KR20200098356A - Composition for preventing or treating diabetes comprising pork rectum extract - Google Patents

Abstract

The present invention provides a health functional food composition and pharmaceutical composition for preventing or treating diabetes containing an extract of pork entrails as an active ingredient. The composition contains functional peptides having antioxidant activities, antidiabetic activities, etc., and can promote the consumption of glucose by involving in the energy metabolism as well as differentiation of adipocytes. Thus, type 2 diabetes caused by a decrease in the usability of blood glucose due to an increase in the insulin resistance can be effectively prevented, alleviated or treated.

Description

Composition for preventing or treating diabetes containing porcine makchang extract {COMPOSITION FOR PREVENTING OR TREATING DIABETES COMPRISING PORK RECTUM EXTRACT}

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

Diabetes mellitus is a type of metabolic disease such as insufficient secretion of insulin, a hormone that regulates glucose metabolism, or inability to function normally.It is characterized by high blood sugar, which increases the concentration of glucose in the blood, and is characterized by various symptoms due to high blood sugar. And causes signs. Diabetes is divided into type 1 and type 2. Type 1 diabetes was previously called'pediatric diabetes', and it is a disease caused by the inability to produce insulin at all. Type 2 diabetes, which is relatively poor in insulin, is characterized by insulin resistance.

Recently, due to the westernization of dietary habits, the transition from a low-fat diet to a high-fat diet is changing, and the obese population is gradually increasing due to the decreased amount of exercise compared to high nutritional intake due to changes in lifestyle. Obesity increases insulin demand and, as a result, the function of cells in the pancreas gradually decreases, leading to diabetes. As a result, diabetes is also on the rise in type 2 rather than type 1.

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

Functional peptides are low molecular weight substances 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 that make up the peptide. Until now, antioxidant, anticancerogenic, antithrombotic, antimicrobial, blood pressure lowering (antihypertensive) has been reported.

For example, a representative functional peptide is collagen, which is isolated from meat and fish, which has a low nutritional value due to the low essential amino acids, but has antioxidant effects and angiotensin converting enzymes when hydrolyzed with trypsin enzyme. Converting Enzyme, ACE) has an inhibitory effect, and in addition, fractions of hemoglobin or hydrolyzate in the blood have been found to have antibacterial activity.

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

Korean Patent Registration 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, including porcine makchang extract.

The present invention provides a pharmaceutical composition for preventing or treating diabetes, comprising a porcine 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.

Porcine makchang extract according to the present invention contains functional peptides having antioxidant activity, antidiabetic activity, and the like, and may promote the consumption of blood glucose by engaging in energy metabolism regulation as well as differentiation of adipocytes.

By using 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 occurs due to decrease in the availability of blood glucose due to an increase in insulin resistance or the like.

1 is a flow chart showing the manufacturing process of pig makchang extract according to an embodiment of the present invention.
Figure 2 is a graph showing the results of cytotoxicity evaluation of porcine 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 changes 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 completed the present invention by extracting the functional peptide from the pig makchang and confirming the antidiabetic effect from the extract.

In the present specification, the term "pig makchang" refers to an internal part of a pig, and may mean a rectum or a lower part of the rectum from the back part of the pig intestine to the anus, that is, the end of the large intestine near the anus, and Can be used interchangeably.

In the present specification, the term "extract" refers to a material obtained by extracting a component of pork makchang regardless of the extraction method, extraction solvent, extracted component, or form of the extract. After extracting the material obtained by extracting the component of pig makchang It may include all materials that can be obtained by processing or processing in other ways.

In the present specification, "prevention" means any action of inhibiting or delaying the onset of at least one symptom of diabetes or diabetes by administration of the pharmaceutical composition or dietary supplement composition according to the present invention. In addition, it includes treatment of a subject with remission of the disease to prevent or prevent recurrence.

In the present specification, "treatment" refers to any act of improving or beneficially altering the symptoms such as alleviating, reducing, or eliminating at least one symptom of diabetes or diabetes by administration of the pharmaceutical composition according to the present invention.

In the present specification, the term "improvement" refers to any act of improving or beneficially altering the symptoms such as alleviating, reducing, or eliminating at least one symptom of diabetes or diabetes by ingestion of the health functional food composition according to the present invention. do.

