KR20210052382A - Peptide(PEPTIDE 8) for anti-obesity and use thereof - Google Patents

Abstract

The present invention relates to a peptide exhibiting anti-obesity efficacy or an anti-obesity composition containing the same. According to the present invention, the composition has showed a significant differentiation inhibitory effect on adipocytes, and thus in the prevention or treatment of obesity, it is expected that a more fundamental approach and target treatment can be performed.

Description

Peptide (Peptide 8) having anti-obesity effect and its use {Peptide (PEPTIDE 8) for anti-obesity and use thereof}

The present invention relates to a peptide exhibiting anti-obesity efficacy and a use thereof.

Obesity is caused by abnormal hypertrophy of the subcutaneous fat tissue caused by the accumulation of excess energy in the body when an imbalance in metabolic processes occurs due to endocrine factors, genetic factors, social and environmental factors, etc. Adipose tissue hypertrophy is a phenomenon in which the size of adipocytes increases (adipocyte hypertrophy) or the number of adipocytes increases (adipocyte hyperplasia), which also affects the stagnation of the local venous-lymphatic system, leading to vascular tissue disease in the dermis-subcutaneous tissue. It can be triggered. Therefore, obesity is recognized as an independent disease by itself, and the World Health Organization treats obesity as a global nutritional problem and recognizes it as a disease to be treated, not a simple risk factor to harm health.

Currently, obesity drugs can be classified into drugs that affect appetite by acting on the central nervous system and drugs that inhibit absorption by acting on the gastrointestinal tract, depending on the mechanism of action. Drugs acting on the central nervous system include fenfluramine and dexfenfluramine, which inhibit serotonin (5-HT) reuptake, ephedrine and caffeine, which inhibit noradrenaline reuptake, and recently, sibutramine, which simultaneously acts on serotonin and noradrenaline nervous system. have. As a drug that acts on the gastrointestinal tract and inhibits obesity, there is orlistat, which is licensed as a treatment for obesity by inhibiting lipase produced in the pancreas and reducing the absorption of fat. However, among the previously used drugs, fenfluramine, an appetite suppressant, was not only classified as a narcotic component, but also was banned because it caused side effects such as primary pulmonary hypertension or heart valve lesions. It can be used in patients with heart failure and kidney disease. Orlistat, a lipolysis inhibitor, also causes gastrointestinal side effects, causing discomfort and inhibiting the absorption of fat-soluble vitamins.Sibutramine, approved by the US FDA for long-term obesity treatment, is marketed because it causes cardiovascular diseases such as heart palpitations or dizziness. This was banned.

One of those that have been suggested as an alternative for reducing the side effects of these drugs and for more fundamental prevention and inhibition of obesity is an adipocyte differentiation inhibitor. Currently identified secreted substances include TNF-α, IL-6, IL-8, plasminogen activator inhibitor-1, angiotensin-II, leptin and adiponectin, but these are various metabolic diseases and cardiovascular It is known to cause disease. Energy storage and secretion of endocrine substances in adipose tissue are closely related to the differentiation and growth of adipocytes, and occur simultaneously with the adipogenesis process. Adipocyte growth is a process in which non-large adipocytes are formed by accumulation of triglycerides in differentiated adipocytes without changing the number of adipocytes. Obesity due to the increase of non-large adipocytes is frequent in adults and is controlled by diet. It is possible.

Under this technical background, research on a new anti-obesity substance is being actively conducted (Korean Patent Registration No. 10-1662062), but it is still insufficient.

It is an object of the present invention to provide a composition for preventing or treating obesity comprising a peptide exhibiting anti-obesity efficacy and the same as an active ingredient.

In addition, another object of the present invention is to provide a composition for external application for skin for preventing or improving obesity comprising the peptide as an active ingredient.

However, the technical problem to be achieved by the present invention is not limited to the problems mentioned above, and other problems that are not mentioned will be clearly understood by those skilled in the art from the following description.

The present invention was conceived to solve the above problems, and the present inventors synthesized peptides having no cytotoxicity and exhibiting anti-obesity efficacy and confirmed their significant adipocyte differentiation inhibitory effect, that is, anti-obesity effect. Was completed.

Accordingly, the present invention provides a peptide exhibiting anti-obesity efficacy.

