KR102265418B1 - Composition for preventing or treating diabetes comprising pork rectum extract - Google Patents

Abstract

The present invention provides a pharmaceutical composition for preventing or treating diabetes and a health functional food composition comprising a pig makchang extract as an active ingredient. The composition contains a functional peptide having antioxidant activity, antidiabetic activity, etc., and can promote glucose consumption by participating in the regulation of energy metabolism as well as differentiation of adipocytes. It is possible to effectively prevent, ameliorate or treat type 2 diabetes that occurs.

Description

A composition for preventing or treating diabetes comprising an extract of pig makchang {COMPOSITION FOR PREVENTING OR TREATING DIABETES COMPRISING PORK RECTUM EXTRACT}

The present invention relates to a composition for preventing or treating diabetes comprising a pig makchang extract.

Diabetes mellitus is a type of metabolic disease in which the secretion of insulin, a hormone that regulates glucose metabolism, is insufficient or a normal function is not achieved. and symptoms. Diabetes mellitus is divided into type 1 and type 2, and type 1 diabetes, previously called 'juvenile diabetes', is a disease caused by the inability to produce insulin at all. Type 2 diabetes, which is relatively deficient in insulin, is characterized by insulin resistance.

Due to the recent westernization of dietary life, a low-fat diet is shifting to a high-fat diet, and due to a change in lifestyle, the obese population is gradually increasing due to the decreased amount of exercise compared to high nutritional intake. Obesity increases the insulin requirement, and as a result, the function of cells in the pancreas gradually declines, leading to diabetes. As a result, type 2 diabetes is increasing more than type 1 diabetes.

In addition to these problems, research on functional substances has been actively conducted as interest in health has recently increased. Functional peptides are also one of them, and many studies have been reported to reveal their activity by isolating them from foods such as soybeans, protein, milk, grains, meat, and fish.

A functional peptide is a low-molecular substance consisting of 2 to 20 amino acids and can be separated through chemical and enzymatic hydrolysis and processing. The efficacy of functional peptides is determined by the type and sequence of amino acids constituting the peptide. So far, antioxidant, anticancerogenic, antithrombotic, antimicrobial, blood pressure lowering (antihypertensive) and other effects have been reported.

For example, a representative functional peptide is collagen isolated from meat and fish, which has low nutritional value due to low essential amino acids, but has an antioxidant effect and angiotensin converting enzyme (Angiotensin) when hydrolyzed by trypsin enzyme. It has an inhibitory effect on converting enzyme (ACE), and in addition, it has been found that a fraction or hydrolyzate of hemoglobin in the blood has antibacterial activity.

As the efficacy of functional peptides has been revealed through these studies, peptides isolated from various foods have been reported. In particular, in the case of livestock products, various peptides are found not only in lean meat but also in by-products generated during slaughter and processing.

Korean Patent No. 10-1142373 (registered on April 26, 2012)

In order to solve the above problems, the present invention provides a composition for preventing or treating diabetes comprising a pig makchang extract.

The present invention provides a pharmaceutical composition for preventing or treating diabetes comprising a pig makchang extract as an active ingredient.

In addition, the present invention provides a health functional food composition for preventing or improving diabetes comprising a pig makchang extract as an active ingredient.

Pig makchang extract according to the present invention contains functional peptides having antioxidant activity, antidiabetic activity, etc., and can promote blood glucose consumption by participating in the regulation of energy metabolism as well as differentiation of adipocytes.

By utilizing the extract as a pharmaceutical composition or a health functional food composition, it is possible to effectively prevent, improve or treat type 2 diabetes, which is caused by a decrease in the availability of glucose in the blood due to increased insulin resistance or the like.

1 is a flowchart showing a process for producing a pig makchang extract according to an embodiment of the present invention.
Figure 2 is a graph of the cytotoxicity evaluation result of the pig makchang extract according to an experimental example of the present invention.
3 is a graph (B) analyzing the adipocyte differentiation process (A) and fat content according to an experimental example of the present invention.
4 shows changes in mRNA expression of adipocyte-related factors according to an experimental example of the present invention.
5 shows a change in the expression of adipocyte-related factors at the protein level according to an experimental example of the present invention.

