NOVEL FAT ACCUMULATION INHIBITORY PEPTIDE AND PHARMACEUTICAL COMPOSITION FOR PREVENTING OR TREATING OBESITY CONTAINING THE SAME

Abstract

The disclosure relates to a fat accumulation inhibitory peptide which essentially comprises an amino acid sequence represented by SEQ ID NO: 1, a pharmaceutical composition for preventing or treating obesity, which contains the peptide, and a health functional food for preventing or alleviating obesity, which contains the peptide. The fat accumulation inhibitory peptide according to the present invention has the function of inhibiting the differentiation of mesenchymal stem cells into adipocytes to thereby inhibit the accumulation of adipose tissue. Thus, the peptide according to the present invention is highly useful for the prevention or treatment of obesity.

Claims

1. A method for inhibiting fat accumulation, comprising administering a fat accumulation inhibitory peptide which essentially comprises the amino acid sequence represented by SEQ ID NO: 1 to a subject in need of fat accumulation inhibition.

2. The method of claim 1, wherein the fat accumulation inhibitory peptide consists of the amino acid sequence represented by SEQ ID NO: 2.

3. The method of claim 1, wherein the peptide acts to inhibit the differentiation of mesenchymal stem cells into adipocytes.

4. A method for preventing or treating obesity, comprising administering a pharmaceutical composition which comprises the amino acid sequence represented by SEQ ID NO: 1 to a subject in need of reducing obesity.

5. The method of claim 4, wherein the obesity is obesity caused by estrogen deficiency.

6. The method of claim 4, wherein the pharmaceutical composition is administered by oral administration, injection administration, or local transplantation in the form of a gelling agent.

7. The method of claim 6, wherein the gelling agent comprises a synthetic polymer or a natural polymer.

8. The method of claim 7, wherein the synthetic polymer is any one selected from the group consisting of polylacticglycolic acid, poloxamer, and propylene glycol.

9. The method of claim 7, wherein the natural polymer is any one selected from the group consisting of collagen, alginic acid, propylene glycol alginic acid, chondroitin sulfate, and chitosan.

10. The method of claim 4, wherein the pharmaceutical composition is administered in an amount of 1-60 mg per kg of body weight of the subject in need of reducing obesity.

11. The method of claim 1, wherein the fat accumulation inhibitory peptide is administered in a health functional food which contains the fat accumulation inhibitory peptide.

12. The method of claim 11, wherein the fat accumulation is caused by estrogen deficiency.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIGS. 1A and 1B show the inhibition of differentiation of mesenchymal stem cells into adipocytes by the fat accumulation inhibitory peptide according to the present invention. Specifically, FIG. 1A is an image of adipocytes stained with Oil Red O, and FIG. 1B shows the absorbance of free Oil Red O.

[0018] FIG. 2 shows the inhibition of differentiation of mesenchymal stem cells into adipocytes by the fat accumulation inhibitory peptide according to the present invention, and shows the results of electrophoresis of PCR products obtained in Example 2.

[0019] FIG. 3 shows the results of measuring the change in body weight caused by the fat accumulation inhibitory peptide according to the present invention, in Example 3.

[0020] FIG. 4 shows the results of measuring the change in total fat weight caused by the fat accumulation inhibitory peptide according to the present invention, in Example 3.

[0021] FIG. 5 shows the results of measuring the change in total fat/body weight caused by the fat accumulation inhibitory peptide according to the present invention, in Example 3.

[0022] FIG. 6 shows the results of measuring the change in subcutaneous fat weight caused by the fat accumulation inhibitory peptide according to the present invention, in Example 3.

[0023] FIG. 7 shows the results of measuring the change in subcutaneous fat/body weight caused by the fat accumulation inhibitory peptide according to the present invention, in Example 3.

[0024] FIG. 8 shows the results of measuring the change in liver/body weight caused by the fat accumulation inhibitory peptide according to the present invention, in Example 3.

[0025] FIG. 9 shows the results of measuring the change in kidney/body weight caused by the fat accumulation inhibitory peptide according to the present invention, in Example 3.