The term "obesity" as used herein means excessive accumulation of body fat in the body.

Specifically, the present invention may be a peptide consisting of one of the amino acid sequences represented by SEQ ID NO: 1 to SEQ ID NO: 23 showing anti-obesity activity.

The term “peptide” as used herein refers to a linear molecule formed by bonding amino acid residues to each other by peptide bonds. The peptides of the present invention are chemically synthesized methods known in the art, in particular solid-phase synthesis techniques; Merrifield, J. Amer. Chem. Soc. 85:2149-54 (1963); Stewart, et al., Solid Phase Peptide Synthesis, 2nd.ed., Pierce Chem. Co.: Rockford, 111 (1984)) or liquid phase synthesis technology (US Patent No. 5,516,891), but is not limited thereto, and known A peptide synthesis method can be used.

The peptide of the present invention may select a part of the amino acid sequence and induce a modification at the N-terminus or C-terminus in order to increase its activity. Through this modification, the peptide of the present invention can have a high half-life by increasing the half-life when administered in vivo.

In addition, the C-terminus of the peptide of the present invention _{is modified with a hydroxy group (-OH), an amino group (-NH 2} ), an azide (-NHNH ₂ ), and the like, and the N-terminus of the peptide is an acetyl group, a fluorenyl group. A protecting group selected from the group consisting of oxycarbonyl group, formyl group, palmitoyl group, myristyl group, stearyl group and polyethylene glycol (PEG) may be bonded.

The above-described amino acid modification acts to greatly improve the stability of the peptide of the present invention. In the present specification, the term “stability” means not only in vivo stability, but also storage stability (eg, room temperature storage stability). The above-described protecting group functions to protect the peptide of the present invention from attack by protein cleavage enzymes in vivo.

In addition, the present invention provides a pharmaceutical composition for preventing or treating obesity comprising the peptide as an active ingredient.

In addition, the present invention provides a composition for external application for skin for preventing or improving obesity comprising the peptide as an active ingredient.

Since the contents related to the peptide in the composition are the same as described above, descriptions of overlapping contents will be omitted.

The composition can be locally reduced by applying directly to topical areas with a lot of subcutaneous fat such as the face, abdomen, thighs, buttocks, arms, etc., and formulated in the form of a skin external preparation suitable for this purpose. However, the formulation is not particularly limited. In addition, in the composition according to an aspect of the present invention, the composition may reduce fat in the body by drinking or taking it for oral administration, and may be a liquid dosage form for oral administration to suit this purpose. In addition to the above ingredients, the composition of the present invention is appropriately formulated with auxiliary ingredients generally used in the field, and is used as a dietary supplement or medicine in formulations such as tablets, capsules, soft capsules, pills, granules, and drinks. Can be used.

The present invention relates to a peptide exhibiting anti-obesity efficacy and an anti-obesity composition comprising the same as an active ingredient. According to the present invention, the peptide and the composition comprising the same exhibited a significant differentiation inhibitory effect on adipocytes, and thus, in the prevention or treatment of obesity, it is expected that a more fundamental approach and target treatment can be performed.

1 is a result of confirming the change in the differentiation rate and fat accumulation from 3T3-L1 adipocytes to adipocytes according to the treatment of peptides.
2 shows the results of a cytotoxicity test by measuring the survival rate of 3T3-L1 adipocytes according to the treatment of peptides.

Hereinafter, a preferred embodiment is presented to aid the understanding of the present invention. However, the following examples are provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

[Experimental Example]

Experimental Example 1. Experimental materials and experimental methods

Synthesis of 1-1 peptide

The peptide was requested to be synthesized by Peptron Co., Ltd. and 23 kinds of peptide samples were obtained. The specific peptide synthesis method is as follows. Peptide synthesis was performed using an organic chemical reaction, using a solid Fmoc Chemistry method through a continuous flow condition. This is a method of synthesizing each amino acid one by one in a resin according to a predetermined sequence. Peptron Co., Ltd. synthesized 48 different peptides at the same time using an automatic synthesizer developed by Peptron Co., Ltd. The synthesized Crude peptide was purified using reverse phase HPLC and dried using a lyophilizer (Table 1).

