KR102489413B1 - Composition for promoting insulin secretion comprising extract of Cornus walteri - Google Patents

Abstract

It relates to a composition for stimulating insulin secretion containing a compound of tirucallane triterpenoid, a pharmaceutical composition for preventing or treating type 2 diabetes (T2D) containing the same, and a health functional food, It can exert excellent effects in maintaining, improving function, controlling insulin, preventing, improving, and treating type 2 diabetes (T2D).

Description

Composition for promoting insulin secretion comprising extract of Cornus walteri}

It relates to a composition for stimulating insulin secretion containing a compound of tirucallane triterpenoid, a pharmaceutical composition for preventing or treating type 2 diabetes (T2D) containing the same, and a health functional food.

Diabetes mellitus, which is one of the most common diseases in modern people, is a kind of metabolic disease such as insufficient secretion of insulin or failure of normal function, and is characterized by high blood glucose concentration, and causes various symptoms and signs due to hyperglycemia.

Diabetes is an abnormal action of insulin secreted by the pancreas, which causes abnormalities in carbohydrate metabolism as well as protein, lipid, and electrolyte metabolism. It can cause complications such as nephropathy, neurosis, nephrotoxicosis and coma, leading to death. Representative symptoms of diabetes include polyuria, polydipsia, and the following, accompanied by chronic fatigue.

Diabetes is largely classified into two types, namely type 1 diabetes and type 2 diabetes. Type 1 diabetes is caused by a lack of secretion of insulin, a hormone that regulates glucose in the blood. It occurs in the age group. Type 2 diabetes usually develops after the age of 40 and accounts for the majority of diabetic patients. As the etiology of type 2 diabetes, both insulin secretion disorders in pancreatic beta cells and insulin action defects in target cells (insulin resistance) are observed.

Insulin resistance is a state in which the action of insulin is reduced in peripheral tissues and is the main cause of type 2 diabetes. Currently, 90% of diabetes is type 2 diabetes, and type 2 diabetes causes persistent hyperglycemia due to insulin resistance, reduced insulin action, and inefficient insulin production in the pancreas (Int J Appl Res Nat Prod, 2016, 9 :33-38), when chronic, it is accompanied by very serious complications such as diabetic retinopathy, renal failure, hypertension, and arteriosclerosis. Therefore, type 2 diabetes has emerged as an urgent problem to be solved both medically and socioeconomically.

Currently known type 2 diabetes treatments, biguanides that move sugar into muscle cells and inhibit sugar synthesis in the liver, and thiazolidinediones that activate PPARγ related to adipocyte differentiation ( thiazolidinediones), sulfonylureas that indirectly induce secretion of insulin, and α-glucosidase inhibitors that inhibit enzymes that make glucose in the small intestine are used, but diabetes using these drugs Treatment has many side effects (Diabetes, 2008, 57:737-745), so the World Health Organization (WHO) strongly recommends using natural products with fewer side effects for diabetes.

Accordingly, while researching a composition for the treatment and prevention of diabetes using natural products, the present inventor found that (-)-Leucophyllone compound obtained from dogwood is non-toxic to pancreatic cells and maintains the function of pancreatic cells. And it was found that there was an effect in improving the function of the gene promoter in insulin, and the invention was completed.

Korean Patent Registration No. 10-1269208

An object of the present invention is to provide a composition for stimulating insulin secretion containing a compound of tirucallane triterpenoid, a pharmaceutical composition for preventing or treating type 2 diabetes (T2D) containing the same, and a health functional food. .

According to one embodiment of the present invention, a composition for stimulating insulin secretion containing a tirucallane triterpenoid-like compound is provided.

In the present invention, the tirucallane triterpenoid compounds may provide a composition for stimulating insulin secretion extracted from Cornus walteri .

In the present invention, the tirucallane triterpenoid compound can provide a composition for stimulating insulin secretion including a (-)-leucophyllone compound represented by Formula 1 below. there is.

[Formula 1]

In the present invention, the composition may provide an insulin secretion stimulating composition having a non-toxic effect on INS-1 cell line-derived cells.