BEST MODE FOR CARRYING OUT THE INVENTION

[0026] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Generally, the nomenclature used herein and the experiment methods, which will be described below, are those well known and commonly employed in the art.

[0027] In the present invention, a novel fat accumulation inhibitory peptide was prepared, and mice were treated with the prepared peptide in order to examine the effects of the peptide on the inhibition of adiopocyte differentiation and fat accumulation. As a result, it was found that the fat accumulation inhibitory peptide has the effect of inhibiting fat accumulation, and thus can be used as an obesity treatment agent.

[0028] In one aspect, the present invention is directed to a fat accumulation inhibitory peptide which essentially comprises an amino acid sequence represented by the following SEQ ID NO: 1:

[0029] SEQ ID NO 1: YGLRSKS

[0030] In the present invention, the fat accumulation inhibitory peptide may comprise an amino acid sequence represented by the following SEQ ID NO 2:

[0031] SEQ ID NO 2: YGLRSKSKKFRRPDIQYPDAT.

[0032] In the present invention, the fat accumulation inhibitory peptide may act to inhibit the differentiation of mesenchymal stem cells into adipocytes.

[0033] In another aspect, the present invention is directed to a pharmaceutical composition for preventing or treating obesity, which contain the above fat accumulation inhibitory peptide.

[0034] In still another aspect, the present invention is directed to a method for preventing or treating obesity, which comprises administering the above pharmaceutical composition containing the fat accumulation inhibitory peptide.

[0035] In yet another aspect, the present invention is directed to the use of the above pharmaceutical composition containing the fat accumulation inhibitory peptide, for the prevention or treatment of obesity.

[0036] As used herein, the term “composition” is intended to include not only a product containing a specific component but also any product made directly or indirectly by the combination of a specific component.

[0037] In the present invention, obesity may be caused by estrogen deficiency.

[0038] In the present invention, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier. The carrier pharmaceutically acceptable may be at least one selected from the group consisting of physiological saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, and ethanol, but is not limited thereto.

[0039] In the present invention, the pharmaceutical composition may further contain at least one additive selected from the group consisting of an excipient, a buffer, an antimicrobial preservative, a surfactant, an antioxidant, a tonicity adjuster, a preservative, a thickener, and a viscosity modifier, but is not limited thereto.

[0040] In the present invention, the pharmaceutical composition may be formulated for oral administration, injection administration or in the form of a gelling agent for local transplantation, but is not limited thereto. The composition of the present invention may be prepared into a suitable formulation using a known technique (Joseph Price Remington, Remington's Pharmaceutical Science, 17th edition, Mack Publishing Company, Easton Pa.).

[0041] The pharmaceutical composition for preventing or treating obesity according to the present invention can be administered through routes that are usually used in the medical field. The composition of the present invention is preferably administered parenterally. The composition according to the present invention may be administered, for example, orally, intravenously, intramuscularly, intraarterially, intramedullarily, intradually, intracardially, transdermally, subcutaneously, intraperitoneally, intrarectally, sublingually or topically.

[0042] In the present invention, the gelling agent for local transplantation comprises a synthetic polymer such as polylacticglycolic acid, poloxamer or propylene glycol, or a natural polymer such as collagen, alginic acid, propylene glycol alginic acid, chondroitin sulfate or chitosan, but is not limited to thereto.

[0043] The dose of the pharmaceutical composition for preventing or treating obesity according to the present invention may vary depending on the patient's weight, age, sex, health condition and diet, the time of administration, the mode of administration, excretion rate, the severity of the disease, or the like, and can be easily determined by those skilled in the art in consideration of the above factors.

[0044] 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.

[0045] In the present invention, the fat accumulation inhibitory peptide may be administered in an amount of preferably 1-60 mg, more preferably 3-30 mg, per kg of body weight of a subject to be treated.

[0046] In a further aspect, the present invention is directed to a health functional food for preventing or alleviating obesity, which contains the above fat accumulation inhibitory peptide.

[0047] As used herein, the term “health functional food” refers to a food is prepared and processed from raw materials or components having functionality useful for the human body pursuant to the law No. 6722 on the health functional food, or refers to a food that is taken for the purpose of controlling nutrients with respect to the structure and function of the human body or obtaining the effects useful for the health purposes such as physiologically functional purpose.