Sequence number Amino acid sequence information Chain length Molecular composition Molecular Weight
(Da) One KLPFQR
(Lys-Leu-Pro-Phe-Gln-Arg) 6 C37H61N11O8 787 2 STELLIR (Ser-Thr-Glu-Leu-Lue-Ile-Arg) 7 C36H66N10O12 830 3 EIAQDFK (Glu-Ile-Ala-Gln-Asp-Phe-Lys) 7 C38H59N9O13 849 4 HLQLAIR (His-Leu-Gln-Leu-Ala-Ile-Arg) 7 C38H67N13O9 849 5 AVQGLLK (Ala-Val-Gln-Gly-Leu-Leu-Lys) 7 C33H61N9O9 727 6 IAQGGVLP (Ile-Ala-Gln-Gly-Gly-Val-Leu-Pro) 8 C34H59N9O10 753 7 NDEELNKLL (Asn-Asp-Glu-Glu-Leu-Asn-Lys-Leu-Leu) 9 C46H78N12O18 1,086 8 AGLQFPVGR (Ala-Gly-Leu-Gln-Phe-Pro-Val-Gly-Arg) 9 C43H69N13O11 943 9 YRPGTVALR (Tyr-Arg-Pro-Gly-Thr-Val-Ala-Leu-Arg) 9 C46H77N15O12 1,031 10 KSTGGKAPR (Lys-Ser-Thr-Gly-Gly-Lys-Ala-Pro-Arg) 9 C37H68N14O12 900 11 KQLATKAAR (Lys-Gln-Leu-Ala-Thr-Lys-Ala-Ala-Arg) 9 C42H79N15O12 985 12 RFQSSAVMA (Arg-Phe-Gln-Ser-Ser-Ala-Val-Met-Ala) 9 C42H69N13O13S1 995 13 TLSDYNIQK (Thr-Leu-Ser-Asp-Tyr-Asn-Ile-Gln-Lys) 9 C47H76N12O17 1,080 14 NKLLSGVTIA (Asn-Lys-Leu-Leu-Ser-Gly-Val-Thr-Ile-Ala) 10 C45H82N12O14 1,014 15 EIAQDFKTDL (Glu-Ile-Ala-Gln-Asp-Phe-Lys-Thr-Asp-Leu) 10 C52H82N12O19 1,178 16 ALNQAWAFLK
(Ala-Leu-Asn-Gln-Ala-Trp-Ala-Phe-Leu-Lys) 10 C56H84N14O13 1,160 17 AERVGAGAPVY Ala-Glu-Arg-Val-Gly-Ala-Gly-Ala-Pro-Val-Tyr 11 C48H76N14O15 1,088 18 RIVDFHMLESR Arg-Ile-Val-Asp-Phe-His-Met-Leu-Glu-Ser-Arg 11 C61H99N19O17S1 1,401 19 SGVTIAQGGVLP
Ser-Gly-Val-Thr-Ile-Ala-Gln-Gly-Gly-Val-Leu-Pro 12 C48H83N13O16 1,097 20 APRKQLATKAAR Ala-Pro-Arg-Lys-Gln-Leu-Ala-Thr-Lys-Ala-Ala-Arg 12 C56H103N21O15 1,309 21 QNIIPASTGAAK
Gln-Asn-Ile-Ile-Pro-Ala-Ser-Thr-Gly-Ala-Ala-Lys 12 C50H87N15O17 1,169 22 HLQLAIRNDEELNK (His-Leu-Gln-Leu-Ala-Ile-Arg-Asn-Asp-Glu-Glu-Leu-Asn-Lys) 14 C72H121N23O24 1,691 23 VTIAQGGVLPNIQA (Val-Thr-Ile-Ala-Gln-Gly-Gly-Val-Leu-Pro-Asn-Ile-Gln-Ala) 14 C61H105N17O19 1,379