Hereinafter, the present invention will be described in detail.

The present inventors have completed the present invention by extracting functional peptides from pig makchang, and confirming the antidiabetic effect from the extract.

As used herein, the term "pig intestine" is a pig's intestines, and it may mean the lower part of the rectum or rectum from the back part of the pig's large intestine to the anus, that is, the end of the large intestine near the anus, and "pig intestine" and can be used interchangeably.

As used herein, the term "extract" refers to a material obtained by extracting the components of pig makchang regardless of the extraction method, extraction solvent, extracted component or the form of the extract, and after extracting the material obtained by extracting the component of pig makchang It may include any material obtainable by processing or processing by other methods.

As used herein, "prevention" refers to any action that suppresses or delays the onset of diabetes or at least one symptom of diabetes by administration of the pharmaceutical composition or health functional food composition according to the present invention. Also included is treatment of a subject in remission of the disease to prevent or prevent recurrence.

As used herein, "treatment" refers to any action that improves or beneficially changes diabetes or at least one symptom of diabetes by administration of the pharmaceutical composition according to the present invention, such as alleviating, reducing, or eliminating the symptoms.

As used herein, "improvement" refers to any action that improves or beneficially changes the symptoms, such as alleviation, reduction, or disappearance of diabetes or at least one symptom of diabetes by ingestion of the health functional food composition according to the present invention do.

As used herein, the term "pharmaceutical composition" refers to a composition administered for a specific purpose, and for the purpose of the present invention, is administered to prevent or treat diabetes or at least one or more symptoms of diabetes.

As used herein, "health functional food" is a food manufactured and processed using raw materials or ingredients useful for the human body, and is a food with high medical and medical effects processed to efficiently exhibit bioregulatory functions in addition to nutrition supply. meaning and may be used interchangeably with terms known in the art, such as functional food.

The present invention provides a pharmaceutical composition for preventing or treating diabetes comprising a pig makchang extract as an active ingredient.

In the pharmaceutical composition according to the present invention, the pig makchang extract may be a hot water extract. More specifically, the hot water extract is 90 to 100 ° C., preferably by adding 4 to 6 times distilled water, preferably 5 times, distilled water of the precipitate to the precipitate produced by crushing and centrifuging the pig makchang It may be extracted at 95° C. for 10 to 15 hours, preferably for 12 to 13 hours. According to an embodiment of the present invention, the pig makchang extract is first to remove the fat of the pig makchang, washed several times, and then pulverized, and after separating the pulverized pig makchang by a centrifuge, the supernatant is removed from the precipitate 5 times its of distilled water was added and extracted at 95 °C for 12 hours, which was again filtered and centrifuged, followed by fractionation. In addition, the pig makchang extract may be prepared by drying, for example, it may be dried by a method such as hot air drying, reduced pressure drying, freeze drying, etc., preferably prepared by freeze drying.

In the pharmaceutical composition according to the present invention, the pig makchang extract may include one or more peptides selected from the group consisting of SEQ ID NOs: 1 to 7, and the peptides may have antidiabetic activity.

SEQ ID NO: 1: Leu-Leu-Thr-Leu-Lys (LLTLK)

SEQ ID NO: 2: Ala-Val-Arg-Pro-Glu-Glu (AVRPEE)

SEQ ID NO:3: Val-Leu-Glu-Asn-Arg (VLENR)

SEQ ID NO: 4: Glu-Leu-Val-Arg (ELVR)

SEQ ID NO: 5: Glu-Val-Arg-Leu (EVRL)

SEQ ID NO: 6: Leu-Ala-Pro-Ser-Thr-Met-Lys (LAPSTMK)

SEQ ID NO: 7: Asn-Leu-Lys-Arg (N(+.98)LKR)

The letters used in the peptide refer to the following amino acids according to standard abbreviation rules in the field of biochemistry.

