KR20210100870A - Pharmaceutical composition for preventing or treating diabetes and food composition having the same - Google Patents

Abstract

The present invention discovers a novel use of heneicosane, a natural pheromone, and provides a pharmaceutical composition containing the heneicosane as an active component to reduce blood sugar levels in the body. The present invention provides a pharmaceutical composition comprising heneicosane to increase mRNA of PPARγ and C/EBP-α, which are major transcription factors for adipocyte differentiation and lipid accumulation.

Description

A pharmaceutical composition for preventing or treating diabetes containing heneicosan, and a food composition comprising the same {Pharmaceutical composition for preventing or treating diabetes and food composition having the same}

The present invention relates to a pharmaceutical composition for glycemic control comprising Heneicosane as an active ingredient and a food composition comprising the same, and more particularly, Heneicosan increases the phosphorylation of Akt, FoxO1 and GSK3β, thereby increasing serum glucose concentration. It relates to a pharmaceutical composition for blood sugar control that reduces and inhibits gluconeogenic metabolism, and a food composition comprising the same.

Diabetes is a subject of much interest because of its high incidence and serious acute and chronic complications. Diabetes is divided into 5 types according to the etiology, but clinically, it is broadly classified into insulin-dependent (or type 1) and non-insulin-dependent (or type 2) diabetes for convenience.

Insulin-dependent diabetes mellitus is a type of autoimmune disease caused by the destruction of β-cells, which are insulin-secreting cells, by infiltrating lymphocytes into the pancreatic islet, and it develops regardless of age. Therefore, it has been reported that in insulin-dependent diabetes mellitus, the amount of insulin in the blood is significantly reduced, and diabetic ketoacidosis occurs due to excessive accumulation of ketone bodies, which are lipolysis products, caused by insufficient insulin secretion.

In non-insulin-dependent diabetes mellitus, insulin is secreted from β cells, but insulin resistance in the peripheral target organs is increased, which means that insulin in the blood does not act. Therefore, ketoacidosis, autoantibodies, etc. cannot be observed, and in humans, it occurs mainly after the age of 40 and is usually accompanied by obesity.

In non-insulin-dependent diabetes mellitus, diet and exercise are combined, and if not treated in this way, oral hypoglycemic agents are sometimes used. As such oral hypoglycemic agents, metformin and biguanide drugs applied to obese patients and sulfonylurea drugs applied to non-obese patients are mainly used. are accompanied by side effects of severe lactic acidemia and hypoglycemia, respectively.

Since side effects exist for each specific drug for each person, the development of various types of blood sugar lowering agents is required.

The present invention discovers a new use of heneicosan, a natural pheromone, and provides a pharmaceutical composition containing the heneicosan as an active ingredient to reduce blood sugar levels in the body.

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

In one embodiment of the present invention, the diabetes may be a pharmaceutical composition for preventing or treating diabetes, which is type 2 diabetes.

In one embodiment of the present invention, the pharmaceutical composition may be a pharmaceutical composition for preventing or treating diabetes that regulates adipocyte differentiation and lipid accumulation through up-regulation of PPARγ and C/EBP-α protein.

In one embodiment of the present invention, the pharmaceutical composition may be a pharmaceutical composition for preventing or treating diabetes by increasing Akt/Fox/GSK3β phosphorylation, thereby reducing serum glucose concentration and inhibiting gluconeogenic metabolism. .

In one embodiment of the present invention, the pharmaceutical composition may be a pharmaceutical composition for preventing or treating diabetes administered by oral administration or injection into the body.

The present invention provides a food composition for preventing or improving diabetes comprising heneicosane as an active ingredient.

In one embodiment of the present invention, the pharmaceutical composition may be a food composition for preventing or improving diabetes that regulates adipocyte differentiation and lipid accumulation through up-regulation of PPARγ and C/EBP-α protein.

In one embodiment of the present invention, the pharmaceutical composition may be a food composition for preventing or improving diabetes by increasing Akt/Fox/GSK3β phosphorylation, thereby reducing serum glucose concentration and inhibiting gluconeogenic metabolism.

In one embodiment of the present invention, the pharmaceutical composition may be a food composition for preventing or improving diabetes orally administered.

An embodiment of the present invention may include a pharmaceutical composition for lowering blood sugar comprising heneicosane as an active ingredient. In addition, an embodiment of the present invention may include a food composition for lowering blood sugar containing heneicosane as an active ingredient. Through these effects, the pharmaceutical and/or food composition according to an embodiment of the present invention may be used as an insulin substitute composition.

The pharmaceutical composition and food composition according to an embodiment of the present invention may be used as an insulin replacement composition, and by not affecting a lipid profile such as cholesterol, may be effective in alleviating symptoms and treating cardiovascular diseases.

One embodiment of the present invention may include a pharmaceutical composition for the treatment of cardiovascular diseases comprising heneicosane as an active ingredient. In addition, it may include a food composition for the treatment of cardiovascular diseases comprising heneicosane as an active ingredient.

The cardiovascular disease may include coronary heart disease, congestive heart failure, peripheral vascular disease, heart attack, stroke or transient ischemic attack, renal vascular disease, and the like.

One embodiment of the present invention may include a pharmaceutical composition for protecting the pancreas comprising heneicosane as an active ingredient. In addition, it may include a food composition for protecting the pancreas comprising heneicosane as an active ingredient.

The present invention provides a pharmaceutical composition comprising heneicosan to increase mRNA of PPARγ and C/EBP-α, which are major transcription factors for adipocyte differentiation and lipid accumulation.

The present invention provides a pharmaceutical composition comprising heneicosan to increase Akt/Fox/GSK3β phosphorylation, thereby reducing serum glucose concentration and inhibiting gluconeogenic metabolism.

The present invention provides a pharmaceutical composition comprising heneichoic acid to regulate glucose metabolism by increasing the phosphorylation level through the Akt-mediated signal transduction pathway and inhibiting FoxO1 and GSK3β activity.