1-2 Cell culture and fat measurement method

The mast cell 3T3-L1 cell line was pre-sold and used by ATCC. Penicillin (100 U/mL) and streptomycin (100 μg/mL) were added to DMEM containing 10% calf serum at 37°C, 5%, Incubated and maintained in CO _2. For the differentiation of 3T3-L1 adipocytes into adipocytes, cells of 5×10 ⁵ cells/well were dispensed into 6 well plates and cultured so that the cells were sufficiently concentrated. After 2 days, differentiation was initiated by incubating for 2 days in DMEM containing 0.5 mM isobutylmethylxanthine (IBMX), 1 μM dexamethasone, 10 μg/mL insulin (MDI solution), and 10% FBS, followed by 10 μg/mL insulin and Differentiation was performed for 3 days by exchange with DMEM containing 10% FBS (fetal bovine serum). After that, the cells were differentiated into adipocytes that form lipid droplets by culturing in DMEM containing only 10% FBS, and the amount of fat was confirmed using Oil Red O. Specifically, after differentiation, the medium was removed and the cells were fixed with 10% formaldehyde solution. After fixing at room temperature for 30 minutes, the solution was removed, washed twice with PBS, washed twice with 70% alcohol, and stained using Oil Red O solution. After observation under a microscope, the stained cells were dissolved using a solution containing 4% NP-40 in isopropyl alcohol, and the absorbance was measured at 510 nm.

1-3 Method of measuring cytotoxicity

3T3-L1 cells (5×105 cells) were dispensed into each well of a 96-well microplate, and a peptide sample was treated at a concentration of 62.5 μg/mL in each well containing 3T3-L1 cells, and the cells were cultured for 24 hours. After that, 50 μL of MTT (1.0 mg/ml) solution was treated in each well and incubated for 4 hours. The cultured cells were centrifuged (3000rpm, 10min, 20℃) to remove the supernatant, and treated with 200 μL of dimethylsulfoxide (DMSO) solution to dissolve the resulting formazan crystal by pipetting, and after 10 minutes, a 96-well microplate reader was used. The absorbance was measured at 540 nm by using. At this time, the result analysis was calculated as a percentage of the average absorbance of the control group by obtaining the average absorbance of the insect sample group.

1-4 Peptide anti-obesity efficacy animal test evaluation (high fat diet mouse feeding experiment)

C57BL/6 mice, 4 weeks old, were purchased at Nara Bio Animal Center (Nara Biotech, Korea, Seoul) and tested while rearing them in disease-free plastic cages. The animal room environment was controlled with a 12-hour dark lighting cycle and 40-45% relative humidity at 20-21°C. All experiments were approved by the Animal Protection and Use Committee of Konkuk University.

One week after adaptation, the mice were randomly divided into two groups, the control group and the experimental group, the control group fed a normal diet with 8 subjects (control, 10% kcal fat content), and the experimental group fed a high-fat diet with 30 subjects (HFD, 60 % kcal fat content). The rats' food intake was recorded daily, and the rats' body weight was measured twice weekly. After 5 weeks, mice whose body weight was 20% higher than that of the control group were selected and randomly divided into three groups (n = 8/group). HFD (high fat diet) mice treated with saline, HFD + peptide (SEQ ID NO: 8 or 15, 100 mg / kg), HFD + Orlistat (Orlistat, 60 mg / kg) treated with orlistat was used as a positive control. After 12 weeks, after one day fasting, they were slaughtered and investigated.

Blood samples were taken for further analysis. Adipose tissue (cortex, subcutaneous, visceral, liver) was weighed, and extradermal adipose tissue and liver tissue were collected, frozen in liquid nitrogen, and stored at -80 °C until further analysis. For histological analysis, percutaneous fat and liver tissue were fixed in 10% formaldehyde.

1-5 body fat composition analysis

In order to compare body fat, dual-energy X-ray absorptiometry (DXA) was conducted, and bone and tissue were measured once in low energy and once in high energy using a whole body scanner in the fourth week of the experiment. Fat and lean body weight were calculated.

1-6 Histological Analysis

Liver and epidural adipose tissue were dissected and fixed with 10% netural buffered formalin, and paraffin was embedded for biopsy. The formalin-fixed and paraffin-embedded tissue was cut into 4 μm thick and stained with hematoxylin and eosin (H&E). The frozen liver area was fixed with 10% formaldehyde solution for 10 minutes and then rinsed with running water. After washing with distilled water for a few seconds, soak in isopropanol solution for 20-30 seconds, dye with oil red O reagent for 15-20 minutes, and rinse with distilled water. Finally, after staining with hematoxylin for 40 seconds, it was immersed in tap water for 5 minutes. Liver tissue was photographed at 200 times or less, and adipocytes were analyzed using a microscope.