In the present invention, the composition may provide an insulin secretion stimulating composition having an effect of regulating PDX-1 expression through IRS-2, PI3K, and Akt signaling pathways.

In the present invention, the composition has the effect of protein expression of IRS-2 (Ser731), P-IRS-2, PI3K, P-PI3K, Akt, P-Akt (Ser473) and PDX-1 in cells derived from the INS-1 cell line. Eggplant can provide an insulin secretion stimulating composition.

In the present invention, the composition may provide an insulin secretion stimulating composition that enhances glucose-stimulated insulin secretion (GSIS).

In the present invention, the composition provides an insulin secretion stimulating composition for any one or more selected from the group consisting of maintenance, improvement, insulin control, prevention, improvement and treatment of pancreatic cell function. can do.

According to another embodiment of the present invention, a pharmaceutical composition for preventing or treating type 2 diabetes (T2D) containing the insulin secretion stimulating composition is provided.

According to another embodiment of the present invention, a health functional food for preventing or improving type 2 diabetes (T2D) containing the insulin secretion promoting composition is provided.

A composition for stimulating insulin secretion containing a tirucallane triterpenoid compound according to an embodiment of the present invention, a pharmaceutical composition for preventing or treating type 2 diabetes (T2D) containing the same, and a health function Food can exert excellent effects on maintaining and improving the function of pancreatic cells, controlling insulin, and preventing, improving, and treating type 2 diabetes (T2D).

Figure 1A is a graph showing the experimental results of analyzing the effect of (-) -leucophyllone using the EZ-Cytox cell viability assay for the viability of INS-1 cells treated with the determined control group (0 μM) and (-)-leucophyllone. , Compared to the control, (-)-leucophyllone treatment at concentrations of 2.5, 5, and 10 μM does not affect cell viability and is an image confirming that it is non-toxic to cells.
Figure 1B shows the effect of (-)-leucophyllone on glucose-stimulated insulin secretion (GSIS, 400,000 ng/mL per cell) after treatment with (-)-leucophyllone at concentrations of 2.5, 5, and 10 μM in INS-1 cells for 1 hour, respectively. It is a result image showing the comparison of the effect and the determined control group (0 μM) by GSIS analysis.
Figure 1C is an image of the comparison of GSIS expressed as a glucose stimulation index (GSI, 16.7 mM glucose to 2.8 mM glucose for 1 hour) fold stimulation.
Figure 2A shows the proteins of P-IRS-2 (Ser731), IRS-2, P-PI3K, PI3K, P-Akt (Ser473), Akt, PDX-1 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in INS. expression level It is an image shown as a result of Western blotting experiment.
2B, 2C, 2D, and 2E are bar graph images showing the density of the western blotting bands of cells treated with (-)-leucophyllone at concentrations of 5 and 10 μM for 24 hours.
Figure 3 is a schematic diagram of the effect of (-) -leucophyllone isolated from dogwood on the insulin receptor substrate IRS-2, phosphatidylinositol 3-kinase (PI3K), Akt, and protein expression levels of the pancreas and duodenum, INS-1 It is an image showing cell homeobox-1 (PDX-1).

Hereinafter, an example will be described in detail for a more detailed explanation. However, the following examples are illustrative, and the scope of the present invention is not limited thereto.

According to one embodiment of the present invention, it relates to a composition for stimulating insulin secretion comprising a tirucallane triterpenoid-type compound.

The tirucallane triterpenoid compounds of the present invention may be a composition for stimulating insulin secretion extracted from cornus walteri .

In the present invention, cornus walteri is a dogwood tree belonging to the deciduous tree of the dicotyledonous plant umbel ( Apiales ) dogwood family ( Cornaceae ), and they may be used alone or in combination, and there is no particular limitation. .