A(Ala): alanine; C(Cys): cysteine; D(Asp):aspartic acid; E(Glu): glutamic acid; F(Phe): phenylalanine; G(Gly): glycine; H(His): histidine; I(IIe): isoleucine; K(Lys): lysine; L(Leu): leucine; M(Met): methionine; N(Asn): asparagine; O(Ply)pyrrolysine; P(Pro): proline; Q(Gln): glutamine; R(Arg): arginine; S(Ser): serine; T(Thr): threonine; U(Sec):selenocysteine, V(Val):valine; W(Trp): tryptophan; Y (Tyr): Tyrosine.

According to an experimental example of the present invention, peptides having antidiabetic activity generally have leucine, valine, tyrosine, and the like in common, and the peptides of SEQ ID NOs: 1 to 7 also include leucine, valine, and the like. , tyrosine, etc. are shown to contain many, and it can be said that it has antidiabetic activity.

The pharmaceutical composition according to the present invention can promote the consumption of blood glucose by inducing adipocyte differentiation. According to an experimental example of the present invention, it was found that in the sample treated with the porcine makchang extract, the formation of small adipocytes, which appeared in the process of differentiation into adipocytes, was actively induced by 3T3-L1 preadipocytes, and this increase in adipogenesis was analyzed to mean that the amount of glucose in plasma was consumed.

In addition, the pharmaceutical composition according to the present invention is at least one selected from the group consisting of GLUT4, PPARγ, adiponectin, FAS, C/EBPβ, C/EBPa, SREBP-1c, perilipin, FABP4, and ACC. expression can be increased. The GLUT4, PPARγ, adiponectin, FAS, C/EBPβ, C/EBPa, SREBP-1c, perilipin, FABP4, and ACC are factors related to adipocyte differentiation and energy metabolism, According to an experimental example, when the change in the mRNA expression of the factor through RT-PCR and the protein expression change of the factor through western blot were confirmed, the expression of the factor in the sample treated with the pig makchang extract was found to increase significantly. Through this, it was analyzed that the pig makchang extract was involved in adipocyte differentiation as well as energy metabolism to promote blood glucose consumption. Due to these effects, the pig makchang extract can be used as a pharmaceutical composition for preventing or treating diabetes. More details will be described later by the following experimental examples.

In the pharmaceutical composition according to the present invention, the diabetes mellitus type 1 diabetes (insulin dependent diabetes mellitus, IDDM), which is a form of diabetes caused by an insufficient absolute amount of insulin, insulin secretion function is partially left, but various causes Type 2 diabetes (Noninsulin dependent diabetes mellitus, NIDDM) caused by relatively increased insulin resistance by Endocrine diseases, certain drugs, chemicals, insulin or insulin receptor abnormalities, diabetes mellitus secondary to a genetic syndrome, etc. may include, preferably insulin resistance (insulin resistance) increase, that is, insulin function to lower blood sugar This could be type 2 diabetes, which is caused by the cells failing to burn glucose effectively.

The pharmaceutical composition according to the present invention may be prepared according to a conventional method in the pharmaceutical field.

The pharmaceutical composition according to the present invention may be combined with an appropriate pharmaceutically acceptable carrier according to the formulation, and if necessary, excipients, diluents, dispersants, emulsifiers, buffers, stabilizers, binders, disintegrants, solvents, etc. can be manufactured.

The "pharmaceutically acceptable" means that there is no toxicity to cells or humans exposed to the pharmaceutical composition, and the appropriate carrier does not inhibit the activity and properties of the pig makchang extract according to the present invention, and administration It may be selected differently depending on the form and formulation.

The pharmaceutical composition according to the present invention may be applied in any dosage form, and more specifically, it may be formulated and used in parenteral dosage forms of oral dosage forms, external preparations, suppositories and sterile injection solutions according to conventional methods.

The solid dosage form among the oral dosage forms is in the form of tablets, pills, powders, granules, capsules, etc., and at least one or more excipients, for example, starch, calcium carbonate, sucrose, lactose, sorbitol, mannitol, cellulose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc may be included. In addition, the capsule formulation may further include a liquid carrier such as fatty oil in addition to the above-mentioned substances.

