KR101576126B1 - A novel pharmaceutical composition for preventing and treating obesity comprising alphaAL14 peptide - Google Patents

Abstract

The present invention relates to a novel pharmaceutical composition comprising an alphaAL14 peptide as an active ingredient for preventing and treating obesity and, more specifically, to a pharmaceutical composition comprising a peptide consisting of amino acid sequences represented by SEQ ID NO. 1 as an active ingredient for preventing and treating obesity.

Description

[0001] The present invention relates to a novel pharmaceutical composition for preventing and treating obesity, which comprises an alpha AL14 peptide as an active ingredient,

TECHNICAL FIELD The present invention relates to a pharmaceutical composition, and more particularly, to a pharmaceutical composition for preventing and treating new obesity which comprises an alpha AL14 peptide as an active ingredient.

Adipose tissue plays an important role in energy storage and lipid homeostasis, but it causes an imbalance between energy intake and consumption, leading to metabolic diseases such as type 2 diabetes, hypertension, hyperlipidemia and atherosclerosis, and a major risk factor for many chronic diseases It induces obesity. Obesity increases adipose tissue mass by adipogenesis and adipocyte proliferation and deposition of cytoplasmic triglycerides, and causes many adult diseases such as diabetes, hyperlipemia and hypertension. Accordingly, various pharmaceutical compositions having an effect of inhibiting lipogenesis have been reported.

Korean Patent No. 1201286 discloses a functional composition having inhibitory effects on fat, carbohydrate and protein absorption, inhibition of fat accumulation and lipolysis, and foods using the functional composition.

However, there are not so many pharmaceutical compositions containing an alpha-helical peptide having an excellent fat-suppressing effect and few side effects as an effective ingredient.

The present invention has been made to solve the above-mentioned problems, and it is an object of the present invention to provide a peptide having an excellent fat-suppressing effect. However, these problems are exemplary and do not limit the scope of the present invention.

According to one aspect of the present invention, there is provided a pharmaceutical composition for preventing and treating obesity, which comprises, as an active ingredient, a peptide consisting of the amino acid sequence of SEQ ID NO: 1.

According to another aspect of the present invention, there is provided a health functional food for obesity improvement comprising, as an active ingredient, a peptide consisting of the amino acid sequence of SEQ ID NO: 1.

According to another aspect of the present invention, there is provided a feed additive for preventing and improving animal obesity, which comprises, as an active ingredient, a peptide consisting of the amino acid sequence of SEQ ID NO: 1.

According to one embodiment of the present invention as described above, an excellent fat formation inhibiting effect can be achieved by treating the fat cells with the peptide. Of course, the scope of the present invention is not limited by these effects.

FIG. 1 is a photograph of a cell treated with αAL14 according to an embodiment of the present invention to determine the effect of inhibiting lipid accumulation by oil-red O staining.
FIG. 2 is a graph showing that αAL14 is treated by concentration in cells cultured according to an embodiment of the present invention, and triglyceride is measured, thereby inhibiting the accumulation of triglycerides in a concentration-dependent manner.

Definition of Terms:

As used herein, the term "alpha AL14" is defined as consisting of 14 amino acids and forms extended strands and random coils at the N-terminal and C-terminal portions of the amino acid sequence of SEQ ID NO: , While the central region (residues 5-10) has an alpha-helical structure and has a molecular weight of 1466.9 Da and C ₇₁ H ₁₂₃ N 19 O ₁₄ .

As used herein, the term "3T3-L1 cell" is a cell line isolated from adipose tissue of mice and is primarily used in biological studies. 3T3-L1 cells are similar to fibroblasts, but when treated with a differentiation-inducing agent, they differentiate into similar phenotypes to adipocytes.

As used herein, the term " triglyceride (TG) "refers to an ester bond of three fatty acids to three hydroxyl groups contained in glycerin. Most of the edible oils (usually over 99%) are composed of triglycerides, More than 95% of the fat is fat and the fat (fat tissue) of the animal is also triglyceride.

DETAILED DESCRIPTION OF THE INVENTION [

 According to one aspect of the present invention, there is provided a pharmaceutical composition for preventing and treating obesity, which comprises, as an active ingredient, a peptide consisting of the amino acid sequence of SEQ ID NO: 1.

In the above composition, the C-terminal of the peptide may have a carboxyl group (COOH) or an amine group (-NH ₂ ).

