KR102161353B1 - Composition for Anti-Diabetes Using an Extract of Lespedeza cuneata - Google Patents

Abstract

The present invention discloses an antidiabetic composition using a non-suri extract that promotes activation (phosphorylation) of AMPK and inactivation (phosphorylation) of ACC and absorption of glucose in muscle cells.

Description

Composition for Anti-Diabetes Using an Extract of Lespedeza cuneata}

The present invention relates to an antidiabetic composition using the non- suri ( Lespedeza cuneata ) extract.

Diabetes is a disease in which blood sugar in the blood is not used as energy because insulin, which is a glucose regulating hormone secreted by the beta cells of the pancreas, is insufficient or does not work properly, and it accumulates in the blood, causing high blood sugar, and sugar is detected in the urine.

Diabetes is largely classified into insulin-dependent type 1 diabetes caused by insulin deficiency caused by the destruction of pancreatic beta cells, and insulin-independent type 2 diabetes caused by defects in insulin secretion and action (A balanced overview. Diabetes Care) 1992, 15: 318-368).

Type 1 diabetes is an insulin-dependent type and is classified into immune-mediated type 1 diabetes in which pancreatic beta cells are destroyed by an immunological mechanism, resulting in impaired insulin production, and idiopathic type 1 diabetes whose cause is not yet known (Lancet, 2006, 367:847-858), type 2 diabetes is a disease in which both insulin resistance and impaired insulin secretion are involved. Insulin resistance plays a major role. Insulin resistance type 2 diabetes and insulin secretion disorder play a major role. It is classified as insulin-deficient type 2 diabetes (BMJ, 2000, 321:405-412).

Currently, 90% of diabetes is type 2 diabetes, and type 2 diabetes causes persistent hyperglycemic symptoms due to insulin resistance, decreased insulin action, and inefficient insulin production in the pancreas (Int J Appl Res Nat Prod, 2016, 9 : 33-38), when it becomes chronic, it is accompanied by very serious complications such as diabetic retinopathy, renal failure, high blood pressure, and atherosclerosis. Therefore, type 2 diabetes is emerging as an urgent task to be solved medically and socio-economically.

As the currently known type 2 diabetes treatment, biguanides, which have the effect of migrating sugar to muscle cells and inhibiting the synthesis of sugar in the liver, thiazolidinediones, which activate PPARγ, which is involved in adipocyte differentiation. ), sulfonylureas, which indirectly induce the secretion of insulin, and α-glucosidase inhibitors, which inhibit enzymes that make glucose in the small intestine, but these drugs are used to treat diabetes. Has many side effects (Diabetes, 2008, 57:737-745), and the World Health Organization (WHO) strongly recommends the use of natural products with fewer side effects for diabetes (Journal of Ethnopharmacology, 2000, 73:461-470). ).

AMPK (AMP-activated protein kinase) is a phosphorylation enzyme that acts as a sensor to maintain energy homeostasis in cells and regulates its activity in response to the AMP/ATP ratio, which is the energy level of the cell (Trends Mol Med, 2008, 14:539). -549; Cell Metab, 2005, 1:15-25). AMPK is known to affect various physiological activities such as glucose transport, fatty acid and cholesterol synthesis by regulating the phosphorylation of various enzymes involved in the energy metabolism of cells (Am J Physiol Endocrinol Metab, 2007, 292:1308 -1317). In particular, according to a recent study, it promotes the absorption of glucose in muscle cells that are promoted by exercise, which is a substance that activates AMPK, which can have the effect of promoting the absorption and consumption of glucose in muscle cells, just like exercise without exercise. It suggests that there is, and therefore, is attracting attention as a new target for the treatment of type 2 diabetes (Therapie, 2007, 62:293-310). In the case of metformin, which is currently widely used as a diabetes treatment, it is known as a substance that phosphorylates AMPK to activate it. When AMPK is activated, ACC (acetyl-CoA carboxylase) is phosphorylated and inactivated.ACC is an enzyme involved in the initial stage of fatty acid synthesis.When ACC is inactivated, it is known that fat oxidation is promoted to lower blood sugar (Trends Biochem. Sci 24:22-25, 1999; Biochem Soc Trans 2003, 31:162-168).

The present invention discloses an antidiabetic composition using a non-suri extract that promotes activation (phosphorylation) of AMPK and inactivation (phosphorylation) of ACC and absorption of glucose in muscle cells.

An object of the present invention is to provide an antidiabetic composition using a non-suri extract.

Other or specific objects of the present invention will be presented below.