[0048] The health functional food according to the present invention may be formulated into a typical health functional food preparation known in the art. The health functional food may be prepared in the form of granules, tablets, pills, suspensions, emulsions, syrups, chewing gums, teas, jellies, various beverages, drinks, alcoholic beverages or the like. There is no particular limitation in the kind of the health functional food.

[0049] The health functional food according to the present invention may be any suitable galenical form for administration to the animal body including the human body, more specifically, any conventional form for oral administration, for example, food or feed, food or feed additives and adjuvants, enhanced food or feed, a solid form such as tablets, pills, granules, capsules and foam formulations, or a liquid form such as solutions, suspensions, emulsions, drinks and pastes. The composition of the present invention may contain nutrients, vitamins, electrolytes, sweeteners, colorants, organic acids, preservatives, etc. These additives may be used independently or in combination.

EXAMPLES

[0050] Hereinafter, the present invention will be described in further detail with reference to examples. It will be obvious to a person having ordinary skill in the art that these examples are illustrative purposes only and are not to be construed to limit the scope of the present invention.

Example 1

Synthesis of Fat Accumulation Inhibitory Peptide

[0051] A peptide represented by the following SEQ ID NO: 2 was synthesized from the C-terminus by an F-moc solid phase chemical synthesis method using a peptide synthesizer:

[0052] SEQ ID NO 2: YGLRSKSKKFRRPDIQYPDAT

[0053] The synthesized peptide sequence was separated from resin, washed, freeze-dried, and then purified by liquid chromatography. The molecular weight of the purified peptide was analyzed by MALDI.

Example 2

Adipocyte Differentiation Inhibitory Effect of Fat Accumulation Inhibitory Peptide

[0054] Using mesenchymal stem cells (MSCs), the effect of the fat accumulation inhibitory peptide, prepared in Example 1, on adipocyte differentiation, was studied.

[0055] Mesenchymal stem cells (MSCs) were cultured in DMEM containing 1% antibiotic-antimycotic and 10% FBS, and were stored. Next, the cells were cultured in adipocyte differentiation induction medium (DMEM containing 10% FBS, 10 μM dexamethasone, 0.5 mM methyl-isobutylxanthine, 10 μg/ml insulin, 10 mM indomethacin, and 1% antibiotic-antimycotic) for 3 days, and were cultured in adipocyte differentiation induction medium (DMEM containing 10 μg/ml insulin, 10% FBS, and 1% antibiotic-antimycotic) for 3 days. The cells were culture for 14 days while the medium was replaced in the above order, thereby inducing differentiation of the cells. During the culture, a mixture of 95% air and 5% CO.sub.2 was continuously supplied while a humidity of 100% and a temperature of 37° C. were maintained. The culture for differentiation was performed for a total of 14 days, and the peptide prepared in Example 1 was added whenever the medium was replaced. The peptide was added at concentrations of 0, 10, 100 and 200 μg/mL.

[0056] The cells cultured in the differentiation medium were washed with PBS and fixed with 10% formalin for 1 hour. 30% Oil red O solution diluted with 60% isopropanol was added to the cells which were then incubated at room temperature for 10 minutes. The cells were washed with purified water and observed with an optical microscope. After observation, isopropanol was added to dissolve the formed fat, and the absorbance at 510 nm was measured.

[0057] As a result, it could be seen that the differentiation of the mesenchymal stem cells into adipocytes was increased by the adipocyte differentiation medium (FIGS. 1A and 1B). In addition, it could be microscopically observed that, as the concentration of the peptide used to treat the cells increased, the accumulation of the fat stained with Oil red O decreased (FIG. 1A). Furthermore, when the produced fat was dissolved and the absorbance at 510 nm was measured, it could be seen that, as the concentration of the peptide increased, the absorbance decreased (FIG. 1B).