In the present invention, "extract" includes all materials obtained by extracting components therein from natural products, regardless of the extraction method or the type of component. For example, it is a concept in a broad sense that includes all of those obtained by extracting a component soluble in a solvent from a natural product using water or an organic solvent, and extracting only a specific component of a natural product. The insulin composition stimulating agent of the present invention contains the extract of Dogchae tree as an active ingredient, and has an effect of stimulating insulin secretion. The extracted part of the dogwood tree may be any one or more parts or outposts selected from the group consisting of leaves, stems, roots and flowers, but is not limited thereto. Preferably, in the present invention, the dogwood extract may be extracted using the stem and stem bark of the dogwood tree.

In the present invention, the tirucallane triterpenoid compound is a concept included in the triterpenoid-based compound, and can be interpreted as a class of chemical compounds composed of three terpene units of molecular formula C ₃₀ H ₄₈ It is easily understood by those skilled in the art.

The tirucallane triterpenoid compound of the present invention may be a composition for stimulating insulin secretion including a (-)-leucophyllone compound represented by Formula 1 below.

[Formula 1]

In the present invention, (-)-Leucophyllone is a chemical compound composed of molecular formula C ₃₁ H ₅₀ O ₂ and serves as a plant metabolite, specifically ether, tyrucalan triterpenoid and cyclic terpene. It may correspond to ketone.

The composition of the present invention may be an insulin secretion stimulating composition having a non-toxic effect on INS-1 cell line-derived cells.

세포로서 인슐린을 합성, 분비하여 포도당 자극에 따라 혈당을 조절하는 역할을 하며, 구체적으로

세포에서의 인슐린 분비는 포도당의 유입, 포도당 대사, TCA회로, 산화적 인산화 과정을 거쳐 ATP/ADP 비율 이 증가되면 KATP channel이 닫히고 그에 따른 탈분극에 의해 전압 의존적인 Ca2+ channel이 열려서 Ca2+ 유입되는 경로와 증가한 [Ca2+]i에 의한 인슐린 분비의 효율을 높이는 신호 전달 경로가 생성되어 이루어지기에, INS-1 세포주를 이용하여 포도당 자극 인슐린 분비 저해 기작 연구를 수행 할 수 있음은 당업계 통상의 실시자에게 쉽게 이해되는 것이다.In the present invention, the INS-1 cell line is a pancreatic islet, islet cell

As a cell, it synthesizes and secretes insulin and plays a role in regulating blood sugar according to glucose stimulation.

Insulin secretion in cells goes through glucose influx, glucose metabolism, TCA cycle, and oxidative phosphorylation process, and when the ATP/ADP ratio increases, the KATP channel closes and the voltage-dependent Ca2+ channel opens due to the depolarization that leads to the Ca2+ influx pathway. Since a signal transduction pathway that increases the efficiency of insulin secretion by increased [Ca2+]i is created and made, it is known to those skilled in the art that research on the mechanism of inhibition of glucose-stimulated insulin secretion can be performed using the INS-1 cell line. It is easy to understand.

The fact that the insulin secretion stimulating composition of the present invention has a non-toxic effect on INS-1 cell line-derived cells can be interpreted as having stability in cell survival without causing cytotoxicity.

The composition of the present invention may be an insulin secretion stimulating composition having an effect of regulating PDX-1 expression through IRS-2, PI3K, and Akt signaling pathways.

In the present invention, "IRS-2" acts as a molecular adapter between various receptors and downstream effectors, encoding cytoplasmic signaling molecules and insulin receptor substrates 2 that mediate the effects of insulin, insulin-like growth factor 1 and other cytokines. includes "PI3K, Akt" mainly plays a role in signal transduction pathways in skeletal muscle, fat, tissue, liver, brain and pancreas, specifically "PI3K" is a family of lipid kinases that phosphorylate phosphatidylinositol, a component of eukaryotic cell membranes, " Akt" is divided into three isoforms (AKT1, AKT2, and AKT3) according to differences in serine and threonine residues. AKT1 is ubiquitously expressed, and AKT2 is mainly expressed in insulin-sensitive tissues such as skeletal muscle, adipose tissue, and liver. AKT3 can be expressed in testis and brain.