Among the oral dosage forms, liquid formulations include suspensions, solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients, for example, wetting agents, sweeteners, fragrances, preservatives, etc. may be included. have.

The parenteral formulation may include a sterile aqueous solution, a non-aqueous solution, a suspension, an emulsion, a freeze-dried formulation, and a suppository. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, Tween 61, cacao butter, laurin fat, glycerogelatin, and the like can be used. Without being limited thereto, any suitable agent known in the art may be used.

In addition, the pharmaceutical composition according to the present invention _{may further add calcium or vitamin D 3} and the like to enhance therapeutic efficacy.

In the pharmaceutical composition according to the present invention, the pharmaceutical composition may be administered in a pharmaceutically effective amount. The "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment and not to cause side effects.

The effective dose level of the pharmaceutical composition is determined by the purpose of use, the age, sex, weight and health status of the patient, the type of disease, severity, drug activity, sensitivity to drug, administration method, administration time, administration route and excretion rate, treatment It may be determined differently depending on factors including the duration, formulation or concurrent use of drugs and other factors well known in the medical field. For example, although not constant, generally 0.001 to 100 mg/kg, preferably 0.01 to 10 mg/kg, may be administered once to several times a day. The above dosage does not limit the scope of the present invention in any way.

The pharmaceutical composition according to the present invention may be administered to any animal capable of developing diabetes, and the animal may include, for example, not only humans and primates, but also livestock such as cattle, pigs, horses, and dogs.

The pharmaceutical composition according to the present invention may be administered by an appropriate administration route according to the form of the formulation, and may be administered through various routes, either oral or parenteral, as long as it can reach the target tissue. The administration method is not particularly limited, and for example, oral, rectal or intravenous, intramuscular, subcutaneous, endobronchial inhalation, intrauterine dural or intracerebroventricular injection, etc. can be administered in a conventional manner. .

The pharmaceutical composition according to the present invention may be used alone for preventing or treating diabetes, or may be used in combination with surgery or other drug treatment.

In addition, the present invention provides a health functional food composition for preventing or improving diabetes comprising a pig makchang extract as an active ingredient. As can be seen in an experimental example of the present invention, the composition can induce adipocyte differentiation and promote blood glucose consumption, and GLUT4, PPARγ, adiponectin, FAS, C/EBPβ, C/EBPa, SREBP -1c, perilipin (Perilipin), FABP4, and can promote the consumption of blood glucose by increasing the expression of one or more selected from the group consisting of adipocyte differentiation and energy metabolism, such as for preventing or improving diabetes It can be used as a health functional food composition.

In the health functional food composition according to the present invention, the health functional food may be prepared in powder, granule, tablet, capsule, syrup or beverage, etc., and there is no limitation in the form that the health functional food can take, It can include any food with meaning. For example, beverages and various drinks, fruits and their processed foods (canned fruit, jam, etc.), fish, meat and their processed foods (ham, bacon, etc.), breads and noodles, cookies and snacks, dairy products (butter, cheese, etc.) ), etc. are possible, and may include all functional foods in a conventional sense. It may also include food used as feed for animals.

The health functional food composition according to the present invention may be prepared by further including pharmaceutically acceptable food additives and other suitable auxiliary ingredients commonly used in the art. For example, flavoring agents, natural carbohydrates, sweetening agents, vitamins, electrolytes, coloring agents, pectic acid, alginic acid, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents, etc. can In particular, as the natural carbohydrate, monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol can be used. , as the sweetener, natural sweeteners such as taumatine and stevia extract or synthetic sweeteners such as saccharin and aspartame may be used.

The health functional food composition according to the present invention has the advantage that there are no side effects that may occur when taking the drug for a long period of time by using food as a raw material, unlike general drugs, and has excellent portability, so that it can be ingested as an adjuvant for preventing or improving diabetes. can

Hereinafter, to help the understanding of the present invention, examples will be described in detail. However, the following examples are merely illustrative of the contents of the present invention, and the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art.