The present invention promotes AMPK activation (phosphorylation) and ACC inactivation (phosphorylation) in L6 myotube cells, and glucose absorption in muscle cells as confirmed in the Examples and Experimental Examples below. It is completed by confirming that it promotes.

In consideration of the above, the present invention can be understood as an antidiabetic composition comprising a non-suri extract as an active ingredient.

In the present specification, the "non-suri extract" refers to water, lower alcohols having 1 to 4 carbon atoms (methanol, ethanol, butanol, etc.), methylene chloride, ethylene, etc. of non-suri stems, branches, leaves, roots, outposts, or a mixture thereof to be extracted Using acetone, hexane, ether, chloroform, ethyl acetate, butyl acetate, N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 1,3-butylene glycol, propylene glycol, or a mixed solvent thereof It refers to an extract obtained by leaching (that is, an extract soluble in the extraction solvent), an extract obtained using a supercritical extraction solvent such as carbon dioxide, pentane, or a fraction obtained by fractionating the extract, and the extraction method is the polarity of the active substance and the degree of extraction , In consideration of the degree of preservation, arbitrary methods such as cold settling, reflux, heating, ultrasonic radiation, and supercritical extraction can be applied. In the case of a fractionated extract, a fraction obtained by suspending the extract in a specific solvent and then mixing and policing it with a solvent having a different polarity, the crude extract adsorbed on a column filled with a polar or non-polar solid phase such as silica gel, and then a hydrophobic solvent, a hydrophilic solvent, or It is meant to include a fraction obtained by using these mixed solvents as a mobile phase. In addition, the meaning of the extract includes a concentrated liquid extract or a solid extract from which the extraction solvent has been removed by a method such as freeze drying, vacuum drying, hot air drying, or spray drying. Preferably, an extract obtained by using water, ethanol or a mixed solvent thereof as an extraction solvent, more preferably an extract obtained by using a mixed solvent of water and ethanol as an extraction solvent, and a high obtained by removing the extraction solvent from the extract. It refers to a fraction of each layer obtained when a mixed solvent extract of water and ethanol in the shape is suspended in water and then sequentially fractionated with normal hexane, chloroform, ethyl acetate and normal butanol. Particularly, among the fractions, a fraction of a normal hexane layer having excellent AMPK activation and ACC deactivation degree as shown in the following examples and experimental examples is preferable. Here, "sequential fractionation" means that the residual water layer is continuously used after fractionation and fractionated with the fractionation solvent in the order listed above.

In addition, in the present specification, "antidiabetic" refers to lowering blood sugar and preventing, treating, improving or delaying the onset of diabetes. Specifically, high blood sugar caused by not producing insulin or insufficient insulin, detecting sugar in the urine, etc. It means preventing, treating, ameliorating, or delaying the onset of such pathological symptoms.

In addition, in the present specification, "diabetes" is meant to include insulin-dependent diabetes and non-insulin-dependent diabetes as described above, and further, diabetes that occurs when the pancreas is damaged due to other diseases, such as hyperthyroidism, hyperactivity of the adrenal cortex. , Growth hormone hyperactivity or catecholamine hyperactivity due to diabetes, gestational diabetes. It means preferably insulin-independent type 2 diabetes.

In addition, in the present specification, the term "active ingredient" refers to a component capable of exhibiting a desired activity alone, or capable of exhibiting activity together with a carrier that is not itself active.

In the composition of the present invention, the active ingredient may be included in an arbitrary amount (effective amount) depending on the use, formulation, etc., as long as it can exhibit an antidiabetic effect, etc., and a typical effective amount is 0.001% by weight based on the total weight of the composition. To 15% by weight. Herein, "effective amount" refers to the intended medical and pharmacological effects such as anti-diabetic effects when the composition of the present invention is administered to a mammal, preferably a human, during the administration period according to the advice of a medical professional, etc. , Refers to the amount of the active ingredient contained in the composition of the present invention. Such effective amounts can be determined empirically within the range of ordinary skill in the art.

The composition of the present invention can be grasped as a food composition in a specific aspect.

The food composition of the present invention may be prepared in any form, such as beverages such as tea, juice, carbonated beverages, ionized beverages, processed oils such as milk and yogurt, gums, rice cakes, Korean confectionery, bread, Foods such as confectionery and noodles, tablets, capsules, pills, granules, liquids, powders, flakes, pastes, syrups, gels, jelly, and health functional food preparations such as bars can be prepared. In addition, the food composition of the present invention can be classified as a product as long as it conforms to the enforcement regulations at the time of manufacture and distribution in terms of legal and functional classification. For example, it is a health functional food in accordance with the Korean "Health Functional Food Act", or confectionery, bean, tea, etc. according to each food type according to the food code of the Korean "Food Sanitation Act" (“Food Standards and Standards” notified by the Ministry of Food and Drug Safety). It may be beverages, special purpose foods, etc.