- 세포 기능을 유지하고 인슐린 내 유전자 프로모터를 활성화시키는 중요한 전사 인자의 발현을 조절하는 것은 인슐린 분비 촉진을 조절할 수 있는 것이며 그것의 결핍은 마우스와 인간 췌장

세포에서 결함있는 GSIS를 일으키는 것일 수 있다.Accordingly, it is easily understood by those skilled in the art that normal metabolism is possible through the IRS-2, PI3K, and Akt signaling pathways, and an imbalance can lead to obesity and type 2 diabetes. Expression of PDX-1 via signaling pathway, that is, pancreas in normal GSIS

- Regulating the expression of important transcription factors that maintain cell function and activate gene promoters in insulin can regulate the promotion of insulin secretion, the deficiency of which is in the mouse and human pancreas

It may be what causes defective GSIS in cells.

The composition of the present invention has a protein expression effect of IRS-2 (Ser731), P-IRS-2, PI3K, P-PI3K, Akt, P-Akt (Ser473) and PDX-1 in INS-1 cell line-derived cells It may be an insulin secretion stimulating composition.

In the present invention, "IRS-2 (Ser731), P-IRS-2, PI3K, P-PI3K, Akt, P-Akt (Ser473) and PDX-1" are insulin receptor substrates, and their protein expression levels are known in the art. A person skilled in the art can understand the effect of (-)-Leucophyllone.

The composition of the present invention may be an insulin secretion stimulating composition that enhances glucose-stimulated insulin secretion (GSIS).

In the present invention, "glucose-stimulated insulin secretion (GSIS)" means that glucose is absorbed from food to stimulate insulin secretion in ß cells of the pancreas, but is not limited thereto.

세포 기능을 유지하거나 향상시킴이 가능하고, 제2형 당뇨병(T2D)의 예방 및 치료를 위한 전략적인 접근 방식이 될 수 있다.In the present invention, by enhancing glucose-stimulated insulin secretion (GSIS), the pancreas

It is possible to maintain or improve cellular function and could be a strategic approach for the prevention and treatment of type 2 diabetes (T2D).

The composition of the present invention may be an insulin secretion stimulating composition for any one or more selected from the group consisting of maintenance, improvement, insulin control, prevention, improvement and treatment of pancreatic cell function. .

In the present invention, "diabetes" is meant to include the above-mentioned insulin-dependent diabetes and non-insulin-dependent diabetes, and furthermore, diabetes caused by damage to the pancreas due to other diseases, such as hyperthyroidism, hyperadrenocortical function, It includes diabetes and gestational diabetes caused by excessive growth hormone or excessive catecholamine. Preferably, it means non-insulin dependent type 2 diabetes.

According to one embodiment of the present invention, it relates to a pharmaceutical composition for preventing or treating type 2 diabetes (T2D) comprising an insulin secretion stimulating composition.

In the present invention, the "prevention" refers to any action that suppresses or delays arthritis, and the "treatment" refers to any action that improves or beneficially changes the symptoms of a suspected or affected subject of arthritis. In addition, the pharmaceutical composition may be provided as a pharmaceutical composition including an active ingredient alone or one or more pharmaceutically acceptable carriers, excipients, or diluents. In the above, 'pharmaceutically acceptable' refers to a composition that is physiologically acceptable and does not usually cause an allergic reaction or similar reaction when administered to humans.

Furthermore, the pharmaceutical composition may be provided by mixing with conventionally known therapeutic substances for diabetes. That is, the pharmaceutical composition may be administered in parallel with a known compound having an effect of preventing or treating diabetes.

The term “administration” refers to introducing a predetermined substance into a subject by an appropriate method, and the term “subject” refers to all animals, such as rats, mice, and livestock, including humans who have or may develop diabetes. As a specific example, it may be mammals including humans.

If necessary, the pharmaceutical composition may additionally include an antidiabetic compound. Examples of such antidiabetic compounds include myricetin, cathetin, galgeun, campreol, saffron, cinchonine, anthocyanin, hesperidin, glutathione derivatives, and the like, but are not limited thereto.