<Example 1> Preparation of pork makchang extract

1 is a flowchart showing a process for producing a pig makchang extract according to an embodiment of the present invention. Referring to Figure 1, after removing the fat of pork makchang supplied from 'Dalguji Food', it was first washed with 0.1N sodium hydroxide (NaOH), and then washed with phosphate buffered saline (hereinafter, PBS). Then, an equal amount (1:1) of PBS was added and the mixture was pulverized using a pulverizer (HMF-3800SS, Hanil, Korea). The pulverized solution was separated using a centrifuge (Centrifuge 5810 R, Eppendorf, Germany) at 9000 g, 10 min, 4°C conditions. After removing the supernatant, 5 times the amount of distilled water was added to the precipitate, followed by extraction at 95°C for 12 hours. After filtration, the extract was centrifuged again (9000g, 10min, 4℃) to obtain a supernatant, which was fractionated using Sephadex G-200, and then freeze-dried to prepare a final pig makchang extract sample.

<Experimental Example 1> Analysis of total protein content of pork makchang extract

A protein is a component of a living body, and is a high molecular weight organic substance made by concatenating several amino acids in a chain. A peptide is a group of compounds in which two or more amino acids are bound by a peptide (peptide) bond, and a protein having 50 or more amino acid residues is called a protein. There are various peptides in living organisms, and some exhibit physiological activities such as metabolic regulation or hormone action. Food-derived proteins and peptides have functions such as antioxidant, antibacterial action, immune regulation, blood pressure lowering, bone protection, and anti-hyperlipidemia. In particular, peptides isolated from meat by-products have a blood pressure lowering effect.

The total protein contents of the pig makchang extract prepared according to Example 1 were measured using a Pierce BCA protein assay kit [Pierce BCA (bicinchoninic acid) Protein assay kit, Thermo Fisher Scientific, USA]. The concentration of the sample was 10 mg/ml, and the absorbance was measured at 562 nm. Total protein content was calculated from a standard curve using bovine serum albumin (BSA).

Total protein concentration (mg/mg) PRE 0.33±0.01

Table 1 is the total protein content of the pig makchang extract according to an experimental example of the present invention. Referring to Table 1, the protein concentration of the porcine makchang extract measured by the BCA method was about 0.33 mg/mg, and it was found that the porcine makchang extract contained more than 30% protein.

<Experimental Example 2> Amino acid analysis of pork makchang extract

The pig makchang extract prepared according to Example 1 was diluted with 70% ethanol to make 15 mL at a concentration of 20 mg/mL, and then 20% trichloroacetic acid (hereinafter, TCA, Sigma Aldrich) 15 mL was added and the precipitated protein was centrifuged at 3,000 rpm for 20 minutes. After adding and mixing 30 mL of ethyl ether to the supernatant, TCA and fat-soluble substances were removed, and the aqueous layer was concentrated under reduced pressure. The concentrate was adjusted to 5 mL with 0.2 M citrate buffer (pH 2.2), filtered through a 0.2 μm membrane filter, and analyzed with an automatic amino acid analyzer (L-8800, GMI Co., USA).

Table 2 below is an amino acid analysis result of pig makchang extract according to an experimental example of the present invention. Functional peptides have different functionalities depending on the size and amino acid composition, and the functionality of the peptide can be estimated by analyzing the amino acid composition. For example, cysteine, methionine, tyrosine, lysine, arginine, serine, proline, phenylalanine, alanine, histidine Amino acids such as (histidine) have a specific antioxidant activity, and the presence of the amino acid can improve the antioxidant properties of the polypeptide.

In addition, the tetrapeptide composed of amino acids alanine-leucine-proline-methionine has angiotensin converting enzyme (Angiotensin Converting Enzyme; hereinafter, ACE) inhibitory ability, tryptophan-tyrosine-methionine or proline-histidine-histidine When composed of, it may have antioxidant activity.