The food composition of the present invention may contain food additives in addition to the active ingredients. Food additives can generally be understood as substances that are added to food and mixed or infiltrated in manufacturing, processing, or preserving food. Since they are consumed daily and for a long time with food, their safety must be ensured. In the Food Additive Code (“Food Sanitation Act” in Korea) governing the manufacture and distribution of food, food additives with guaranteed safety are limited in terms of ingredients or functions. In the Korean Food Additives Code (KFDA notice "Food Additive Standards and Specifications), food additives are classified into chemical synthetic products, natural additives, and mixed preparations in terms of ingredients. These food additives are sweeteners and flavoring agents in terms of function. , Preservatives, emulsifiers, acidulants, thickeners, etc.

Sweeteners are used to impart a suitable sweetness to foods, and both natural and synthetic ones can be used in the food composition of the present invention. Preferably, a natural sweetener is used, and examples of the natural sweetener include sugar sweeteners such as corn syrup solids, honey, sucrose, fructose, lactose, and maltose.

Flavoring agents are used to improve taste or flavor, and both natural and synthetic flavors can be used. Preferably, it is the case of using a natural one. In the case of using natural ingredients, the purpose of nutrient enhancement can be combined in addition to flavor. As a natural flavoring agent, it may be obtained from apples, lemons, tangerines, grapes, strawberries, peaches, etc., or may be obtained from green tea leaves, roundtails, bamboo leaves, cinnamon, chrysanthemum leaves, jasmine, and the like. In addition, you can use those obtained from ginseng (red ginseng), bamboo shoots, aloe vera, and ginkgo. The natural flavoring agent may be a liquid concentrate or a solid extract. In some cases, synthetic flavoring agents may be used. As the synthetic flavoring agents, esters, alcohols, aldehydes, terpenes, and the like may be used.

As a preservative, calcium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate, EDTA (ethylenediaminetetraacetic acid), etc. can be used, and as an emulsifier, acacia gum, carboxymethylcellulose, xanthan gum, pectin Etc. may be used, and as the acidulant, arithmetic, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, phosphoric acid, and the like can be used. The acidulant may be added so that the food composition has an appropriate acidity for the purpose of suppressing the growth of microorganisms in addition to the purpose of enhancing taste. As the thickening agent, a suspending agent, a settling agent, a gel-forming agent, a swelling agent, and the like may be used.

In addition to the food additives described above, the food composition of the present invention may contain a physiologically active substance or minerals known in the art for the purpose of supplementing and reinforcing functionality and nutritional properties and ensuring stability as a food additive.

Examples of such physiologically active substances include catechins contained in green tea, vitamins such as vitamin B1, vitamin C, vitamin E, and vitamin B12, tocopherol, dibenzoyl thiamine, etc., and minerals include calcium preparations such as calcium citrate, magnesium stearate. Magnesium preparations, such as magnesium preparations, iron preparations such as iron citrate, chromium chloride, potassium iodide, selenium, germanium, vanadium, and zinc.

In the food composition of the present invention, the food additive as described above may be included in an appropriate amount to achieve the purpose of addition according to the product type.

With regard to other food additives that may be included in the food composition of the present invention, reference may be made to the food code or food additive code according to the laws of each country.

The composition of the present invention may be understood as a pharmaceutical composition in other specific embodiments.

The pharmaceutical composition of the present invention may be prepared in an oral dosage form or a parenteral dosage form according to an administration route by a conventional method known in the art, including a pharmaceutically acceptable carrier in addition to the active ingredient. Here, the meaning of "pharmaceutically acceptable" means that the application (prescription) does not have toxicity beyond adaptable without inhibiting the activity of the active ingredient.

When the pharmaceutical composition of the present invention is prepared in an oral dosage form, powders, granules, tablets, pills, dragees, capsules, solutions, gels, syrups, suspensions, wafers according to a method known in the art together with a suitable carrier It can be prepared in such a formulation. Examples of suitable pharmaceutically acceptable carriers include sugars such as lactose, glucose, sucrose, dextrose, sorbitol, mannitol, and xylitol, corn starch, potato starch, wheat starch, and other starches, cellulose, methylcellulose, ethylcellulose, Celluloses such as sodium carboxymethylcellulose and hydroxypropylmethylcellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, mineral oil, malt, gelatin, talc, polyol, vegetable Yu, etc. are mentioned. In the case of formulation activity, if necessary, it may be formulated including diluents and/or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