In the present invention, "anti-diabetic" refers to lowering blood sugar and preventing, treating, improving, or delaying the onset of diabetes, specifically, hyperglycemia caused by not producing insulin or insufficient insulin, detecting sugar in urine, etc. It means preventing, treating, ameliorating or delaying the onset of pathological symptoms.

The route of administration of the pharmaceutical composition is, but is not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual or work is included

The pharmaceutical composition can be administered orally or parenterally, and in the case of parenteral administration, it is preferable to select an injection method for external skin application or intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, intramuscular injection or intrathoracic injection. , but is not limited thereto, and oral administration may be preferably selected from the viewpoint of selecting a more effective absorption route.

In the case of preparations for oral administration, the composition of the present invention may be formulated into powders, granules, tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc. using methods known in the art. can For example, preparations for oral use may be obtained by combining the active ingredient with a solid excipient, which is then milled and, after adding suitable auxiliaries, processed into a mixture of granules to obtain tablets or dragees. Examples of suitable excipients include sugars including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol and maltitol, starches including corn starch, wheat starch, rice starch and potato starch, cellulose, Celluloses including methyl cellulose, sodium carboxymethylcellulose and hydroxypropylmethyl-cellulose, and the like, fillers such as gelatin, polyvinylpyrrolidone, and the like may be included. In addition, cross-linked polyvinylpyrrolidone, agar, alginic acid or sodium alginate may be added as a disintegrant, if desired. Furthermore, the pharmaceutical composition of the present invention may further include an anticoagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifier, and a preservative.

In the case of preparations for parenteral administration, they may be formulated in the form of injections, creams, lotions, external ointments, oils, moisturizers, gels, aerosols and nasal inhalants by methods known in the art. These formulations are described in Remington's Pharmaceutical Science, 15th Edition, 1975. Mack Publishing Company, Easton, Pennsylvania 18042, Chapter 87: Blaug, Seymour, which is a generally known formula for all pharmaceutical chemistry.

A preferred dosage of the pharmaceutical composition varies depending on the condition and body weight of the patient, the severity of the disease, the type of drug, the route and duration of administration, but can be appropriately selected by those skilled in the art. However, for the desired effect, the pharmaceutical composition of the present invention may vary the content of the active ingredient according to the degree of disease. Preferably, the preferred total dose of the dogwood extract of the present invention may be about 0.01 μg to 1,000 mg, most preferably 0.1 μg to 100 mg per 1 kg of patient body weight per day. However, the dosage of the dogwood extract is effective for the patient in consideration of various factors such as the age, weight, health condition, sex, severity of the disease, diet and excretion rate, as well as the route of administration of the pharmaceutical composition and the number of treatments. Since the amount is determined, considering this point, those skilled in the art will be able to determine an appropriate effective dosage according to the specific use of the dogwood extract as a preventive or therapeutic agent for diabetes. The pharmaceutical composition according to the present invention is not particularly limited in its formulation, administration route and administration method as long as it exhibits the effects of the present invention. When formulating the pharmaceutical composition, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations contain at least one excipient, for example, starch, calcium carbonate, sucrose, etc. Alternatively, it is prepared by mixing lactose and gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, solutions for oral use, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base for the suppository, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used.

Furthermore, the pharmaceutical composition may further include an anticoagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifier, and a preservative.

In the case of preparations for parenteral administration, they can be formulated in the form of injections by a method known in the art, and these formulations are generally known prescriptions in all pharmaceutical chemistry (Remington's Pharmaceutical Science, 15th Edition, 1975. Mack Publishing Company, Easton, Pennsylvania 18042, Chapter 87: Blaug, Seymour).

According to one embodiment of the present invention, it relates to a health functional food for preventing or improving type 2 diabetes (T2D) containing an insulin secretion promoting composition.

The health functional food may be formulated as one selected from the group consisting of tablets, pills, powders, granules, powders, capsules and liquid formulations by further including one or more of carriers, diluents, excipients and additives. Foods to which the acteoside or its food-acceptable salt can be added include various foods, powders, granules, tablets, capsules, syrups, beverages, gums, teas, vitamin complexes, nutritional supplements, health functional foods, and food additives, etc.