[0058] In addition, in the same manner as described above, mesenchymal stem cells were treated with the peptide prepared in Example 1. RNA was extracted from the cells and subjected to reverse transcription polymerase chain reaction (RT-PCR) using primers for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) which is a control, and adipocyte protein 2 (aP2) and peroxisome proliferators activated receptor γ (PPARγ), which are adipocyte differentiation markers. The PCR products were electrophoresed on agarose gel in order to compare the expression levels of the control and the differentiation markers.

[0059] As a result, it could be seen that, as the concentration of the peptide used to treat the cells increased, the expression of the control GAPDH gene did not change, but the expression of the adipocyte differentiation marker genes (aP2 and PPARγ) decreased (FIG. 2).

Example 3

Fat Accumulation Inhibitory and Toxic Effects of Fat Accumulation Inhibitory Peptide

[0060] ICR mice (Orient Bio, Korea) were purchased and acclimated, and the ovary was removed from the mice to induce estrogen deficiency, thereby inducing bone loss and an increase in the body weight of the mice. For ovariectomy, 6-week-old ICR mice were generally anesthetized by intramuscularly injecting a mixture of 10 mg/kg of xylazine (Rompun®, Bayer, Korea) and 100 mg/kg of ketamine (Ketalar®, Yuhan Corp., Korea) into the femoral region of the mice. The ovary present below both kidneys was carefully removed, and the mice were sutured according to a conventional method and injected intramuscularly with 3 mg/kg of gentamicin (Gentamicin®, Choongwae Pharma Corp., Korea), and then kept. From 3 months after ovariectomy, the peptide prepared in Example 1 was administered intraabdominally to the mice twice a week for a total of 8 weeks. The test animals were divided into 7 test groups as shown in Table 1 below, and the change in body weight and the fat accumulation of the mice were measured.

TABLE-US-00001 TABLE 1 Division of Test Animals Sham Sham (Pseudo- (Pseudo- OVX Normal OVX)-PBS OVX)-HP Control LP HP Treatment materials PBS PBS Peptide of PBS Peptide of Peptide of (concentration, μg/25 g SEQ ID NO: 2 SEQ ID SEQ ID NO: 2 mice) (600 μg) NO: 2 (60 μg) (600 μg) Number (heads) 10 5 5 10 10 10 [Normal: normal group; Sham (Pseudo-OVX): non-ovariectomized test group; OVX: ovariectomized test group; Control: control group; LP: low-concentration peptide (60 μg/25 g mouse); HP: high-concentration peptide (600 μg/25 g mouse)]

[0061] When the change in the body weight of the mice was observed, it could be seen that the body weight and total fat weight of the ovariectomized test group increased and that the OVX-PBS group showed a significant increase in the body weight compared to the normal group. In addition, it could be seen that the total fat weight and the total fat/body weight were significantly higher in the OVX-PBS group than in the normal group, but significantly decreased in the test group treated with the peptide, suggesting that the peptide inhibits fat accumulation (FIGS. 3 to 5).

[0062] When the change in subcutaneous fat weight of the mice was observed, it could be seen that the subcutaneous fat weight changed more greatly than the total fat weight. Also, it could be seen that, in the ovariectomized test group, the subcutaneous fat weight significantly increased, but in the test group treated with the peptide, the subcutaneous fat weight decreased (FIGS. 6 and 7).

[0063] In addition, as the indices of the hepatotoxicity and nephrotoxicity of the peptide, the weights of the liver and kidney of the mice were measured after autopsy. As a result, it could be seen that the weights of liver and kidney/body weight of the test group treated with the peptide were all similar to those of the normal group, suggesting that the hepatotoxicity and nephrotoxicity of the peptide were insignificant (FIGS. 8 and 9).

INDUSTRIAL APPLICABILITY

[0064] As described above, the fat accumulation inhibitory peptide according to the present invention has the function of inhibiting the differentiation of mesenchymal stem cells into adipocytes to thereby inhibit the accumulation of adipose tissue, similar to estrogen. Thus, the peptide according to the present invention is highly useful for the prevention or treatment of obesity.

[0065] Although the present invention has been described in detail with reference to the specific features, it will be apparent to those skilled in the art that this description is only for a preferred embodiment and does not limit the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.