing or improving obesity characterized by decreasing the expression of at least one mRNA. 13. The health functional food for preventing or improving obesity according to claim 12, wherein the health functional food comprises mexin at a concentration of 25-100 占 퐂 / ml. 13. The health functional food for preventing or improving obesity according to claim 12, wherein the health functional food is a composition for inhibiting growth of adipocytes, reducing triglyceride content in adipocytes and reducing lipid accumulation in adipocytes.

Images