Additives that may be further included in the health functional food include natural carbohydrates, flavors, nutrients, vitamins, minerals (electrolytes), flavors (synthetic flavors, natural flavors, etc.), coloring agents, fillers (cheese, chocolate, etc.) , pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, antioxidants, glycerin, alcohols, carbonation agents and at least one component selected from the group consisting of fruit flesh may be used. there is.

Examples of the natural carbohydrates include monosaccharides such as glucose, fructose, and the like; disaccharides such as maltose, sucrose and the like; and polysaccharides such as conventional sugars such as dextrins, cyclodextrins, and the like, and sugar alcohols such as xylitol, sorbitol, and erythritol. As the flavoring agent, natural flavoring agents (thaumatin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. In addition, various Nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and enhancers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, It may contain a pH adjusting agent, a stabilizer, a preservative, glycerin, alcohol, a carbonating agent used in carbonated beverages, etc. In addition, the health functional food may contain fruit flesh for the production of natural fruit juice and vegetable beverages. These components may be used independently or in combination.

Specific examples of the carrier, excipient, diluent, and additive include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, erythritol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium phosphate, calcium Silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, polyvinylpyrrolidone, methylcellulose, water, sugar syrup, methylcellulose, methyl hydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate And at least one selected from the group consisting of mineral oil is preferably used.

When formulating the health functional food, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

Hereinafter, examples will be described in detail for more detailed description.

Example

Preparation Example 1. Extraction and purification of (-)-leucophyllone from dogwood

2.5 kg of dogwood stem and stem bark were dried, chopped, extracted twice in 80% MeOH aqueous solution for 6 hours, and the resulting extract was filtered. The filtrate was concentrated using an evaporator under vacuum to give 220 g of MeOH extract, which was applied to 7.2 L of distilled H ₂ O, followed by solvent fractionation successively with three organic solvents including hexane, CHCl ₃ and n-BuOH. 9.5, 25.0 and 43.0 g of the residue were provided respectively.

9.5 g of the hexane fraction was chromatographed on a 300 g silica gel column with hexane-EtOAc (3:1 to 1:1, gradient solvent system) to obtain F1-F5 fractions. Next, 3.3 g of the F1 fraction was chromatographed with 100% MeOH on a 100 g RP-C18 silica gel column to obtain F11-F15 fractions. Next, 300 mg of the F14 fraction was subjected to medium pressure liquid chromatography (MPLC) using a LiChroprep Lobar-A Si gel 60 column (n-hexane-EtOAc, 16:1), followed by normal phase semi-preparative HPLC and n-hexane: 30mg (0.0136%) of (-)-leucophyllone was purified using a solvent system containing EtOAc (12:1).

Preparation Example 2. INS-1 cell culture

- 세포(INS-1)는 11mM D-포도당(D-glucose), 10 % 태아 소 혈청(fetal bovine serum), 1% 페니실린/스트렙토마이신(penicillin/streptomycin) (Invitrogen Co., Grand Island, NY, USA), 0.05 mM2-메르캅토에탄올(2-mercaptoethanol), 2 mM L-글루타민(L-glutamine), 10 mM 하이드록시에틸피페라진에테인설폰산(HEPES) 및 1 mM 나트륨피루브산(sodium pyruvate)를 포함하는 RPMI-1640 (Cellgro, Manassas, VA, USA)에서 37℃, 5 % CO₂ 및 95 % 습도의 조건으로 배양하였다.Rat pancreas purchased from Biohermes (Shanghai, China)

- Cells (INS-1) were prepared with 11mM D-glucose, 10% fetal bovine serum, and 1% penicillin/streptomycin (Invitrogen Co., Grand Island, NY; USA), 0.05 mM 2-mercaptoethanol, 2 mM L-glutamine, 10 mM hydroxyethylpiperazineethanesulfonic acid (HEPES) and 1 mM sodium pyruvate. were cultured in RPMI-1640 (Cellgro, Manassas, VA, USA) at 37°C, 5% CO ₂ and 95% humidity.

Experimental example 1.