Fig. 1a shows that when adipocytes were observed for 5 days in the presence of insulin, IBMX and dexamethasone, treatment with heneichoic acid on day 5 had a greater dependence on lipid concentration than control cells.
Figure 1b shows that when adipocytes were observed for 10 days in the presence of insulin, IBMX and dexamethasone, treatment with heneichoic acid on day 10 had a greater dependence on lipid concentration than control cells.
Figure 2a shows that treatment with heneichoic acid increases the mRNA of PPARγ, a key transcription factor for adipocyte differentiation and lipid accumulation.
Figure 2b shows that treatment with heneichoic acid increases the mRNA of C/EBP-α, a key transcription factor for adipocyte differentiation and lipid accumulation.
3a shows the results of electrophoresis of Heneiko acid in PPARγ, C/EBP-α, ACC, FAS, aP2, AdipoQ, and GAPDH.
Figure 3b is a bar graph showing PPARγ/GAPDH of the control group, H25 and H50.
Figure 3c is a bar graph showing C/EBP-α/GAPDH of the control group, H25 and H50.
Figure 3d is a bar graph of ACC/GAPDH of the control group, H25 and H50.
Figure 3e is a bar graph showing the FAS/GAPDH of the control group, H25 and H50.
Figure 3f is a bar graph showing the aP2/GAPDH of the control group, H25 and H50.
Figure 3g is a bar graph of AdipoQ /GAPDH of the control group, H25 and H50.
Figure 4a shows the electrophoresis results for the control group, H50 and R1.
Figure 4b is a bar graph showing PPARγ/GAPDH of the control group, H50 and R1.
Figure 4c is a bar graph showing C/EBP-α/GAPDH of the control group, H50 and R1.
Figure 5a shows the electrophoresis results for the control group, H25, H50.
Figure 5b is a bar graph showing ^{Akt S473} /Akt of the control group, H25, H50 and R1.
Figure 5c is a bar graph showing ^{Akt T308} /Akt of the control group, H25, H50 and R1.
Figure 6a is fed to rats that have increased their body weight for 9 weeks by mixing heneichoic acid with a feed, and comparing the weight of the rats with a control group.
Figure 6b is a bar graph showing the glucose levels of the control group, H25 and H50.
Figure 6c is a bar graph showing the AST values of the control group, H25 and H50.
Figure 6d is a bar graph showing the ALT levels of the control group, H25 and H50.
Figure 6e is a bar graph showing the creatinine levels of the control group, H25 and H50.
Figure 6f is a bar graph showing the cholesterol levels of the control group, H25 and H50.
Figure 6g is a bar graph showing the LDL levels of the control group, H25 and H50.
Figure 6h is a bar graph showing the triglyceride levels of the control group, H25 and H50.
Figure 6i is a bar graph showing the HDL levels of the control group, H25 and H50.
Figure 7a shows the electrophoresis results of the control group, H25 and H50 for pAkt ^S473 , pAkt ^T308 , Akt, pFoxO1 ^S256 , tFoxO1 , pGSK ^S9 , tGSK, and GAPDH in muscle.
Figure 7b is a bar graph showing ^{pAkt S473} /tAkt of control, H25 and H50 in muscle.
^{Figure 7c is a histogram of pAkt T308} /tAkt of control, H25 and H50 in muscle.
Figure 7d is a bar graph showing ^{pFoxO1 S256} /tFoxO1 of control, H25 and H50 in muscle.
^{Figure 7e is a histogram of pGSK S9} /tGSK of control, H25 and H50 in muscle.
8A shows the results of electrophoresis of control, H25 and H50 in the liver for pAkt ^S473 , pAkt ^T308 , Akt, pFoxO1 ^S256 , tFoxO1 , pGSK ^S9 , tGSK, and GAPDH.
Figure 8b is a bar graph showing ^{the pAkt S473} /tAkt of the control, H25 and H50 in the liver.
Figure 8c is a bar graph ^{of pAkt T308} /tAkt of control, H25 and H50 in liver.
Figure 8d is a bar graph ^{of pFoxO1 S256} /tFoxO1 of control, H25 and H50 in liver.
^{Figure 8e is a histogram of pGSK S9} /tGSK of control, H25 and H50 in liver.
9 is a bar graph showing the comparison of fasting blood glucose levels of diabetic rats treated with henicosan and control diabetic rats according to time period.
10 is a bar graph showing the comparison of the body weight of diabetic rats treated with henicoic acid and diabetic rats as a control group according to time period.
11 is a line graph showing the weekly measurement of fasting blood glucose of LC, DC, H12.5, H25 and H50.
12 is a line graph showing postprandial blood glucose measurements of LC, DC, H12.5, H25 and H50 weekly.
13 is a graph showing the OGTT values of LC, DC, H12.5, H25 and M100 measured every 20 minutes as a line graph.
Figure 14 is a graph showing the measured insulin levels of LC, DC, H12.5, H25 and M100 as a line graph.
Figure 15a shows the results of electrophoresis of LC, DC, H12.5, H25 and M100 for pAkt ^S473 , pAkt ^{T308 , Akt in muscle.
Figure 15b shows pAkt S473} /tAkt of LC, DC, H12.5, H25 and M100 in muscle. is shown as a bar graph.
^{Figure 15c shows pAkt T308} /tAkt of LC, DC, H12.5, H25 and M100 in muscle. is shown as a bar graph.
Figure 16a shows the results of electrophoresis of LC, DC, H12.5, H25 and M100 in the liver for pAkt ^S473 , pAkt ^{T308 , Akt.
Figure 16b shows pAkt S473} /tAkt of LC, DC, H12.5, H25 and M100 in liver. is shown as a bar graph.
^{Figure 16c shows pAkt T308} /tAkt of LC, DC, H12.5, H25 and M100 in liver. is shown as a bar graph.
17A shows the results of electrophoresis of LC, DC, H25 and M100 in muscle for pFoxO1 ^S256 , tFoxO1 , pGSK3β ^{S9 , and tGSK3β .}
Figure 17b is a bar graph showing ^{pFoxO1 S256} /tFoxO1 of LC, DC, H25 and M100 in muscle.
Figure 17c is a bar graph showing ^{pGSK3β S9} /tGSK3β of LC, DC, H25 and M100 in muscle.
18A shows the results of electrophoresis of LC, DC, H25 and M100 in the liver for pFoxO1 ^S256 , tFoxO1 , pGSK3β ^{S9 , and tGSK3β .}
Figure 18b is a bar graph ^{of pFoxO1 S256} /tFoxO1 of LC, DC, H25 and M100 in the liver.
Figure 18c is a bar graph ^{of pGSK3β S9} /tGSK3β in LC, DC, H25 and M100 in liver.
Figure 19a shows the histopathological picture in the LC.
Figure 19b shows the histopathological picture in DC.
Figure 19c shows the histopathological picture at H12.5.
Figure 19d shows the histopathological picture in H25.
Figure 19e shows the histopathological picture in M100.