When the pharmaceutical composition of the present invention is prepared in a parenteral dosage form, it may be formulated in the form of eye drops, injections, transdermal administration, nasal inhalation, and suppositories according to a method known in the art together with a suitable carrier. When formulated as an eye drop, suitable carriers may include sterile water, saline, and isotonic solutions such as 5% dextrose, and benzalkonium chloride, mefilparaben, ethylparaben, etc. may be added for preservative purposes if necessary. When formulated as an injection, sterile water, ethanol, polyols such as glycerol or propylene glycol, or mixtures thereof may be used as suitable carriers, preferably Ringer's solution, PBS (phosphate buffered saline) containing triethanol amine, or sterilization for injection Water, isotonic solutions such as 5% dextrose can be used. When formulated into a transdermal dosage form, it can be formulated in the form of an ointment, cream, lotion, gel, external solution, pasta, liniment, air roll, and the like. In the case of nasal inhalants, suitable propellants such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, and carbon dioxide can be used to form an aerosol spray.When formulated as a suppository, the base is Withepsol ( witepsol), tween 61, polyethylene glycols, cacao butter, laurin paper, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, sorbitan fatty acid esters, and the like.

Regarding the specific formulation of the pharmaceutical composition is known in the art, for example, Remington's Pharmaceutical Sciences (19th ed., 1995) and the like can be referred to. These documents are considered as part of this specification.

The preferred dosage of the pharmaceutical composition of the present invention is in the range of 0.001 mg/kg to 10 g/kg per day, preferably 0.001 mg/kg to 1 g, depending on the patient's condition, weight, sex, age, patient severity, and route of administration. May be in the /kg range. Administration can be made once a day or divided into several times. These dosages should not be construed as limiting the scope of the invention in any aspect.

As described above, according to the present invention, it is possible to provide an antidiabetic composition using a non-suri extract. The composition of the present invention can be commercialized as foods such as health functional foods or drugs such as natural medicines.

1 schematically shows the manufacturing process of non-suri extract.
2 and 3 are results of Western blot evaluation of the extent to which non-suri extract and normal hexane fraction affect activation of APMK and inactivation of ACC in L6 muscle cells, respectively.
4 is a result of evaluating the effect of the normal hexane fraction of the non-suri extract on the glucose uptake ability in L6 muscle cells.

Hereinafter, the present invention will be described with reference to Examples and Experimental Examples. However, the scope of the present invention is not limited to these Examples and Experimental Examples.

<Example> Preparation of non-suri extract

To 500 g of a mixture of Lespedeza cuneata stems and leaves, 500 mL of 70% ethanol was added to the dry powder, extracted at room temperature for 24 hours, and then ultrasonically radiated for 1 hour. Centrifuge at 10,000×g for 20 minutes to take the supernatant, extract the remaining residue 3 times in the same way as above, mix these extracts, filter with filter paper (Whatman No 2), and concentrate the filtrate under reduced pressure. Freeze-dried to obtain 220 g of a solid extract. Next, the extract was suspended in water, and the same amount of n-hexane, chloroform, ethyl acetate, and n-butanol were sequentially fractionated, hexane fraction (LC-H) 32.77 g, chloroform fraction (LC-C) 4.05 g , 8.50 g of ethyl acetate fraction (LC-E), 30.80 g of butanol fraction (LC-B), and 167.51 g of residual water layer fraction (LC-W) were obtained. These non-suri extract manufacturing processes are schematically shown in FIG. 1.

<Experimental Example> Antidiabetic Activity of Bisuri Extract

<Experimental Example 1> AMPK and ACC activity evaluation-Western Blot

The example samples were treated on L6 muscle cells, and the degree of activation of AMPK and ACC enzymes was measured by Western blot method. Western blot was performed using the method described in Schlattner U, et al., J Biol Chem. 279, pp 43940-51, 2004; Smwar R et al., Clin Ther. 20, 125-40, 1998). , Specifically, after culturing the L6 muscle cells and treating the non-repaired extract for each concentration for 2h, the cells were subjected to a RIPA buffer solution (10) to which a protease inhibitor cocktail (Sigma, st. Louis, MO USA) was added. mM Tris-HCl (pH 7.5), 1% NP-40, 0.1% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 1 mM EDTA). Thereafter, the obtained cell lysate was centrifuged at 12,000 × g for 5 min at 4° C., and the protein concentration was measured using a BCA protein kit. A sample buffer was added to the protein extracted from the cells, heated at 100° C. for 5 min, and then SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) was performed. After electrophoresis, the proteins separated by molecular weight were transferred from the gel to the nitrocellulose (NC) membrane. At this time, in order to minimize the non-specific reaction, a buffer solution (phosphate buffered saline; PBS (pH 7.4), 0.1% Tween 20, 5% skimmed milk) was reacted at room temperature for 1 h, and then the primary antibody was diluted as follows. Used. Antibody dilution was diluted by reacting for 1 h at room temperature using a 5% Skim milk solution. After the reaction, washed 3 to 4 times for 5 min with a washing buffer (0.1% Tween-20, PBS) and then 1:5000. It was reacted at room temperature for 1 h with a diluted secondary antibody (HRP-conjugated anti-goat IgG antibody) solution. After the secondary antibody reaction, the protein band to be analyzed was colored and quantified using an ECL (enhanced chemiluminescence) reagent.