1. Cell viability measurement ( Evaluate the non-toxic dose range of (-)-leucophyllone)

The protective effect against pancreatic toxicity was evaluated using the rat insulinoma cell line INS-1 (Biohermes, Shanghai, China) cells as follows. The INS-1 cells cultured in Preparation Example 2 were dispensed in a 96-well plate at 1Х10 ⁴ cells per well, and then cultured under the same conditions for 24 hours to stabilize the cells. Then, after treatment with (-)-leucophyllone at a concentration of 2.5, 5 or 10 μM, they were cultured under the same conditions for 24 hours. Then, EZ-Cytox cell viability assay solution (100 μL; Daeil Lab Service Co., Ltd., Seoul) was added to the plate and incubated for 40 minutes. Next, the absorbance of the sample in the well plate was measured using a PowerWave XS microplate reader (Bio-Tek Instruments, Winooski, VT, USA) at a wavelength of 450 nm to determine cell viability.

As shown in FIG. 1A, it can be confirmed that the difference between the number of INS-1 cells in the control group (0 μM) and the number of INS-1 cells treated with (-)-leucophyllone at a concentration of 2.5, 5 or 10 μM is minute. 2.5 μM, 5 μM, and 10 μM of (-)-leucophyllone were non-toxic to INS-1 cells.

Experimental Example 2. Evaluation of insulin secretion promotion

1. GSIS (Glucose Stimulated Insulin Secretion) assay

INS-1 cells were dispensed in a 12-well plate at 4 Х 10 ⁵ cells per well and cultured under the same conditions for 24 hours to stabilize the cells. Then 4.8mM KCl, 129mM NaCl, 1.2mM KH ₂ PO ₄ , 1.2mM MgSO ₄ , 2.5mM CaCl ₂ , 10mM HEPES, 5mM NaHCO ₃ and 0.1% bovine serum albumin at 34° C. to 37° C. INS-1 cells were washed twice using Krebs-Ringer's Krebs-Ringer bicarbonate HEPES buffer (KRBB) (pH 7.4). Afterwards, INS-1 cells are transferred to fresh KRBB and starved for 2 h. Next, they were treated with (-)-leucophyllone at concentrations of 2.5, 5, and 10 μM for 1 hour, respectively. INS-1 cells were then stimulated with fresh KRBB containing 3.3 mM or 16.7 mM glucose for 1 hour. Next, the culture supernatant was immediately collected, and GSIS was measured using the rat insulin ELISA kit (Gentaur, Shibayagi Co. Ltd., Gunma, Shibukaw Japan).

The bar presented in Fig. 1B shows the effect on glucose-stimulated insulin secretion (GSIS, 400,000 ng/mL per cell) of INS-1 cells treated with 2.5, 5, and 10 μM concentrations of (-)-leucophyllone for 1 hour, respectively, and the determined control group ( 0 μM) is a result image showing the comparison with GSIS analysis.

According to FIG. 1B, when INS-1 cells treated with 2.5, 5, and 10 μM concentrations of (-)-leucophyllone for 1 hour, respectively, were stimulated with 3.3 mM glucose, there was no difference from the amount of insulin secretion in the control group (0 μM). On the other hand, when stimulated with 16.7mM glucose, it can be seen that the amount of insulin secretion is significantly different from that of the control group (0 μM). Therefore, these results suggest that (-)-leucophylllone has an effect of stimulating insulin secretion in response to hyperglycemia without actually exhibiting toxicity to INS-1 cells.

1C shows that (-)-leucophyllone improves insulin secretion in response to hyperglycemia in terms of GSI values, and the resulting GSI values were 5.16 ± 0.12 and 5.16 ± 0.12 for (-)-leucophyllone at 5 μM and 10 μM, respectively. It is suggested to be 13.11 ± 0.17. Therefore, it was confirmed that (-)-leucophyllone has an effect of stimulating insulin secretion in response to hyperglycemia without actually exhibiting toxicity to INS-1 cells.