In the present specification, the term "pharmaceutical composition" means a composition administered for a specific purpose, and for the purposes of the present invention, it is meant to be administered to prevent or treat at least one symptom of diabetes or diabetes.

In the present specification, "health functional food" refers to foods manufactured and processed using raw materials or ingredients that have useful functionalities for the human body, and foods with high medical and medical effects processed to effectively show the biological control function in addition to supplying nutrition. It means and can be mixed with terms known in the art such as functional food.

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

In the pharmaceutical composition according to the present invention, the porcine 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, to the precipitate generated by pulverizing and centrifuging the pig makchang. It may be extracted for 10 to 15 hours, preferably 12 to 13 hours at 95 ℃. According to an embodiment of the present invention, the pig makchang extract was first removed the fat of the pig makchang, washed several times, and then pulverized, and after separating the pulverized pig makchang with a centrifuge, the supernatant was removed in a sediment 5 times that Distilled water was added and extracted for 12 hours at 95°C, which was filtered and centrifuged again, and then fractionated. In addition, the pig makchang extract may be prepared by drying, for example, may be dried by a method such as hot air drying, reduced pressure drying, freeze drying, and preferably freeze drying.

In the pharmaceutical composition according to the present invention, the porcine makchang extract may include one or more peptides selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 7, and the peptide 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 mean the following amino acids according to standard abbreviation regulations 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(Gin): 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, and the peptides of SEQ ID NOs 1 to 7 are also leucine, valine. , Tyrosine and the like appear to contain a large number, and can be said to have 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, in the sample treated with the porcine makchang extract, it was found that the formation of adipose particles appearing in the process of differentiation of 3T3-L1 adipocytes into adipocytes is actively induced, and such an 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 one or more selected from the group consisting of GLUT4, PPARγ, adiponectin, FAS, C/EBPβ, C/EBPα, SREBP-1c, perilipin, FABP4, and ACC. Expression can be increased. The GLUT4, PPARγ, adiponectin, FAS, C/EBPβ, C/EBPα, SREBP-1c, Perilipin, FABP4, and ACC are factors related to the differentiation and energy metabolism of adipocytes. According to an experimental example, when the change in mRNA expression of the factor through RT-PCR and the change in protein expression of the factor through western blot were confirmed, the expression of the factor in a sample treated with the porcine makchang extract It has been shown to increase significantly. Through this, it was analyzed that the porcine makchang extract promotes the consumption of blood glucose by engaging in energy metabolism as well as adipocyte differentiation. Due to these effects, the porcine 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 is type 1 diabetes (insulin dependent diabetes mellitus, IDDM), which is a form of diabetes that occurs due to a lack of absolute amount of insulin, and some of the insulin secretion function remains, but various causes Type 2 diabetes (NIDDM, Noninsulin dependent diabetes mellitus, NIDDM), which is caused by relatively increased insulin resistance, gestational diabetes and pancreatic disease caused by abnormal glucose control that was first detected during pregnancy or occurred at the beginning of pregnancy, Endocrine diseases, specific drugs, chemicals, insulin or insulin receptor abnormalities, diabetes, etc., which are secondaryly caused by genetic syndrome, etc., preferably, insulin resistance (insulin resistance) increase, that is, insulin function to lower blood sugar This can be a type 2 diabetes caused by cells not burning glucose effectively.

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

The pharmaceutical composition according to the present invention may be blended with an appropriate pharmaceutically acceptable carrier according to the above formulation, and further includes excipients, diluents, dispersants, emulsifiers, buffers, stabilizers, binders, disintegrants, solvents, etc., if necessary. It 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, etc., does not inhibit the activity and properties of the porcine makchang extract according to the present invention, 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 particularly, may be formulated and used in parenteral dosage forms of oral dosage forms, external preparations, suppositories, and sterile injectable solutions according to conventional methods.