Name ng/mL P-Serine 105.3 Taurin 220.2 PEA 48.7 Urea 84.7 Aspatate 204.1 Threonine 221.6 Serine 333.1 Glutamate 828.9 Glycine 258.8 Alanine 14.4 Citrullin 6.6 a-ABA 355.7 Cystein 34.0 g-ABA 99.6 EOHNH2 163.6 NH3 42.0 Hylys 3.1 Ornitin 27.4 Lysine 330.4 3Mehis 30.2 Ariginin 334.1

Referring to Table 2, as a result of analyzing the amino acid composition contained in the porcine makchang extract according to Experimental Example 2, the porcine makchang extract contained abundantly glutamate, glycine, taurin ), aspartic acid, threonine, lysine, etc. are also found to be contained, which is thought to have physiological activities such as antioxidant activity.

Table 3 below shows the amino acid sequences of the peptides present in the pig makchang extract. Among the functional peptides reported so far, antioxidant activity was found to be high when the sequence had the sequence of cysteine, histidine, and phenylalanine, and proline-histidine-histidine had high antioxidant activity in the linoleic acid system.

The amino acid sequence that increases the expression of glucose receptor type 4 (Glucose transporter type 4; hereinafter, GLUT4) consists of glycine-alanine-glycine-valine-tyrosine or glycine-glutamic acid-tyrosine, glycine-tyrosine-glycine. In addition, in the case of peptides having an antidiabetic effect, leucine, valine, tyrosine, and the like have in common.

Peptide, amino acid sequence
Length Molecular
weight
(MW)
ppm
Purity (%) LLTLK Leu-Leu-Thr-Leu-Lys 5 586.1 4.8 97 AVRPEE Ala-Val-Arg-Pro-Glu-Glu 6 699.4 7.6 93 VLENR Val-Leu-Glu-Asn-Arg 5 630.3 10.5 92 ELVR Glu-Leu-Val-Arg 4 515.3 6.1 88 EVRL Glu-Val-Arg-Leu 4 515.3 7.8 84 LAPSTMK Leu-Ala-Pro-Ser-Thr-Met-Lys 7 746.4 6.1 82 N(+.98)LKR Asn-Leu-Lys-Arg 4 530.3 6.7 81

Referring to Table 3, it was found that all seven types of peptides were present in the extract of pig makchang. Since a large number of peptides including leucine, valine, tyrosine, etc. exist in the pig makchang extract, these peptides are considered to be molecules having antidiabetic activity in the extract.

<Experimental Example 3> Cytotoxicity evaluation for 3T3-L1 adipocytes

To determine the concentration range of the porcine makchang extract sample to be used for cell experiments, toxicity evaluation was performed on 3T3-L1 preadipocytes. The cytotoxicity of the pig makchang extract sample prepared according to Example 1 was analyzed using the MTT assay method.

Cells were cultured in 100 μL of Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) at ^{1×10 4} cells per well of a 96-well plate. Each was dispensed, 37 ℃, 5% CO ₂ Incubated in an incubator (incubator). After culturing for 24 hours for cell adhesion, samples diluted by concentration (125, 250, 500, 1000 μg/mL) were treated and cultured by 150 μL each. After incubation, 7.5 μL of MTT reagent of 5 mg/mL was treated in each well, the medium was removed after 3 hours, and 100 μL of dimethyl sulfoxide (DMSO) was added to measure the absorbance at 570 nm. Cytotoxicity was compared as a percentage relative to the control.

Figure 2 is a graph of the cytotoxicity evaluation result of the pig makchang extract according to an experimental example of the present invention.

Referring to FIG. 2, it was confirmed that no cytotoxicity was found at all treatment concentrations of porcine makchang extract 125, 250, 500, and 1000 μg/mL, and also did not affect the morphological aspects of cells. Therefore, it is considered that there is no cytotoxicity to 3T3-L1 preadipocytes, and the concentration standard of the sample to be used for the cell experiment was 1000 μg/mL.