Since the present invention can have various changes and can have various embodiments, specific embodiments are illustrated in the drawings and will be described in detail in the text. However, this is not intended to limit the present invention to specific embodiments, and it should be understood to include all modifications, equivalents and substitutes included in the spirit and scope of the present invention. In describing the present invention, if it is determined that a detailed description of a related known technology may obscure the gist of the present invention, the detailed description thereof will be omitted.

Hereinafter, embodiments disclosed in the present specification will be described in detail with reference to the drawings. Exemplary embodiments described above in the detailed description, drawings, and claims are not intended to be limiting, and embodiments other than the described embodiments may be used, and other changes may be made without departing from the spirit or scope of the technology disclosed herein. are also possible

All terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed technology belongs, unless defined otherwise. Terms defined in the dictionary should be interpreted as being consistent with the meaning of the context of the related art, and cannot be interpreted as having an ideal or excessively formal meaning unless explicitly defined in the present application.

The present invention provides a pharmaceutical composition for preventing or treating diabetes comprising the heneicosane as an active ingredient. The pharmaceutical composition may be formulated in various oral or parenteral dosage forms. For example, it may be any dosage form for oral administration, such as tablets, pills, hard/soft capsules, solutions, suspensions, emulsifiers, syrups, granules, elixirs, and the like. Formulations for oral administration include, in addition to the above active ingredients, according to the conventional composition of each formulation, for example, diluents such as lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine, silica, talc , stearic acid and its magnesium or calcium salt and/or a lubricant such as polyethylene glycol.

In addition, when the dosage form for oral administration is a tablet, a binder such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidine may be included. Depending on the application, it may contain a disintegrant such as starch, agar, alginic acid or its sodium salt, a boiling mixture and/or an absorbent, a coloring agent, a flavoring agent or a sweetening agent, and the like.

In addition, the pharmaceutical composition may be formulated in a parenteral dosage form, and in this case, it is administered by a parenteral administration method such as subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection. At this time, in order to formulate the formulation for parenteral administration, the pharmaceutical composition is prepared as a solution or suspension by mixing the active ingredient, ie, a derivative of Formula I or a pharmaceutically acceptable salt thereof, in water with a stabilizer or buffer. and such solutions or suspensions may be prepared in unit dosage form in ampoules or vials.

In addition, the pharmaceutical composition may further include adjuvants such as sterilized, preservatives, stabilizers, wetting agents or emulsification promoters, salts and/or buffers for regulating osmotic pressure, and may further include other therapeutically useful substances. , mixing, granulating or coating according to conventional methods.

The present invention provides a food composition for preventing or improving diabetes comprising the heneicosane as an active ingredient. The food composition may be used as it is, or may be used together with other conventional food composition ingredients, and may be appropriately used according to a conventional method.

The food composition may be contained in health food for the purpose of preventing and improving diabetes, and there is no particular limitation on the type thereof. Examples of foods to which the above substances can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, tea, drinks, There are alcoholic beverages, vitamin complexes, and the like, and includes all health foods in the ordinary sense.

In addition to the above, the food composition of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, It may contain alcohol, a carbonation agent used in carbonated beverages, and the like. In addition, the food composition of the present invention may be contained as flesh for the production of natural fruit juice, fruit juice beverage and vegetable beverage. These components may be used independently or in combination. The proportion of these additives is not very important, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

Diabetes is divided into five types according to the etiology, but clinically, it is broadly classified into insulin-dependent (or type 1) and non-insulin-dependent (or type 2) diabetes for convenience.

Insulin type 2 diabetes means that insulin is secreted from β cells, but insulin in the blood does not act due to increased resistance to insulin in peripheral target organs. Therefore, ketoacidosis, autoantibodies, etc. cannot be observed, and in humans, it occurs mainly after the age of 40 and is usually accompanied by obesity.

In non-insulin-dependent diabetes mellitus, diet and exercise are combined, and if not treated in this way, oral hypoglycemic agents are sometimes used. As such oral hypoglycemic agents, metformin and biguanide drugs applied to obese patients and sulfonylurea drugs applied to non-obese patients are mainly used. are accompanied by side effects of severe lactic acidemia and hypoglycemia, respectively.

The pharmaceutical composition according to an embodiment of the present invention can be used as a substitute for insulin. For example, insulin can be prescribed and used instead of insulin for a variety of ailments that can relieve symptoms. In particular, the pharmaceutical composition is effective for preventing or treating type 2 diabetes.

In addition, in one embodiment of the present invention, the pharmaceutical composition can regulate adipocyte differentiation and lipid accumulation through up-regulation of PPARγ and C/EBP-α protein.

The PPARγ is a peroxisome proliferator activated receptor, a group of nuclear receptor proteins that function as a transcription factor regulating the expression of genes. PPARγ plays an essential role in cellular differentiation, regulation of development and metabolism, and tumorigenesis of higher organisms.

The C/EBP-α (Ccaat-enhancer-binding proteins-α) protein is CCAAT/enhancer-binding protein-α is a protein encoded by the CEBPA gene in humans, and CCAAT/enhancer-binding protein-α is a specific blood protein. It is a transcription factor involved in cell differentiation.

In one embodiment of the present invention, the pharmaceutical composition may decrease serum glucose concentration by increasing Akt/Fox/GSK3β phosphorylation and inhibit gluconeogenic metabolism.