1-7 biological analysis of blood

Blood samples were obtained from the heart of mice, and serum was separated by centrifugation (3000 rpm per 20 minutes) and stored at -80°C until further analysis. Automatic analysis of serum levels of blood urea nitrogen, alanine aminotransferase (ALT), alkaline phosphate, aspartate aminotransferase (AST), total protein, serum albumin, and electrolytes (Abaxis VETSVAN VS2 Chemical Analyzer, CA, USA) It was measured as. Total cholesterol, low-density lipoprotein cholesterol (LDL), high-density lipoprotein cholesterol (HDL), and TG were measured with a high-speed blood lipid analyzer (OSAN Healthcare Lipid Pro, Anyang City, Korea). Adiponectin was analyzed by western blotting, and leptin was quantified using a leptin enzyme-linked immuno-oncology (ELISA) kit (MERCK, DHB, Germany).

[Example]

Example 1. Confirmation of anti-obesity efficacy (In vitro)

In order to evaluate the anti-obesity effect of the peptide according to the present invention, after treating a total of 23 kinds of peptide samples disclosed in Experimental Example 1-1 on 3-L1 adipocytes, the formation of lipid droplets according to the oil red O test method It was confirmed through. On the other hand, a separate peptide treatment was not performed as a control group (hereinafter, the control group is referred to as an untreated group).

As a result, it was possible to confirm the experimental results in Table 2 below or FIG. 1.

Adipocyte differentiation rate (%) CON. 99.8 ± 0.36 One 57.9 ± 0.60 2 59.0 ± 0.60 3 52.2 ± 0.72 4 63.3 ± 0.12 5 48.4 ± 0.36 6 38.0 ± 0.72 7 39.4 ± 0.36 8 33.7 ± 0.84 9 40.6 ± 0.60 10 48.4 ± 0.36 11 94.3 ± 0.24 12 57.5 ± 0.60 13 68.0 ± 0.60 14 77.8 ± 0.24 15 30.2 ± 1.44 16 57.5 ± 0.60 17 35.1 ± 0.84 18 41.9 ± 0.24 19 68.0 ± 0.60 20 94.8 ± 0.60 21 44.4 ± 1.32 22 83.1 ± 1.80 23 74.4 ± 0.96

In the experiment treated with a sample concentration of 62.5 μg/mL, the growth of adipocytes was suppressed in most of the 23 samples. In the control group, adipocytes were differentiated at 99.8%, whereas 15 times (30.2%), 8 times (33.7%), 17 times (35.1%), 6 times (38%), and 7 times (39.4%) were 40%. Adipocytes differentiated below and showed excellent ability to inhibit differentiation of adipocytes. No. 9 (40.6%), No. 18 (41.9%), No. 21 (44.4%), No. 5 (48.4%), No. 10 (48.4%), No. 3 (52.2%), No. 12 (57.5%), No. 16 (57.5%), No. 12 (57.5%), No. 1 (57.9%), No. 2 (59%) account for 40~60% of adipocytes Showed hwaryul. Others 4 (63.3%), 13 (68%), 19 (68%), 23 (74.4%), 14 (77.8%), 22 (83.1%), 11 (94.3%) , No. 20 (94.8%) showed similar or slightly superior ability to inhibit adipocyte differentiation than the control.

Example 2. Confirmation of cytotoxicity of peptide samples

In order to evaluate the anti-obesity effect of the peptide according to the present invention, a total of 23 kinds of peptide samples disclosed in Experimental Example 1-1 were treated with 3-L1 adipocytes, and the survival rate of adipocytes was measured accordingly. On the other hand, a separate peptide treatment was not performed as a control group (hereinafter, the control group is referred to as an untreated group.)

As a result, it was possible to confirm the experimental results in Table 3 below or FIG. 2.