The pharmaceutical composition may additionally comprise one or more pharmaceutically acceptable carriers.

The pharmaceutical composition may be in the form of an oral preparation, a granule, a lotion, a liniment, a remonade, a handmade, a powder, a syrup, an ointment, a liquid, an aerosol, May be any one selected from among suspending agents, precursors, infiltrates, eye drops, tablets, suppositories, injections, main tablets, cataplasms, capsules, creams, troches, tinctures, pastes, pills, soft or hard gelatine capsules have.

The pharmaceutical composition of the present invention may further comprise an appropriate carrier, excipient or diluent according to a conventional method. The carrier, excipient and diluent may be selected from lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose , Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil. In addition, each of the above compositions may be formulated in the form of oral, granule, tablet, capsule, suspension, emulsion, syrup, aerosol or the like oral preparation, external preparation, suppository or sterilized injection solution according to a conventional method. In the case of formulation, it may be prepared using diluents or excipients such as fillers, extenders, binders, humectants, disintegrants, and surfactants that are commonly used, and solid preparations for oral administration may contain tablets, pills, powders, granules, capsules And the excipient may be prepared by mixing starch, calcium carbonate, sucrose, lactose, gelatin, and the like.

The pharmaceutical composition according to the present invention may vary depending on the age, sex, and body weight of the patient, but is generally administered in an amount of 0.01 to 500 mg / kg, preferably 0.1 to 100 mg / kg, And the dose may be increased or decreased depending on the route of administration, degree of disease, sex, weight, age, etc., but is not limited thereto.

The pharmaceutical composition according to the present invention can be administered to mammals such as rats, mice, livestock, humans, and the like in a variety of routes, and can be administered orally or parenterally at the time of clinical administration and can be administered intraperitoneally, , Subcutaneous injection, intravenous injection, intramuscular injection, intra-uterine injection, intracerebral injection or intrathoracic injection, and may be used in the form of a general pharmaceutical preparation.

According to another aspect of the present invention, there is provided a health functional food for obesity improvement comprising, as an active ingredient, a peptide consisting of the amino acid sequence of SEQ ID NO: 1.

The health functional food may further include various pharmaceutically acceptable additives such as emulsifiers, pigments, flavoring agents, coloring agents, antioxidants, and the like.

The health functional foods may be provided in the form of formulations of commonly used health functional foods such as tablets, granules, pills, capsules, powders, and beverages.

In recent years, not only human beings but also companion animals such as dogs and cats with human beings have suffered from obesity and various diseases accompanied by excessive consumption of nutrients and lack of exercise. Accordingly, the peptide of the present invention can be used not only as a human but also as a feed additive for a companion animal or a zoo animal that requires weight management.

Therefore, according to another aspect of the present invention, there is provided a feed additive for preventing and ameliorating obesity in an animal containing a peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

The feed additive may further comprise additives such as vitamins, amino acids, and other inorganic nutrients that are allowed to be added to the feed.

According to another aspect of the present invention, there is provided a feed composition comprising the feed additive.

Hereinafter, the present invention will be described in more detail by way of examples. It should be understood, however, that the invention is not limited to the disclosed embodiments, but may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, Is provided to fully inform the user.

Example 1: Peptide synthesis < RTI ID = 0.0 >

According to one embodiment of the present invention, a peptide showing the effect of inhibiting fat formation was synthesized.

Specifically, the designed peptides were synthesized by solid-phase synthesis method in ASP48S Pepsynthesizer using 9-fluorenylmethoxycarbonyl (Fmoc) -polypeptide active ester chemistry (Peptron Inc, Daejeon, Korea). The molecular weight and the formula of the synthesized peptide were confirmed to be 1466.9 Da and C ₇₁ H ₁₂₃ N19O ₁₄ , and the synthesized peptide sequence was AAWKLLKALAKAAL-NH ₂ (SEQ ID NO: 1). This substance is named αAL14 because it is composed of 14 amino acids taking α-helical structure. The peptides were dissolved in distilled water to make stock solutions and further diluted in the same solution for testing.

Example 2: Cell culture < RTI ID = 0.0 >

Cell culture was performed in order to observe lipid formation inhibitory effect of the synthesized peptide, [alpha] AL14, according to an embodiment of the present invention.