The present invention provides a pharmaceutical or food composition for lowering blood sugar comprising the heneicosane as an active ingredient. In the pharmaceutical or food composition, henicoic acid exhibits a blood sugar lowering effect by affecting insulin or canonical insulin signaling pathways that enhance glycogen synthesis in muscle or liver.

The present invention provides a pharmaceutical composition or a food composition for the treatment of cardiovascular diseases comprising the heneicosane as an active ingredient. In the pharmaceutical composition or food composition, henicoic acid may show an effect in alleviating symptoms and treating cardiovascular diseases by not affecting a lipid profile such as cholesterol.

The present invention provides a pharmaceutical or food composition for protecting the pancreas comprising the heneicosane as an active ingredient. In the pharmaceutical composition or food composition, henicoic acid can protect the pancreas.

[Experimental example]

<Materials and Methods>

The heneicosane solution of the present invention was prepared as follows. DSPE-PEG (SigmaAldrich # 880128P) 10mg, 1,2-dipalmitoyl-Sn-glycero-3-phosphoethanolamine (1,2-dipalmitoyl-Sn-glycero-3-phosphoethanolamine ( SigmaAldrich # P1348))) 4 mg, L-α-phosphatidylcholine (L-α-phosphatidylcholine (Sigma Aldrich # P3556)) 6 mg, and henicoic acid (Sigma Aldrich, St. Louis, USA #51523) 2-4 mg were completely dissolved.

The solution was placed in a small round bottom flask and the organic solvent was evaporated under reduced pressure using a rotary evaporator at 40° C. to 50° C. for 30 minutes. Thereafter, deionized water was added to the flask containing the solution, followed by ultrasonication at 30 times per second for 30 seconds. The solution subjected to the above process was lyophilized and stored at 4° C. or lower until use. Empty liposomes were prepared according to the same procedure as above in the absence of heneichoic acid.

The morphological observation of the liposome was performed using a transmission electron microscope (TEM) (H-7600, Hitachi Instruments Ltd., and Tokyo, Japan). The aqueous liposome solution was dropped onto a carbon film coated copper grid and then dried overnight at room temperature. Thereafter, observation was made at 80 kV and the particle size was measured with nano-ZS at room temperature. At this time, the nanoparticle content in the water was less than 0.1% by weight.

Since heneicosan has strong lipophilicity, liposomes loaded with heneicosan were prepared, and their physicochemical properties were analyzed using TEM and particle size measurement. The empty liposome and the liposome filled with henicoic acid were typical bilayer structures having a small diameter of about 100 nm, and their shapes were similar to each other. As the content of heneicosan increased, the particle size (<300 nm) also gradually increased.

<Differentiation of adipocytes by Heneiko acid>

3T3-L1 cells were thawed and grown for 48 hours in a T175 flask containing a growth medium (DMEM-30-2002) containing 10% fetal bovine serum (FBS). The cultured cells were harvested, seeded on a cell culture plate, incubated at 5% CO ₂ , 37° C., and grown for 3 days until the cells were confluent. After the cells were fused, the cells were maintained in growth medium for an additional 48 hours. Then, the cells were differentiated for 48 hours in differentiation induction medium (DMI) containing insulin 1 μg/mL, dexamethasone 1 μM and IBMX 0.5 mM, then the medium was replaced with insulin medium and cultured for 48 hours. did.

The cells subjected to the above process were cultured in a growth medium for 10 days. When differentiation induction started at the indicated concentration, Heneicosan was treated. For the heneichoic acid experiments, cells were differentiated in DMI in the presence of different concentrations (12.5-50uM) of heneichoic acid for 48 hours. Thereafter, the cells were cultured in post differentiation medium (1 ug/mL insulin) in the presence of heneichoic acid for 48 hours. Cells were placed in normal growth medium containing heneichoic acid every 2 days until the end of the experimental period.

<Quantification of lipids using Oil Red O staining>

Differentiated 3T3-L1 adipocytes in 6 well plates were fixed with 2 mL of 10% formalin for 1 hour and then washed with 40% isopropanol (Fisher Scientific #67630). After washing, 3 mL of Oil Red O (Sigma Aldrich #O9755) staining solution was added to each well plate. 3T3-L1 adipocytes in the well plate were incubated at room temperature for 15 minutes and washed 3 times with distilled water. The stained cells were photographed with an inverted microscope [CKX41, Olympus Corporation, Tokyo Japan]. Additionally, lipids stained through Oil Red O were extracted with 99% isopropyl alcohol from control and Heneicosan-treated cells and measured at 490 nm on days 5 and 10.

<Quantification of gene expression by qPCR>

Total RNA was extracted from cells using the RNeasy Lipid Mini Kit (Qiagen, MD, USA#74804) and quantified by Spectramax i3 (Molecular Devices, CA, USA). Total RNA (500 ng) was reverse transcribed using the iScript cDNA synthesis kit (Hercules, CA, USA# 1708891). Gene transcripts in experimental samples were quantified with a Universal probes supermix (Biorad #172-5130) by a CFX96 real-time PCR detection system (Hercules, CA, USA) with FAM mode using specific probes. (PPARγ (#4331182), C/EBPa (#4331182), and β (#4331182), Thermofisher Scientific, USA). All gene expression was generalized to the housekeeping gene β-actin.

<Protein extraction and immunoblotting from experimental cells>

Protein lysates were prepared using radioimmunoprecipitation assay buffer (RIPA-MB0300050) (Rockland, Limerick, PA) with 1X protease and phosphatase inhibitors (Quartette, Berlin, germany #PPI1015 and Sigma Aldrich, St. Louis, USA#5870). and prepared from cells.

After washing the cells 3 times with PBS, the required volume of RIPA lysis buffer was added based on the plate type, and then the cells were incubated at 4°C for 5 minutes. The cells were rapidly scraped with a cell scraper (TPP, Trasadingen, Switzerland #99010) to remove residual cells and lysed. The cell lysate was transferred to a 2 mL tube and centrifuged at 4 °C for 10 min at 8,000 g. Protein concentration was quantified by a Pierce BCA protein assay kit (Thermofisher Scientific, Massachusetts, USA #23227).