Among the oral dosage forms, the solid dosage form may be in the form of tablets, pills, powders, granules, capsules, etc., and at least one excipient such as starch, calcium carbonate, sucrose, lactose, sorbitol, mannitol, cellulose, gelatin, etc. It can be prepared by mixing, and in addition to simple excipients, lubricants such as magnesium stearate and talc may also be included. In addition, in the case of the capsul formulation, in addition to the above-mentioned substances, a liquid carrier such as fatty oil may be further included.

Among the oral dosage forms, liquid dosage forms correspond to suspensions, liquid solutions, emulsions, syrups, and the like.In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweetening agents, fragrances, preservatives, etc. may be included. have.

The parenteral formulation may include a sterilized aqueous solution, a non-aqueous solvent, a suspension, an emulsion, a lyophilized formulation, and a suppository. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc. may be used as the non-aqueous solvent and suspension. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used. The present invention is not limited thereto, and any suitable agent known in the art may be used.

In addition, the pharmaceutical composition according to the present invention may further include calcium or vitamin D ₃ to improve 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 cause side effects.

The effective dosage level of the pharmaceutical composition is the purpose of use, the patient's age, sex, weight and health condition, the type of disease, the severity, the activity of the drug, the sensitivity to the drug, the method of administration, the administration time, the administration route and the rate of excretion, the treatment It may be determined differently depending on the duration, factors including drugs used in combination or concurrently 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 in any way limit the scope of the present invention.

The pharmaceutical composition according to the present invention may be administered to any animal that may develop diabetes, and the animal may include, for example, humans and primates as well as livestock such as cattle, pigs, horses, and dogs.

The pharmaceutical composition according to the present invention can be administered by an appropriate route of administration according to the form of the formulation, and can be administered through various routes, either oral or parenteral, as long as it can reach the target tissue. The method of administration is not particularly limited and may be administered by conventional methods such as, for example, oral, rectal or intravenous, intramuscular, subcutaneous, intrabronchial inhalation, intrauterine dura mater or intracere-broventricular injection. .

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 to promote the consumption of blood glucose, GLUT4, PPARγ, adiponectin, FAS, C/EBPβ, C/EBPα, SREBP -1c, perilipin (Perilipin), FABP4, and by increasing the expression of one or more selected from the group consisting of ACC, etc., involved in adipocyte differentiation and energy metabolism, thereby promoting the consumption of blood glucose, 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 as a powder, granule, tablet, capsule, syrup or beverage, and there is no limitation on the form that the health functional food can take. It can include all foods of meaning. For example, beverages and various drinks, fruits and processed foods thereof (canned fruit, jam, etc.), fish, meat and processed foods thereof (ham, bacon, etc.), bread and noodles, cookies and snacks, dairy products (butter, cheese, etc.) ), and the like, and may include all functional foods in the usual sense. In addition, foods used as feed for animals may also be included.

The health functional food composition according to the present invention may be prepared further comprising a food pharmaceutically acceptable food additive and other appropriate auxiliary ingredients commonly used in the art. For example, flavoring agents, natural carbohydrates, sweetening agents, vitamins, electrolytes, colorants, pectic acids, alginic acids, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents, etc. I can. In particular, as the natural carbohydrates, monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, sugar alcohols such as xylitol, sorbitol, and erythritol may be used. , As the sweetener, natural sweeteners such as taumatin 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 of not having side effects that may occur when taking the drug for a long period of time, unlike general drugs, using food as a raw material, and is excellent in portability, and can be ingested as an adjuvant for preventing or improving diabetes. I can.

Hereinafter, examples will be described in detail to aid understanding of the present invention. However, the following examples are for illustrative purposes only, 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> Pig makchang extract preparation

1 is a flow chart showing the manufacturing process of pig makchang extract according to an embodiment of the present invention. Referring to Figure 1, after removing the fat of the pig makchang supplied from'Dalguji Food', the first washing with 0.1N sodium hydroxide (NaOH), and the second washing with a phosphate buffered saline (hereinafter, PBS). Then, the same amount (1:1) of PBS was added and pulverized using a grinder (HMF-3800SS, Hanil, Korea). The pulverized liquid was separated at 9000g, 10min, 4°C using a centrifuge (Centrifuge 5810 R, Eppendorf, Germany). 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. The extract was filtered and then centrifuged again (9000g, 10min, 4°C) to obtain a supernatant, which was fractionated using Sephadex G-200, and then freeze-dried to prepare a final porcine makchang extract sample.