3T3-L1 (American Type Culture Collection, Manassas, Va.) Cells were ^treated with 10% fetal calf serum (Gibco, Grand Island, NY, USA) and penicillin (Cellgro, Mediatech, Inc., Manassas, Va., USA) supplemented with 100 μg / ml streptomycin. The cells were then incubated at 5% CO ₂ , 37 ° C, and 95% humidity conditions. To induce adipogenesis, cells were confluence (day 0) and cultured for another day. The cells were cultured in DMEM supplemented with 1 μg / ml insulin, 0.5 mM 3-isobutyl-1-methylxanthine, 1 μM dexamethasone (Sigma-Aldrich, St. Louis, Mo., USA) and 10% fetal bovine serum (MDI). ≪ / RTI >

On the third day of culture, the initial culture medium was replaced with the progressive culture medium (1 μg / ml insulin DMEM supplemented with 10% fetal bovine serum), and on the sixth day of culture, the progressive medium was replaced with maintenance culture medium (DMEM supplemented with 10% fetal bovine serum) Respectively. The cells were treated with 0, 5, 10 and 15 μM of αAL14 at 0 to 6 days of culture.

Example 3: Oil-red O staining

Oil-red O staining was performed to measure the fat accumulation of cultured cells according to one embodiment of the present invention.

Specifically, αAL14 was treated with the differentiation medium of 3T3-L1 cells from 0 to 6 days of culture, and the differentiation medium was replaced with a culture medium containing insulin every 2 days. On the 6th day of culture, the cells were stained using Oil-red O (Sigma-Aldrich) staining reagent. First, the cells were fixed on 10% formaldehyde (Junsei Chemical Co., Ltd., Tokyo, Japan) and stained with filtered Oil-red O staining solution (60% isopropanol and 40% distilled water) for 30 minutes. After staining lipid droplets, the Oil-red O staining solution was removed and the plates were washed and dried. The stained fat granules were visualized by an inverted microscope (magnification, × 200).

As a result, lipid granules were not observed in undifferentiated 3T3-L1 cells, but many fat granules were observed in fully differentiated cells treated with differentiation medium (MDI). The presence of fat granules was used as a marker of fat accumulation and the amount of fat granules decreased by αAL14 in a concentration dependent manner. Thus, treatment with 15 μM, the highest concentration of αAL14, strongly inhibited the differentiation of 3T3-L1 cells and prevented fat accumulation (FIG. 1).

Example 4: Measurement of triglyceride

The triglyceride of 3T3-L1 cells was measured to observe the effect of alpha AL14 on the accumulation of cellular triglyceride according to one embodiment of the present invention.

Specifically, cells treated with αAL14 for 6 days in differentiated 3T3-L1 cells were washed with PBS and homogenized by sonication for 1 minute. Total triglycerides in cell lysates were measured using a commercially available triglyceride quantification kit (BioVision, Inc., CA, USA) according to the manufacturer's instructions.

As a result, αAL14 inhibited intracellular triglyceride accumulation of 3T3-L1 adipose precursor cells and decreased triglyceride contents under a concentration-dependent condition (FIG. 2).

From the above results, it can be seen that the αAL14 peptide according to an embodiment of the present invention inhibits accumulation of intracellular triglyceride in lipoprotein cells, and thus it can be very usefully used for prevention and treatment of obesity. Therefore, the peptide according to an embodiment of the present invention may be useful as a pharmaceutical composition for preventing or treating obesity or a health functional food for obesity improvement.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims. Accordingly, the true scope of the present invention should be determined by the technical idea of the appended claims.

<110> Pukyong National University Industry-University Cooperation Foundation <120> A novel pharmaceutical composition for prevention and treating          obesity <130> PD14-5111 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> alpha AL14 <400> 1 Ala Ala Trp Lys Leu Leu Lys Ala Leu Ala Lys Ala Ala Leu   1 5 10

Claims (7)

1. A pharmaceutical composition for the prevention and treatment of obesity comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient. The method according to claim 1,
Wherein the C-terminal of the peptide is an amino group or a carboxyl group. The method according to claim 1,
A pharmaceutical composition for the prevention and treatment of obesity, which further comprises a pharmaceutically acceptable carrier. 1. A health functional food for improving obesity comprising a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 as an active ingredient. 5. The method of claim 4,
Wherein the C-terminal of the peptide is an amine group or a carboxyl group.
6. The method of claim 5,
A health functional food for obesity improvement, further comprising at least one food additive. A feed additive for preventing and improving animal obesity, which contains, as an active ingredient, a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1.

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