Protein samples were separated by SDS-PAGE (4-12 %, Biorad, California, USA # 456-1094; 456-1095) under Tris glycine (Biorad #161-0734) and polyvinylidene difluoride (PVDF; Polyvinylidene difluoride) membrane (Trans-blot Turbo transfer system, Biorad#1704156;170-4155). Immunoblotting was performed using rabbit monoclonal and polyclonal antibodies according to the Western Breeze Chemiluminescence Kit (Invitrogen, Massachusetts, USA#WB7106). All primary antibody reactions were performed overnight at 4 °C for the specific protein (cell signal technology antibody 1:1000; Abcam antibody 1:1000). HRP-conjugated secondary antibody was used for detection of primary antibody. The band signal was analyzed with an excellent chemiluminescence kit (Bio-Rad# 170-5061) in a chemiluminescence imaging system (Davinch gel imaging system, Seoul, South Korea#CAS400MF). The intensity of the immunoreactive bands was quantified with ImageJ software-1.49 version (32-bit) (Wayne Rasband, National Institute of Health, USA).

<Antibody and reagent>

Antibodies to the targeted protein were rabbit monoclonal PPARγ (# 2435), C/EBP-α (# 8178), FAS (# 3180), ACC (# 3676), GAPDH (# 5174), aP2 (# 2120) , Adiponectin (# 2789), Akt (Pan # 4685), Phospho Akt S473 (# 4060), Phospho AktT308 (# 13038), Total FoxO1 (# 2880), Phospho FoxO1 S256 (# 9461), Total GSK-3β (9315), phospho GSK-β (#9336) were purchased from (Cell Signaling Technology, MA, Danvers, USA).

<In Vivo - Normal Mouse Model>

Animal experiments were performed under the ethical management of animals and the use of laboratory animals (PNUIACUC for Ethical Procedures and Scientific Controls; Approval No.: PNU-2017-1610). ICR rats (10 weeks old, weighing 35-40 g) were used in the study. All rats were housed without specific pathogens under 12 hours of light and 12 hours of dark room, and allowed free access to water and food.

The rats were divided into three groups of 11 rats. Group 1 was a control group, group 2 was a group fed 25 mg/kg of heneichoic acid for 12 weeks, and group 3 was a group fed 50 mg/kg of heneichoic acid for 12 weeks. The heneicosan was mixed with food powder at a food concentration of 2.5 g/kg and provided in granules. The control group was given food pellets without heneichoic acid. Body weight and calorie intake were monitored throughout the experiment. After the experiment was finished, the mice were anesthetized with chloroform, and then killed by cervical dislocation, and whole blood and organs were stored for further analysis.

<Anti-diabetes experiment in vivo- Streptozotosin induction model>

Animal experiments were conducted under ethical animal care and use of laboratory animals. ICR rats (10 weeks old, weighing 35-40 g) were used in the study. All rats were housed without specific pathogens under 12 hours of light and 12 hours of dark room, and allowed free access to water and food.

Intraperitoneal injection was injected into animals with freshly prepared streptozotocin (Merck, USA # S0130) in sodium citrate buffer (50 mg/kg) at pH 4.5, 0.01 M after fasting for 6 hours for 5 consecutive days. Diabetes mellitus in the fasting state was confirmed with a sugar level of >250 mg/dl. After diabetes was confirmed, diabetic rats were randomly divided into 3 subgroups with 10 rats per group. Group 1 is a diabetic control group in which only excipients are administered to rats, Group 2 is an experimental group in which 12.5 mg/kg of henicoic acid is administered per rat, and group 3 is a control group in which henicoic acid is administered at 25 mg/kg for 4 weeks. is the experimental group. The blood glucose level of the mice was checked using a blood glucose meter (OneTouch UltraEasy, USA#). Heneicosan (Sigma Aldrich, USA) was mixed with food powder at a concentration of 2.5 g/kg and food granules were provided. The control group was given food pellets without heneichoic acid. Body weight and calorie intake were monitored throughout the experiment.

<In vivo diabetes test - db/db animal model>

The db/db mice used in the present invention naturally increased blood glucose by genetic manipulation. Animal experiments were conducted under ethical animal care and use of laboratory animals. db/db xenogeneic mice (C57BLKS / J Lar-m + / Leprdb mice and db / db mice) were purchased from SLC Experimental Animal Co., Ltd., Japan. All mice were acclimatized for 1 week to a temperature of 23 ± 3 °C, relative humidity of 55 ± 15%, ventilation 10 to 20 times per hour, and a light-dark cycle of 12 hours (8 am to 8 pm). After one week, they were divided into five groups of 11 rats per group.

Group 1 was fed normal food and water to control db/db xenogeneic mice. Group 2 was fed normal food and water to control db/db mice. Group 3 was administered 12.5 mg/kg of heneichoic acid to db/db mice. Group 4 was administered 25 mg/kg of heneichoic acid to db/db mice. Group 5 was administered metformin at 100 mg/kg to db/db mice for 6 weeks. Heneicosan and metformin (Sigma Aldrich, USA# PHR1084) were mixed with food powder at a food concentration of 2.5 g/kg, and then provided as food granules. The control group was given food pellets without heneichoic acid. Body weight and calorie intake were monitored throughout the experiment. To measure postprandial blood glucose in rats, blood was collected from the tail. Fasting blood glucose was measured the next morning after fasting for about 16 hours using a blood glucose meter (Accu-Check, Performa, Roche Co). After the end of the experiment, whole blood and organs were stored for further analysis.

<Tissue homogenization and quantification of protein>

Protease inhibitor (sodium azide), 1.0 mM phenylmethylsulfonyl fluoride (PMSF; phenylmethylsulfonyl fluoride), 10 μM leupeptin, 0.1 μM aprotinin, 1.0 μM peptin to lyse cells statin (pepstatin) and a phosphatase inhibitor (1.0 mM Na3VO4 and 1.0 mM NaF) in a ratio of 1:4 (150 mM NaCl, 50 mM Tris HCl, 1.0 mM EDTA, 1.0% (v/v) NP-40; Tissues were homogenized in protein lysate containing 0.5% (w/v) sodium deoxycholate, and 0.1% (w/v) SDS). Samples were separated at 9,000 g and 4 °C for 10 min. Proteins were quantified by Biconchoninic acid (BCA) protein assay (Thermofisher Scientific, USA) and used for immunoblot analysis as described above.