sample Cell viability (%) 24hr 48hr No treatment 100.0 ± 0.01 100.0 ± 0.01 1 (KLPFQR) 90.2 ± 0.95 81.5 ± 0.84 2 (STELLIR) 95.6 ± 0.35 81.3 ± 0.92 3 (EIAQDFK) 94.1 ± 0.94 84.7 ± 0.78 4 (HLQLAIR) 97.1 ± 1.38 91.0 ± 1.20 5 (AVQGLLK) 97.4 ± 0.93 89.9 ± 1.67 6 (IAQGGVLP) 96.0 ± 1.23 86.1 ± 1.60 7 (NDEELNKLL) 89.1 ± 0.69 82.6 ± 0.77 8 (AGLQFPVGR) 97.8 ± 0.49 87.6 ± 0.93 9 (YRPGTVALR) 91.7 ± 0.34 86.5 ± 0.19 10 (KSTGGKAPR) 90.6 ± 0.76 79.8 ± 1.43 11 (KQLATKAAR) 95.7 ± 0.67 86.7 ± 0.40 12 (RFQSSAVMA) 90.7 ± 1.14 74.7 ± 0.64 13 (TLSDYNIQK) 86.8 ± 0.15 80.3 ± 0.97 14 (NKLLSGVTIA) 93.8 ± 0.45 87.4 ± 1.12 15 (EIAQDFKTDL) 96.6 ± 0.26 82.7 ± 0.60 16 (ALNQAWAFLK) 109.6 ± 0.52 90.0 ± 0.42 17 (AERVGAGAPVY) 105.4 ± 0.59 88.0 ± 1.95 18 (RIVDFHMLESR) 89.7 ± 0.40 88.6 ± 0.53 19 (SGVTIAQGGVLP) 91.0 ± 1.99 83.1 ± 0.66 20 (APRKQLATKAAR) 95.0 ± 1.24 85.4 ± 0.91 21 (QNIIPASTGAAK) 91.7 ± 0.94 76.9 ± 0.94 22 (HLQLAIRNDEELNK) 94.7 ± 0.79 86.2 ± 0.70 23 (VTIAQGGVLPNIQA) 88.7 ± 0.30 76.5 ± 0.76

At a sample concentration of 62.5 μg/mL, 3T3-L1 adipocytes used in the analysis of obesity inhibitory activity were treated to perform cell toxicity tests. As a result of the experiment, the cell viability was 85% or higher until 24 hours after treatment, and 80% or higher even after 48 hours, and the toxicity of the sample to the cells was judged to be insignificant. Summarizing the above experimental results, the peptides of the present invention were found to be insignificant. Since each of them could effectively inhibit the differentiation of adipocytes, they could be used for prevention, improvement, or treatment of obesity.

Example 3. Changes in body composition of mice fed high fat nutrition

Changes in body weight and fat composition of mice fed high fat diet according to peptide feeding Treatment Weight (g/horse) Fat weight
(g/mari) Before the experiment Week 5 Increase Normal diet group (control) 22.37 28.82 6.45 4.0 High fat diet 22.95 43.16 20.21 ^## 18.2 ^## High fat diet+
Orlistat (positive control) 23.46 36.08 12.62 ^** 13.7 ^** High fat diet + peptide (SEQ ID NO: 8) 23.85 35.15 11.30 ^** 12.7 ^** High fat diet + peptide (SEQ ID NO: 15) 25.06 36.97 11.91 ^** 14.5 ^**

Significant differences at ^## p <0.01 compared with the Con group. Significant differences at ^* p <0.05, ^** p <0.01 compared with the HFD group.

As shown in Table 4, the weight gain rate increased by 6.45g for 5 weeks in the normal diet rats, while the rats fed the high-fat diet increased by 20.21g, and the rats ingested the high-fat diet and the positive control Orlistat at the same time were 12.62g compared to the high-fat diet. The weight gain inhibition rate was 37.6%. Rats taking both high-fat diet and peptide (SEQ ID NO: 8) at the same time showed a 44.1% weight gain inhibition rate with an increase of 11.3 g, and rats that consumed peptide (SEQ ID NO: 15) at the same time showed a 41.1% weight gain inhibition rate with an increase of 11.91 g. As shown, the obesity suppression effect was excellent in the group that consumed the peptide at the same time.