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

Protein is a component of a living body, and is a high molecular organic material made by a series of amino acids. Peptide refers to a compound in which two or more amino acids are bound by a peptide (peptide) bond, and a protein having an amino acid residue of 50 or more 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 antihyperlipidemia. In particular, peptides isolated from meat by-products have a blood pressure lowering effect.

The total protein contents of the porcine makchang extract prepared according to Example 1 were measured using the 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. The 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 pig makchang extract measured by the BCA method was about 0.33mg/mg, and it was found that the pig makchang extract contained 30% or more of protein.

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

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

Table 2 below shows the results of amino acid analysis of porcine makchang extract according to an experimental example of the present invention. Functional peptides differ in their functionality according to their size and amino acid composition, and analysis of the amino acid composition can estimate the functionality of the peptide. For example, cysteine, methionine, tyrosine, lysine, arginine, serine, proline, phenylalanine, alanine, histidine Amino acids such as (histidine) have 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 acid alanine-leucine-proline-methionine has an angiotensin converting enzyme (ACE) inhibitory activity, and 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 pig makchang extract according to Experimental Example 2, the pig makchang extract was rich in glutamate, glycine and taurin. ), aspartic acid, threonine, and lysine are also found to be contained, which is thought to have physiological activities such as antioxidant activity.

Table 3 below shows the amino acid sequence of the peptides present in the porcine makchang extract. Among the functional peptides reported so far, antioxidant activity has been shown a lot in the order of cysteine, histidine, and phenylalanine, and proline-histidine-histidine has high antioxidant activity in the linoleic acid system.

The amino acid sequence that increases the expression of glucose receptor type 4 (hereinafter, GLUT4) is composed 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 are 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 there are all 7 kinds of peptides in the porcine makchang extract. Since a number of peptides containing leucine, valine, tyrosine, etc. are present in the porcine makchang extract, these peptides are considered to be molecules having antidiabetic activity in the extract.

<Experimental Example 3> Evaluation of cytotoxicity to 3T3-L1 adipocytes

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

100 μL of cells using 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 and cultured in an incubator at 37° C. and 5% CO ₂ . After culturing for 24 hours for cell adhesion, samples diluted at different concentrations (125, 250, 500, 1000 μg/mL) were treated by 150 μL and cultured. After incubation, 5 mg/mL of MTT reagent was treated with 7.5 μL in each well, and after 3 hours, the medium was removed, and 100 μL of dimethyl sulfoxide (DMSO) was added to measure absorbance at 570 nm. Cytotoxicity was expressed as a percentage of the control and compared.

Figure 2 is a graph showing the results of cytotoxicity evaluation of porcine makchang extract according to an experimental example of the present invention.

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

<Experimental Example 4> Confirmation of lipid accumulation through oil red O staining

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

Therefore, in order to confirm the effect of the hot water extract of porcine makchang on the generation of adipose particles in the process of differentiation of 3T3-L1 adipocytes into adipocytes, each sample was treated at an appropriate concentration to induce differentiation, and then oil red O (Oil Red O) staining was performed to observe the degree of fat particle formation.

After dispensing 1x10 ⁵ cells per well into a 6-well plate, the cells were differentiated and used in the experiment. The medium was removed from the cells in which differentiation was completed (day 13), and washed 2-3 times with cooled PBS. After fixing for 1 hour at room temperature using 10% formalin (formalin in PBS), washed with 60% isoprophanol (isoprophanol) and completely dried at room temperature. 0.35% Oil red O in isopropanol was diluted with H ₂ O and 3:2, filtered through a 0.2 μm membrane filter, and used. After staining with Oil Red O for 20 minutes, washing 3 to 4 times with distilled water and observing under a microscope, 100% isopropanol was added to the wells to dissolve oil droplets, and the triglyceride content was measured using 492 nm absorbance.

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, in the adipocyte differentiation process of (A), the differentiation status of the group treated with Pork rectum extract (PRE) and the group without treatment were compared with the control group (CON: Control only is differentiated in 3T3-L1). ), it was found that the formation of liposomes in the cytoplasm was actively induced in the group treated with the porcine makchang extract. NOR means undifferentiated 3T3-L1.