<Statistical Analysis>

Data generated from the experiment were applied to one-way ANOVA and multivariate comparative analysis using the Statistical Package for Social Sciences (SPSS-16). <0.05 was considered statistical significance between treatment and no treatment.

<Experiment result>

<Effect of Heneiko acid on adipocyte differentiation and lipid accumulation in 3T3-L1 pre-adipocytes>

FIG. 1A shows that when adipocytes were observed for 5 days in the presence of insulin, IBMX and dexamethasone, treatment with heneichoic acid on day 5 had a greater dependence on lipid concentration than control cells.

Figure 1b shows that when adipocytes were observed for 10 days in the presence of insulin, IBMX and dexamethasone, treatment with heneichoic acid on day 10 had a greater dependence on lipid concentration than control cells.

The graph shows the mean ± SEM for 6 replicates, and statistical analysis was performed using post-hoc tests, comparison with each control group, and a general multivariate linear model.

In addition, as shown in Fig. 2a, treatment with heneichoic acid increased the mRNA of PPARγ, a major transcription factor for adipocyte differentiation and lipid accumulation. As shown in FIG. 2B , treatment with henicoic acid increased the mRNA of C/EBP-α, a major transcription factor for adipocyte differentiation and lipid accumulation. For further confirmation, the expression of proteins involved in differentiation and lipid accumulation in adipocytes was analyzed.

Expression analysis of the protein was determined after normalization to β-actin in the expression of PPARγ and C/EBP-α mRNA (Bars mean±SEM, n=8, p<0.05). Total RNA was extracted and inverted and analyzed via qPCR equipped with specific Taqman probes for quantification of key transcription factors for differentiation and lipid accumulation in experimental adipocytes on days 5 and 10. The above analytical data demonstrated that treatment with heneichoic acid positively regulates adipocyte differentiation and lipid accumulation through upregulation of the major adipogenic factors PPARγ and C/EBP-α protein.

3a shows the results of electrophoresis of Heneiko acid in PPARγ, C/EBP-α, ACC, FAS, aP2, AdipoQ, and GAPDH, and FIG. 3b shows the PPARγ/GAPDH of the control group, H25 and H50 as a histogram. 3c is a bar graph showing C/EBP-α/GAPDH of the control group, H25 and H50, FIG. 3d is a bar graph showing ACC/GAPDH of the control group, H25 and H50, and FIG. 3e is a control group, H25 and FAS/GAPDH of H50 as a histogram, FIG. 3f is a bar graph of aP2/GAPDH of control, H25 and H50, and FIG. 3g is a bar graph of AdipoQ/GAPDH of control, H25 and H50. .

Protein lysates were prepared on day 10 and immunoblotted using specific antibodies against PPARγ, C/EBP-α, ACC, FAS, aP2, AdipoQ and GAPDH (bar mean±SEM, n=5, p<0.05). used for ting. The intensity of protein response bands was determined by densitometry using Image J software.

As shown in FIGS. 3A to 3G , the Heneicosan treatment group up-regulated FAS, ACC and aP2 protein expression compared to the control cells. Low expression of adiponectin was recorded in the control group. However, adiponectin protein was significantly increased in adipocytes in response to heneichoic acid treatment compared to the control group (p<0.05).

As shown in FIGS. 4A to 4C , the effect of heneiko acid on the expression of PPARγ and C/EBP-α protein is similar to that of rosiglitazone treatment. As sequential phosphorylation of Akt at Ser473 and Thr308 is required for activation of differentiation and production in adipocytes, Akt phosphorylation levels on each amino acid residue were determined by immunoblotting with specific anti-phospho Akt antibodies.

As shown in Figs. 5a to 5c, treatment with heneichoic acid enhanced the phosphorylation of Akt at Thr308 and Ser473.

The overall results confirmed that heneichoic acid promotes differentiation and adipogenesis in adipocytes through the Akt-mediated signaling pathway (Bars mean±SEM, n=3, p<0.05). Different a, b, c, and d within the columns indicate significant differences between groups (p<0.05), and each experiment was performed at least 2 or 3 times.

Unlike treatment with rosiglitazone, this positive modulation by heneichoic acid is similar to insulin activity, with Akt phosphorylated heneichoic acid at Ser473 and Thr308 in adipocytes compared to control and rosiglitazone treatment. as demonstrated by

<Effect of heneichoic acid on mice in general>

Heneicosan-treated 3T3-L1 adipocytes showed increased differentiation and lipid accumulation, and at this time, the role of heneicosan was confirmed in a normal mouse model. Heneicosan was orally administered to rats at concentrations of 25 mg and 50 mg/kg (body weight).

Heneicosan 25mg/kg and 50mg/kg concentrations were orally administered to rats, and after treatment with heneicosan, the weight of the rats was analyzed over time compared to the control group. Figure 6a is fed to rats that have increased their body weight for 9 weeks by mixing heneichoic acid with a feed, and comparing the weight of the rats with a control group.

As shown in FIG. 6b , treatment with henicoic acid reduced the serum glucose level compared to control mice. As shown in Figures 6c-6e, even after long-term treatment, mice treated with the indicated concentrations of heneichoic acid did not induce any toxic signals identified by serum liver markers such as AST, ALT and renal marker creatinine levels. . Also, as shown in FIGS. 6f to 6i , heneichoic acid did not significantly affect lipid profiles such as cholesterol, LDL, triglycerides and HDL than the control mice.

In the above experiment, the bars represent the mean ± SEM for 5 repetitions, and statistical analysis was performed using a post-hoc test and comparison with each control and a general multivariate linear model. Different letters a, b, c, and d within the column indicate significant differences between groups (p<0.05). The experiment was performed at least twice.