Composition of body fat in mice according to a high fat diet (dual energy x-ray absorption measurement method) Treatment Sieve composition (%) Fat Fat free Normal diet group (control) 13.9 80.4 High fat diet 41.7 53.6 High fat diet+
Orlistat (positive control) 36.9 59.0 High fat diet + peptide (SEQ ID NO: 8) 35.6 59.9 High fat diet + peptide (SEQ ID NO: 15) 38.5 57.3

As shown in Table 5, as a result of the analysis of the body fat composition of mice measured by dual energy x-ray absorption measurement, the fat composition was very high in 13.9% of the mice on a normal diet, while 41.7% of the mice on a high-fat diet, and a high fat diet. Rats that consumed the over-positive control, Orlistat at the same time, showed the effect of lowering the fat composition to 11.5% compared to the high-fat diet at 36.9%. Rats taking both a high-fat diet and peptide (SEQ ID NO: 8) at the same time had a fat composition of 35.6%, which lowered the fat composition by 14.6% compared to the high-fat diet. Rats that consumed the peptide (SEQ ID NO: 15) at the same time had a fat composition of 38.5%. As compared to the high-fat diet, it showed the effect of lowering fat composition by 7.7%, and the peptide showed the effect of suppressing obesity by suppressing the increase in fat composition.

Example 4. Blood Biological Analysis

Comparison of blood components in high-fat diet mice fed with peptides Treatment Changes in blood composition TG ^a
(mg/dL) TC ^b
(mg/dL) HDL ^c
(mg/dL) LDL/VLDL ^d
(mg/dL)
Normal diet group (control) 108.3 96.1 79.8 16.3
High fat diet 232.6 ^## 119.7 ^## 47.4 ^## 72.3 ^##
High fat diet+
Orlistat (positive control) 117.9 ^** 119.3 ^** 77.8 ^** 44.2 ^**
High fat diet+
peptide (SEQ ID NO: 8) 138.8 ^** 120.5 ^** 75.1 ^** 42.7 ^**
High fat diet+
peptide (SEQ ID NO: 15) 140.3 ^** 107.5 ^** 74.5 ^** 33.0 ^**

Significant differences at ^## p <0.01 compared with the Con group. Significant differences at ^* p <0.05, ^** p <0.01 compared with the HFD group.

^a triglyceride (TG), ^b total cholesterol (TC), ^c high density lipoprotein (HDL), ^d low density lipoprotein (LDL)

As shown in Table 6, the triglyceride (TG) of the mice reared on a high fat diet is 232.6 mg/dL, the total cholesterol (TC) is 119.7 mg/dL, the high-density lipoprotein (HDL) is 47.4 mg/dL, and the density Lipoprotein (LDL) was 72.3mg/dL. The triglyceride (TG) of 138.8mg/dL, total cholesterol (TC) of 120.5mg/dL, high-density lipoprotein (HDL) of 75.1mg/dL, density of mice that consumed both high-fat diet and peptide (SEQ ID NO: 8) at the same time. Lipoprotein (LDL) was 42.7mg/dL, which reduced triglycerides and low-density lipoproteins according to peptide intake, and increased HDL, which reduces vascular diseases such as arteriosclerosis.

In the case of mice that ingested a high-fat diet and peptide (SEQ ID NO: 15) at the same time, triglyceride (TG) was 140.3 mg/dL, total cholesterol (TC) was 107.5 mg/dL, and high-density lipoprotein (HDL) was 74.5 mg/dL, LDL was 33.0mg/dL, which decreased triglycerides, total cholesterol, and low-density lipoprotein, and HDL increased.

Example 5. Comparison of Histological Characteristics of High Fat Nutrient Intake Mice

<peptide (SEQ ID NO: 8)>

Mice fed on a high fat diet increased their liver size when compared to the normal diet group (FIG. 3A) and increased the amount of white adipose tissue (FIG. 3B ), and increased red adipose tissue when stained with oil red o (FIG. 3C ). , It can be seen that the size of the fat is also very large compared to the normal mice (Fig. 3D). Mice fed both high-fat diet and positive control orlistat were significantly smaller in size compared to those in the high-fat diet group, and the amount of white adipose tissue was also small, and red fat cells were relatively small even when stained with oilred o. It can be seen that the size is also relatively small. Similar to the group fed with peptide 8 at the same time as the orilistat intake group, the shape and size of the group compared to the high fat diet group were close to normal, and the number of white adipose tissues and the size of fat were also close to normal. Showed.

<peptide (SEQ ID NO: 15)>

The group fed with the high fat diet and peptide (SEQ ID NO: 15) at the same time also suppressed the increase in fat so that the shape and size of the group compared with the high fat diet group were close to normal, and the number of white adipose tissues and the size of fat were also close to normal. One effect was shown (FIG. 4).