<Experimental Example 4> Confirmation of lipid accumulation through Oil Red O (Oil Red O) staining

Lipid droplets are inactive vesicles composed of triglycerides surrounded by a phospholipid monolayer, where cells store fat. Adipocytes appear in the process of differentiation from precursor fibroblasts to adipocytes, and are regulated by important adipogenic transcription factors such as peroxisome proliferator-activated receptor γ (PPARγ). Liposome maintenance in mature adipocytes is dependent on the influx of triglyceride by lipoprotein lipase (LPL) and adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL). It is controlled by triglyceride efflux by

Therefore, in order to check the effect of the hot water extract of pork makchang on the production of small fat particles that appear in the process of 3T3-L1 preadipocyte differentiation into adipocytes, each sample was treated with an appropriate concentration to induce differentiation, and then, Oil Red O (Oil Red O) was stained to observe the degree of liposome formation.

Cells were seeded in a 6-well plate at 1×10 ⁵ per well, and then differentiated and used for the experiment. The medium was removed from the cells for which differentiation was completed (day 13) and washed 2-3 times with cold PBS. After fixing for 1 hour at room temperature using 10% formalin (formalin in PBS), it was washed with 60% isopropanol and completely dried at room temperature. 0.35% Oil red O (Oil red O in isopropanol) _{was diluted with H 2} O 3:2, filtered through a 0.2 μm membrane filter, and then used. After staining with Oil Red O for 20 minutes, washing with distilled water 3-4 times and observing under a microscope, 100% isopropanol was added to the well to dissolve oil droplets, and the triglyceride content was measured using absorbance at 492 nm.

3 is a graph (B) analyzing the adipocyte differentiation process (A) and fat content according to an experimental example of the present invention.

Referring to FIG. 3 , the differentiation status of the group treated with and not treated with Pork rectum extract (PRE) in the adipocyte differentiation process of (A) was compared with the control group (CON: Control only is differentiated in 3T3-L1). ), it was found that the formation of small fat particles in the cytoplasm was actively induced in the group treated with the pig makchang extract. NOR means undifferentiated 3T3-L1.

Insulin-induced glucose consumption is the first step leading to several pathways leading to the storage of nutrients in the form of triglycerides, where levels of glucose act as substrates and stimulate adipogenesis through several mechanisms. Glucose is converted to acetyl-CoA, thereby promoting fatty acid synthesis and increasing adipogenesis by stimulating the release of insulin and inhibiting the release of glucagon. That is, in the graph of (B), the increase in fat production in the group treated with the porcine makchang extract indirectly shows that the amount of glucose in the plasma was consumed and glucose uptake increased. are giving

<Experimental Example 5> Confirmation of changes in mRNA and protein expression

In order to confirm the effect of pig makchang extract on lipid metabolism, changes in adipogenesis and lipogenesis-related factors of adipocytes were confirmed. Adipogenesis refers to the generation (differentiation) of mature adipocytes from preadipocytes, and lipogenesis refers to the process of fat accumulation and fat synthesis.

Adipogenesis, which is differentiated from preadipocytes into adipocytes, is induced by the stepwise regulation of many types of adipogenesis factors. Among these fat transcription factors, CCAAT enhancer-binding-proteins (C/EBPs) and Peroxisome-proliferators-activated receptor (PPAR) families play an important role. In particular, peroxisome proliferator-activated receptor gamma (hereinafter, PPARγ) belonging to the nuclear receptor superfamily plays a role in regulating adipogenesis as a whole, and It is an essential factor in maintaining a differentiated state. When early adipocytes are exposed to differentiation inducers, within a few hours, expression of CCAAT enhancer-binding-proteins β (hereinafter, C/EBPβ) is induced and differentiation begins, and CCAAT enhancer-binding-protein Alpha (enhancer-binding-proteins α; hereinafter, C/EBPα) plays an important role in the early differentiation process of preadipocytes by promoting the expression of PPARγ, while C/EBPβ is insulin-sensitive glucose uptake. ), the differentiation process is finally completed by promoting the expression of C/EBPa and PPARγ, which are involved in increasing the characteristics of mature mast cells.