Akt, FoxO1 and GSK3β phosphorylation levels were analyzed by western blot in muscle and liver tissues of control and henicoic acid-treated animals. Akt phosphorylation levels on Ser473 and Thr308 were significantly increased in muscle and liver tissues of animals treated with henicoic acid compared with control mice.

7A to 7E, as determined by western blot, immunoblots of cell lysates of muscle tissue treated with heneichoic acid showed the levels of Akt (Ser473 and Thr308), FoxO1 (S256) and GSK3β (S9). It shows that the phosphorylation level is increased. Data represent mean ± SEM (n=3, p value 0.05). Different letters a and b within the columns indicate significant differences between the experimental and control groups (p<0.05).

8A to 8E , the phosphorylation levels of Akt (Ser473 and Thr308), FoxO1 (S256) and GSK3β (S9) as determined by western blot immunoblots of cytolysates of liver tissues treated with henicoic acid. shows that it has increased. Data represent mean ± SEM (n=3, p value 0.05). Different letters a and b within the columns indicate significant differences between the experimental and control groups (p<0.05).

From the above results, the phosphorylation levels of FoxO1 and GSK3β(GSK3) were significantly increased ^{in pFoxO1 Ser256} and pGSK ^{S9, respectively, in muscle and liver tissues of animals treated with henicoic acid than in the control group.} Phosphorylating levels varied according to the concentration-dependent treatment with heneichoic acid.

<Effect of Heneico Acid on Chemical-Induced and Genetically Modified Diabetic Animals>

Heneiko acid treatment decreased serum glucose concentration and inhibited gluconeogenic metabolism by increasing Akt/Fox/GSK3β phosphorylation in a normal mouse model. The effect of heneichoic acid on blood glucose levels in the fasting state, which plays an important role in maintaining blood glucose homeostasis, was confirmed. Streptozotocin is a widely used chemical in the pathogenesis of diabetes in rodents (Lenzen, 2008). In the present invention, diabetic rats induced by streptozotocin (multiple intraperitoneal injections, inducing diabetic state in rats) were orally treated with henicosan for 4 weeks and used. Fasting blood glucose levels were measured weekly for 4 weeks. In the blood glucose measurement process, the blood glucose level was monitored using a blood glucose meter (OneTouch UltraEasy, USA#).

As shown in FIG. 9 , the diabetic rats treated with henicoic acid had significantly lower fasting blood glucose levels compared to the control diabetic rats. Heneicosan more intensely decreased the fasting blood glucose concentration in STZ-induced diabetic rats compared to the diabetic control group (p<0.05). In addition, STZ treatment reduced the body weight of diabetic rats throughout the experiment. Heneicosan treatment did not affect body weight until 3 weeks. In addition, the fasting blood glucose concentration was lower in the group receiving Heneicosan for 4 weeks. Data represent mean ± SEN (n=9 rats/group 1, p-value 0.05). Different letters a and b within the columns indicate significant differences between the experimental and control groups (p<0.05).

As shown in FIG. 10 , at the 4th week, the mice administered with 12.5 and 25 mg of Heneicosan slightly increased in body weight compared to the diabetic control group (p<0.07 and p<0.28). Both normal and STZ-induced diabetic rats potentially responded to 12.5 mg/kg of heneichoic acid. Data represent mean ± SEN (n=9 rats/group 1, p values were 0.07 & 0.28 for 12.5 mg and 25 mg heneichoic acid compared to diabetic animals).

<Heneicoic acid reduces fasting blood glucose, insulin, and glucose homeostasis in Lar-Leprdb/Leprdb rats>

Since Lar-Leprdb/Leprdb mice are the most widely used animal model of type 2 diabetes (Zahraa et al., 2017), the efficacy of heneichoic acid in lowering blood glucose in animals with STZ-induced diabetes was analyzed in a leptin receptor-deficient animal model. . Here, C57BLKS/J Lar-Leprdb/Leprdb mice were treated for 5 weeks by oral administration of heneikosan.

11 is a line graph showing the weekly measurement of fasting blood glucose of LC, DC, H12.5, H25 and H50. Treatment with heneichoic acid for 5 weeks significantly reduced fasting blood glucose concentrations in a concentration-dependent manner compared to the diabetic control and metformin groups. Different letters a and b within the line indicate significant differences between the experimental group and the control group. The line graph represents the mean ± SEM (n=7 rats/1 group, p value 0.05).

In the above description, LC is a skinny control rat, DC is a db/db control rat, H12.5 is a rat treated with 12.5 mg of heneichoic acid/kg (body weight), and H25 is a rat with 25 mg of heneichoic acid. rats treated with /kg (body weight), M100 represents rats treated with 100 mg metformin/kg.

12 is a line graph showing postprandial blood glucose measurements of LC, DC, H12.5, H25 and H50 weekly. Treatment with heneichoic acid for 5 weeks significantly reduced the fasting blood glucose concentration in a concentration-dependent manner compared to the diabetic control and metformin groups. Different letters a and b within the line indicate significant differences between the experimental group and the control group. The line graph represents the mean ± SEM (n=7 rats/1 group, p value 0.05).

In addition, as shown in FIG. 13 , an oral glucose tolerance test (OGTT) performed in fasting Leprdb/Leprdb rats showed that the blood glucose concentration in rats treated with henicoic acid was higher in rats treated with henicoic acid, compared to rats administered only with excipients in a time-dependent manner. It was found to be significantly (p<0.05) reduced. Data represent mean ± SEM (n=5 rats/1 group, p value 0.05).

From the above results, heneicosan is more effective in lowering fasting blood sugar than metformin.

In the present invention, insulin secretion was quantified in db/db mice treated with control and henicosan/metformin using an enzyme-linked immunoprecipitation assay (ELISA). Figure 14 is a graph showing the measured insulin levels of LC, DC, H12.5, H25 and M100 as a line graph. Data represent mean ± SEM (n=5 rats/1 group, p value 0.05). As shown in FIG. 14 , basal blood insulin levels at the end of the experimental period were significantly elevated as a result of treatment with heneichoic acid and metformin.