Insulin-induced glucose consumption is the first step leading several pathways to store nutrients in the form of triglycerides, where glucose levels act as substrates and stimulate fat formation through several mechanisms. Glucose is converted to acetyl-CoA, which promotes fatty acid synthesis, and increases fat production through stimulation of insulin release and inhibition of glucagon release. That is, in the graph of (B), it was indirectly shown that the amount of glucose in the plasma was consumed, indicating that the production of fat was increased in the group treated with the porcine makchang extract, and glucose uptake increased. Are giving.

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

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

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

Porcine makchang extract is related to these adipocyte differentiation and energy metabolism, C/EBPβ, sterol regulatory element-binding protein 1c (SREBP-1c), C/EBPα, PPARγ, adiponectin , GLUT4, fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC), fatty acid binding protein 4 (FABP4) and ferry It was confirmed at the mRNA level how it affects the expression of perilipins. The change in mRNA expression was confirmed through RT-PCR, and the change in protein expression was 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 generally increased in the pig makchang extract treatment group (PRE). PPARγ, FAS, FABP4, C/EBPα, and adiponectin showed a tendency to increase slightly at high concentration, and in the case of SREBP-1c, Perilipin, and GLUT4, their expression was significantly increased by treatment with porcine makchang extract. I confirmed that.

However, it was confirmed that the expression of leptin, which is an antagonistic fasting hormone, was reduced by treatment with the porcine makchang extract. Based on the above results, it is believed that porcine 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.

5, expression at the protein level of adipogenic and lipogenic factors such as C/EBPα, PPARγ and GLUT4, FAS, ACC, FABP4, and periripines in adipocytes in a pig makchang extract treatment group (PRE) Changes in GLUT4, FAS, and periripine showed similar results to mRNA expression, but in the case of C/EBPα, PPARγ and FABP4, the change was more pronounced than in mRNA. This difference is thought to be related to the difference in the time difference in which the messenger signal is expressed as a protein.

Taking the above results together, it is thought that the treatment of porcine makchang extract on 3T3-L1 adipocytes increases the availability of glucose by controlling energy metabolism as well as differentiation of adipocytes. It is believed that the porcine makchang extract can be used for type 2 diabetes, which is caused by decrease in insulin sensitivity, etc., resulting in a decrease in the availability of blood glucose.

As described above, specific parts of the present invention have been described in detail, and for those of ordinary skill in the art, it is obvious that these specific techniques are only preferred embodiments, 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)

A pharmaceutical composition for preventing or treating diabetes, comprising a porcine makchang extract as an active ingredient. The method of claim 1,
The porcine makchang extract,
A pharmaceutical composition for preventing or treating diabetes, characterized in that it is a hot water extract. The method of claim 2,
The hot water extract,
A pharmaceutical composition for preventing or treating diabetes, characterized in that extracting for 10 to 15 hours at 90 to 100° C. by adding 4 to 6 times of distilled water of the sediment to the sediment produced by pulverizing and centrifuging the pig makchang. The method of claim 1,
The porcine makchang extract,
A pharmaceutical composition for preventing or treating diabetes, comprising one or more peptides selected from the group consisting of SEQ ID NOs: 1 to 7. The method of claim 4,
The peptide,
A pharmaceutical composition for preventing or treating diabetes, characterized in that it has anti-diabetic activity. The method of claim 1,
The composition,
A pharmaceutical composition for preventing or treating diabetes, characterized in that to promote the consumption of blood glucose by inducing adipocyte differentiation. The method of claim 1,
The composition,
Diabetes, characterized by increasing the expression of one or more selected from the group consisting of GLUT4, PPARγ, adiponectin, FAS, C/EBPβ, C/EBPα, SREBP-1c, Perilipin, FABP4, and ACC Pharmaceutical composition for prophylaxis or treatment. The method of claim 1,
The diabetes,
A pharmaceutical composition for preventing or treating diabetes, characterized in that it is type 2 diabetes. A health functional food composition for preventing or improving diabetes, comprising pig makchang extract as an active ingredient.

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