2. Western blotting

After dispensing INS-1 cells at 8 Х 10 ⁵ cells per well in a 6-well plate, they were cultured under the same conditions for 24 hours to stabilize the cells. Then, the cells were treated with (-)-leucophyllone at a concentration of 5 or 10 μM and cultured under the same conditions for 24 hours. Next, to extract the total protein lysate, INS-1 cells were lysed on ice for 20 minutes using RIPA buffer (Cell Signaling, Danvers, MA, USA) containing protease inhibitors. . Then, 20 μg of protein sample was separated and tested.

2A shows that the insulin receptor substrates P-IRS-2 (Ser731), IRS-2, P-PI3K, PI3K, P-Akt (Ser473), Akt, PDX-1 and glyceraldehyde 3-phosphate dehydrogenase It is an image of the result of Western blotting experiment on the protein expression level of (GAPDH). Specifically, in the proteins of P-IRS-2 (Ser731), P-PI3K, P-Akt (Ser473), and PDX-1, 5 or 10 μM concentration of (-)-leucophyllone compared to the control group (0 μM concentration of (-)-leucophyllone) It can be seen that the band in the leucophyllone-treated group is clearer and thicker.

2B, 2C, 2D, and 2E show Western blots of P-IRS-2 (Ser731), P-PI3K, P-Akt (Ser473), and PDX-1 cells whose effects were confirmed according to the experimental results of Western blotting. A graph showing the densitometric quantification of the lotting bands. Treatment with (-)-leucophyllone at concentrations of 5 μM and 10 μM in IN S-1 cells significantly increased the protein levels of IRS-2, PI3K, Akt, and PDX-1 compared to untreated controls. It can be seen that the expression increases. These results suggest that (-)-leucophyllone upregulates PDX-1 expression in INS-1 cells through the IRS-2, PI3K, and Akt signaling pathways.

Figure 3 shows a schematic diagram of the effect of (-)-leucophyllone isolated from dogwood on the insulin receptor substrates IRS-2, PI3K, Akt, and the protein expression levels of the pancreas and duodenum. 1 (PDX-1), suggesting the mechanism by which (-)-leucophyllone affects INS-1 cell protein expression in a hyperglycemic environment.

Therefore, through FIGS. 1A to 1C, 2A to 2D, and 3, (-)-leucophyllone promotes insulin secretion in response to hyperglycemia without exhibiting toxicity to INS-1 cells, thereby improving GSIS and IRS. It was confirmed that there was an effect of upregulating PDX-1 expression through activation of -2, PI3K, and Akt signaling pathways.

The scope of the present invention is indicated by the following claims, and all changes or modifications derived from the meaning and scope of the claims and equivalent concepts should be interpreted as being included in the scope of the present invention.

Claims (10)

A composition for stimulating insulin secretion comprising a tirucallane triterpenoid compound extracted from Cornus walteri. delete According to claim 1,
The tirucallane triterpenoid compound is a composition for stimulating insulin secretion comprising a (-)-leucophyllone compound represented by Formula 1 below.
[Formula 1]

According to claim 1,
The composition is an insulin secretion stimulating composition having a non-toxic effect in cells derived from the INS-1 cell line. According to claim 1,
The composition promotes insulin secretion having an effect of regulating PDX-1 expression through IRS-2, PI3K, and Akt signaling pathways. According to claim 1,
The composition promotes insulin secretion having protein expression effects of IRS-2 (Ser731), P-IRS-2, PI3K, P-PI3K, Akt, P-Akt (Ser473) and PDX-1 in INS-1 cell line-derived cells. composition. According to claim 1,
The composition is an insulin secretion stimulating composition that enhances glucose-stimulated insulin secretion (GSIS). According to claim 1,
The composition is an insulin secretion stimulating composition for any one or more selected from the group consisting of maintenance, improvement, insulin control, prevention, improvement and treatment of pancreatic cell function. A pharmaceutical composition for preventing or treating type 2 diabetes (T2D), comprising the insulin secretion stimulating composition according to any one of claims 1 to 8. A health functional food for preventing or improving type 2 diabetes (T2D) comprising the composition for promoting insulin secretion according to any one of claims 1 to 8.

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