In the present invention, proteins were extracted from the muscles and livers of laboratory animals, and rabbit mAb antibodies against Akt, FoxO1 and GSK3β were separated by SDS-PAGE for immunoblotting.

Figure 15a shows the electrophoresis results for LC, DC, H12.5, H25, and M100. 15B is a graphical representation of pAkt Ser473/tAkt in the muscles of mice of LC, DC, H12.5, H25, and M100. Figure 15c is a graphical representation of pAkt Thr308/tAkt in the muscles of mice of LC, DC, H12.5, H25, and M100.

Figure 16a shows the electrophoresis results for LC, DC, H12.5, H25, and M100. Figure 16b is a graphical representation of pAkt Ser473/tAkt in the liver of mice of LC, DC, H12.5, H25, and M100. Figure 16c is a graphical representation of pAkt Thr308/tAkt in the liver of mice of LC, DC, H12.5, H25, and M100.

As shown in FIGS. 15A-15C and 16A-16C , Akt phosphorylation levels in Akt Ser473 and Akt Thr308 were increased in muscle and liver. The intensity of the protein response band was determined by densitometry using images. Different letters a, b and c within the column indicate significant differences between groups (p<0.05).

Figure 17a shows the electrophoresis results for LC, DC, H25, and M100. Figure 17b is a graphical representation of pFoxO1/tFoxO1 in the muscles of mice of LC, DC, H25, and M100. Figure 17c is a graph showing the pGSK / tGSK in the muscles of mice of LC, DC, H25, and M100.

18A shows the electrophoresis results for LC, DC, H25, and M100. Figure 18b is a graphical representation of pFoxO1/tFoxO1 in the liver of mice of LC, DC, H25, and M100. Figure 18c is a graphical representation of pGSK / tGSK in the liver of experimental mice of LC, DC, H25, and M100.

As shown in FIGS. 17A-17C and 18A-18C , the regulation of GSK3β and FoxO1 expression among glycogenic gene expression was inhibited by Heneicosan in muscle and liver tissues of mice.

From the above results, heneicosic acid exhibits a blood sugar lowering effect by affecting the insulin or canonical insulin signaling pathway that enhances glycogen synthesis in muscle or liver.

Considering the above experimental results, heneiko acid affects the regulatory mechanism underlying diabetes along with the enhancement of adipocyte differentiation. Heneicosan treatment enhanced differentiation and lipid accumulation in 3T3-L1 adipocytes in a concentration-dependent manner. In addition, henicoic acid not only increased the weight of rats without side effects but also lowered the level of serum glucose, and heneichoic acid can regulate gluconeogenesis-related proteins in the muscle and liver of normal rats. Fasting blood glucose concentration was lowered by chemically induced heneichoic acid treatment in the genetically modified diabetic mouse model. Heneicosan regulates glucose metabolism by inhibiting FoxO1 and GSK3β activity by increasing phosphorylation levels through the Akt-mediated signaling pathway.

<pancreatic protective effect>

After completion of the test substance, the mice were anesthetized and histopathology (H&E staining) of the pancreatic tissue was performed to confirm inflammation and lesions in the following manner.

The pancreas was evaluated by assigning 0-3 points to the extent to which the borders of the Langerhans Islets are unclear and some of the endocrine cells are replaced by acinar cells, atrophy, or hypertrophy. That is, the degree of degeneration of Langerhans Island was evaluated as 0-3 points.

The results are as shown in Table 1 below. The LC (normal) group maintained a normal histological structure in the pancreas, but in the DC, H12.5, H25, and M100 groups, degeneration of Langerhans islets was observed in the pancreas. Representative histopathological photographs are shown in FIGS. 19a to 19e below.

army Pancreas Degeneration LC 0.18±0.40 DC 2.27±0.47 H12.5 2.00±0.77 H25 1.64±0.67 M100 1.64±0.50

<Scoring results of histopathological lesions of the liver and pancreas>

19A to 19E show histopathological photographs in LC, DC, H12.5, H25, and M100, respectively. As shown, H12.5 showed a mild pancreatic protective effect compared to DC, but in H25, the pancreas The protective effect was remarkable, and the effect was similar to that of M100.

Claims (15)

A pharmaceutical composition for preventing or treating diabetes comprising heneicosane as an active ingredient. According to claim 1,
The diabetes is type 2 diabetes, a pharmaceutical composition for preventing or treating diabetes. 3. The method of claim 2,
The pharmaceutical composition is a pharmaceutical composition for preventing or treating diabetes that regulates adipocyte differentiation and lipid accumulation through up-regulation of PPARγ and C/EBP-α protein. 4. The method of claim 3,
The pharmaceutical composition is a pharmaceutical composition for preventing or treating diabetes by increasing Akt/Fox/GSK3β phosphorylation, thereby reducing serum glucose concentration and inhibiting gluconeogenic metabolism. 5. The method according to any one of claims 1 to 4,
The pharmaceutical composition is a pharmaceutical composition for preventing or treating diabetes administered by oral administration or injection into the body. A food composition for preventing or improving diabetes comprising heneicosane as an active ingredient. 7. The method of claim 6,
The food composition is a food composition for preventing or improving diabetes by regulating adipocyte differentiation and lipid accumulation through upregulation of PPARγ and C/EBP-α protein. 8. The method of claim 7,
The food composition is a food composition for preventing or improving diabetes by increasing Akt / Fox / GSK3β phosphorylation, thereby reducing serum glucose concentration and inhibiting gluconeogenic metabolism. 9. The method according to any one of claims 6 to 8,
The food composition is a food composition for preventing or improving diabetes that is administered orally. A pharmaceutical composition for lowering blood sugar comprising heneicosane as an active ingredient. A food composition for lowering blood sugar comprising heneicosane as an active ingredient. A pharmaceutical composition for the treatment of cardiovascular diseases comprising heneicosane as an active ingredient. A food composition for the treatment of cardiovascular diseases comprising heneicosane as an active ingredient. A pharmaceutical composition for protecting the pancreas comprising heneicosane as an active ingredient. A food composition for protecting the pancreas comprising heneicosane as an active ingredient.

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