Porcine makchang extract is associated with adipocyte differentiation and energy metabolism-related C/EBPβ, sterol regulatory element-binding protein 1c (hereinafter, SREBP-1c), C/EBPa, PPARγ, adiponectin , GLUT4, fatty acid synthase (fatty acid synthase; hereinafter, FAS), acetyl-CoA-carboxylase (hereinafter, ACC), fatty acid binding protein 4 (fatty acid binding protein 4; hereinafter, FABP4) and ferri What effect on the expression of lipin (Perilipin) was confirmed at the mRNA level. Changes in mRNA expression were confirmed through RT-PCR, and changes in protein expression were confirmed through western blot.

4 shows changes in mRNA expression of adipocyte-related factors according to an experimental example of the present invention.

Referring to FIG. 4 , it was found that factors related to adipogenesis and energy metabolism were overall increased in the porcine makchang extract treatment group (PRE). PPARγ, FAS, FABP4, C/EBPa and adiponectin showed a tendency to slightly increase at high concentrations, and in the case of SREBP-1c, Perilipin, and GLUT4, their expression significantly increased by treatment with porcine makchang extract. confirmed that

However, it was confirmed that the expression of leptin, an antagonistic fasting hormone, was reduced by the treatment of the pig makchang extract. Based on the above results, it is considered that the pig makchang extract promotes the use of blood glucose by increasing factors related to adipogenesis and lipogenesis.

5 shows changes in the expression of adipocyte-related factors at the protein level according to an experimental example of the present invention.

Referring to FIG. 5, in adipocytes C/EBPa, PPARγ and GLUT4, FAS, ACC, FABP4, and perilipins in adipogenic and lipogenic factors in adipocytes in the pig makchang extract treatment group (PRE) expression at the protein level Changes in GLUT4, FAS, and perilipin showed similar results to mRNA expression, but in C/EBPa, PPARγ and FABP4, the change was more pronounced than in mRNA. This difference is thought to be also involved in the difference in time difference in which messenger signals are expressed as proteins.

Summarizing the above results, it is considered that the treatment of 3T3-L1 adipocytes with the porcine makchang extract is involved in the regulation of energy metabolism as well as differentiation of adipocytes to increase the availability of glucose. It is thought that the pig makchang extract can be utilized for type 2 diabetes, which occurs due to a decrease in the availability of glucose in the blood due to a decrease in insulin sensitivity and the like.

As described above in detail a specific part of the content of the present invention, for those of ordinary skill in the art, it is clear that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. Do. That is, the substantial scope of the present invention is defined by the appended claims and their equivalents.

<110> Research Cooperation Foundation of Yeungnam University <120> COMPOSITION FOR PREVENTING OR TREATING DIABETES COMPRISING PORK RECTUM EXTRACT <130> ADP-2019-0075 <150> KR 10-2019-0015829 <151> 2019-02-12 <160> 7 <170> KoPatentIn 3.0 <210> 1 <211> 5 <212> PRT <213> Sus scrofa <400> 1 Leu Leu Thr Leu Lys 1 5 <210> 2 <211> 6 <212> PRT <213> Sus scrofa <400> 2 Ala Val Arg Pro Glu Glu 1 5 <210> 3 <211> 5 <212> PRT <213> Sus scrofa <400> 3 Val Leu Glu Asn Arg 1 5 <210> 4 <211> 4 <212> PRT <213> Sus scrofa <400> 4 Glu Leu Val Arg One <210> 5 <211> 4 <212> PRT <213> Sus scrofa <400> 5 Glu Val Arg Leu One <210> 6 <211> 7 <212> PRT <213> Sus scrofa <400> 6 Leu Ala Pro Ser Thr Met Lys 1 5 <210> 7 <211> 4 <212> PRT <213> Sus scrofa <400> 7 Asn Leu Lys Arg One

Claims (9)

delete delete delete delete delete delete delete Contains pork makchang extract as an active ingredient
characterized in that the extract increases the expression of one or more selected from the group consisting of GLUT4, PPARγ, adiponectin, FAS, C/EBPβ, C/EBPa, SREBP-1c, Perilipin, FABP4, and ACC A reagent composition for promoting adipogenesis and lipogenesis of adipocytes. delete

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