The results are shown in FIGS. 2 and 3. Referring to FIG. 2, when the sample was treated at 100 μg/ml, the hexane fraction (LC-H) activated AMPK very clearly (increased expression level of p-AMPK) and inactivated ACC (p- ACC expression level increase) is shown, and referring to FIG. 3 in which the hexane fraction is treated by concentration, it is shown that the hexane fraction activates AMPK and inactivates ACC in a concentration-dependent manner.

<Experimental Example 2> Evaluation of glucose absorption ability

Glucose uptake evaluation was performed using a glucose uptake assay kit (colorimetric; ab136955, abcam) after 2 hours of treatment with the most active hexane fraction (LC-H) in L6 muscle cells. Then, it was evaluated according to the manufacturer's protocol.

Specifically, L6 muscle cells were subcultured in a DMEM culture medium containing 10% FBS using a 37°C CO ₂ incubator, and then an appropriate amount of cells (5×10 ⁴ cells) was cultured in a 96 well culture plate. After incubation for 24 hours, it was differentiated by replacing it with a DMEM culture solution containing 2% FBS (9-15 days). After cell differentiation, the extract was replaced with a DMEM culture medium containing no FBS and starvation (4-10 hours) before treatment. After washing three times with PBS, it was pretreated with KRPH/2% BSA buffer (100 μL/well) for 40 minutes. After the extracts were treated by concentration (total volume 100 μL), 10 mM 2-deoxyglucse (2-DG) was treated with 10 μL each and reacted for 20 minutes. After washing 3 times with PBS to remove 2-DG, 80 μL of Extraction buffer was added and reacted at 85° C. for 40 minutes to degrade the cells. After cooling on ice for 5 minutes, neutralization was performed by adding 10 μL of Neutralization buffer each. After centrifugation at 500 rpm for 2 minutes, 5 μL of the supernatant was dispensed into a new 96 well plate to which the assay buffer was added at each 45 μL/well. Reaction mix A was added at a time of 10 μL/well and then reacted at 37° C. for 1 hour. After the reaction, 90 μL of extraction buffer was added and reacted at 90° C. for 40 minutes. After cooling on ice for 5 minutes, neutralization is performed by adding 12 μL of neutralization buffer. After neutralization, 38 μL of Reaction mix B was added. Completely mixed and measured at OD 412 nm using a microplate reader. The activity was measured by comparing the band size of the extract and the control compared to the control.

The results are shown in Figure 4, the hexane fraction (LC-H) promoted intracellular glucose uptake in a concentration-dependent manner.

Claims (6)

It contains as an active ingredient an extract of a mixture of non-repaired stem and leaves, which promotes activation of AMPK (AMP-activated protein kinase) and inactivation of acetyl-CoA carboxylase (ACC) and absorption of glucose in muscle cells,
The extract is a pharmaceutical composition for treating or preventing diabetes, characterized in that it is a normal hexane fraction obtained when a mixed solvent extract of water and ethanol of non-suried stems and leaves is suspended in water and fractionated with normal hexane.
The method of claim 1,
The antidiabetic composition, characterized in that the prophylactic or therapeutic activity of insulin-independent type 2 diabetes.
The method of claim 1,
The composition, characterized in that the mixed solvent of water and ethanol is 70% ethanol.
It contains as an active ingredient an extract of a mixture of non-repaired stem and leaves, which promotes activation of AMPK (AMP-activated protein kinase) and inactivation of acetyl-CoA carboxylase (ACC) and absorption of glucose in muscle cells,
The extract is a food composition for lowering blood sugar, characterized in that it is a normal hexane fraction obtained when a mixed solvent extract of water and ethanol of non-suried stems and leaves is suspended in water and fractionated with normal hexane.
The method of claim 4,
The composition, characterized in that the mixed solvent of water and ethanol is 70% ethanol.









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