<110> Republic of Korea <120> Peptide (PEPTIDE 8) for anti-obesity and use thereof <130> P20G19C2081 <150> KR 10-2019-0136552 <151> 2019-10-30 <160> 23 <170> KoPatentIn 3.0 <210> 1 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> peptide 1 <400> 1 Lys Leu Pro Phe Gln Arg 1 5 <210> 2 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> peptide 2 <400> 2 Ser Thr Glu Leu Leu Ile Arg 1 5 <210> 3 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> peptide 3 <400> 3 Glu Ile Ala Gln Asp Phe Lys 1 5 <210> 4 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> peptide 4 <400> 4 His Leu Gln Leu Ala Ile Arg 1 5 <210> 5 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> peptide 5 <400> 5 Ala Val Gln Gly Leu Leu Lys 1 5 <210> 6 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> peptide 6 <400> 6 Ile Ala Gln Gly Gly Val Leu Pro 1 5 <210> 7 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> peptide 7 <400> 7 Asn Asp Glu Glu Leu Asn Lys Leu Leu 1 5 <210> 8 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> peptide 8 <400> 8 Ala Gly Leu Gln Phe Pro Val Gly Arg 1 5 <210> 9 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> peptide 9 <400> 9 Tyr Arg Pro Gly Thr Val Ala Leu Arg 1 5 <210> 10 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> peptide 10 <400> 10 Lys Ser Thr Gly Gly Lys Ala Pro Arg 1 5 <210> 11 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> peptide 11 <400> 11 Lys Gln Leu Ala Thr Lys Ala Ala Arg 1 5 <210> 12 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> peptide 12 <400> 12 Arg Phe Gln Ser Ser Ala Val Met Ala 1 5 <210> 13 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> peptide 13 <400> 13 Thr Leu Ser Asp Tyr Asn Ile Gln Lys 1 5 <210> 14 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> peptide 14 <400> 14 Asn Lys Leu Leu Ser Gly Val Thr Ile Ala 1 5 10 <210> 15 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> peptide 15 <400> 15 Glu Ile Ala Gln Asp Phe Lys Thr Asp Leu 1 5 10 <210> 16 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> peptide 16 <400> 16 Ala Leu Asn Gln Ala Trp Ala Phe Leu Lys 1 5 10 <210> 17 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> peptide 17 <400> 17 Ala Glu Arg Val Gly Ala Gly Ala Pro Val Tyr 1 5 10 <210> 18 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> peptide 18 <400> 18 Arg Ile Val Asp Phe His Met Leu Glu Ser Arg 1 5 10 <210> 19 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> peptide 19 <400> 19 Ser Gly Val Thr Ile Ala Gln Gly Gly Val Leu Pro 1 5 10 <210> 20 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> peptide 20 <400> 20 Ala Pro Arg Lys Gln Leu Ala Thr Lys Ala Ala Arg 1 5 10 <210> 21 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> peptide 21 <400> 21 Gln Asn Ile Ile Pro Ala Ser Thr Gly Ala Ala Lys 1 5 10 <210> 22 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> peptide 22 <400> 22 His Leu Gln Leu Ala Ile Arg Asn Asp Glu Glu Leu Asn Lys 1 5 10 <210> 23 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> peptide 23 <400> 23 Val Thr Ile Ala Gln Gly Gly Val Leu Pro Asn Ile Gln Ala 1 5 10

Claims (7)

Peptide showing anti-obesity activity consisting of an amino acid sequence represented by SEQ ID NO: 8. A pharmaceutical composition for preventing or treating obesity comprising the peptide of claim 1. The pharmaceutical composition for preventing or treating obesity according to claim 2, wherein the composition further comprises a peptide consisting of one or more amino acid sequences of SEQ ID NOs: 1 to 7, and 9 to 15. A composition for external application for skin for preventing or improving obesity comprising the peptide of claim 1. The composition for topical skin application for preventing or improving obesity according to claim 4, wherein the composition further comprises a peptide consisting of one or more amino acid sequences of SEQ ID NOs: 1 to 7, and 9 to 15. A food composition for preventing or improving obesity comprising the peptide of claim 1. The food composition for preventing or improving obesity according to claim 6, wherein the composition further comprises a peptide consisting of one or more amino acid sequences of SEQ ID NOs: 1 to 7, and 9 to 15.

Images