KR20200057973A - Composition for Anti-Diabetes Using an Extract of Lespedeza cuneata - Google Patents

Abstract

Disclosed in the present invention is an anti-diabetic composition using a Lespedeza cuneata extract which promotes activation (phosphorylation) of AMPK and inactivation (phosphorylation) of ACC, and promotes glucose absorption in muscle cells. According to the present invention, it is possible to provide the anti-diabetic composition using the Lespedeza cuneata extract.

Description

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

The present invention relates to an anti-diabetic composition using an extract of Lespedeza cuneata .

Diabetes refers to a disease in which blood glucose in the blood is not used as energy, but is accumulated in the blood, causing hyperglycemia, and sugar is detected in the urine because insulin, a glucose regulating hormone secreted from the beta cells of the pancreas, is insufficient or does not function properly.

Diabetes is largely classified into insulin-dependent type 1 diabetes caused by insulin deficiency by 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 divided into immune-mediated type 1 diabetes, where pancreatic beta cells are destroyed by an immunological mechanism, and insulin-producing disorder occurs, and idiopathic type 1 diabetes, which has not yet been identified (Lancet, 2006, 367: 847-858), type 2 diabetes is a disease in which insulin resistance and insulin secretion disorder are both related. 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, hypertension, and atherosclerosis. Therefore, type 2 diabetes is emerging as an urgent task to be solved medically and socio-economically.

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

AMPK (AMP-activated protein kinase) is a phosphorylation enzyme whose activity is regulated in response to the cell's energy level, AMP / ATP ratio, by acting as a sensor in maintaining energy homeostasis in cells (Trends Mol Med, 2008, 14: 539 -549; Cell Metab, 2005, 1: 15-25). AMPK is known to influence various physiological activities such as glucose transport, fatty acid and cholesterol synthesis by regulating phosphorylation of various enzymes involved in 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 promoted by exercise, which can promote the absorption and consumption of glucose from muscle cells, like AMPK-activated substances exercise without exercise. Suggests the presence (J Appl Physiol, 2001, 91: 1073-1083). This suggests that the substance that activates AMPK can have the effect of promoting the absorption and consumption of glucose in muscle cells as if exercising without exercise, and thus is attracting attention as a new target for the treatment of type 2 diabetes. (Therapie, 2007, 62: 293-310). Metformin, which is currently used as a therapeutic agent for diabetes, is known as a substance that phosphorylates and activates AMPK. When AMPK is activated, ACC is phosphorylated and inactivated. ACC is an enzyme involved in the initial stage of fatty acid synthesis. It is known that when ACC is inactivated, 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 anti-diabetic composition using an unrepaired extract that promotes activation of AMPK (phosphorylation) and inactivation of ACC (phosphorylation) and promotes glucose absorption in muscle cells.

An object of the present invention is to provide an anti-diabetic composition using an unrepaired extract.

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

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

Considering the above, the present invention can be grasped with an anti-diabetic composition comprising an unrepaired extract as an active ingredient.

In the present specification, "unrepaired extract" refers to unrefined stems, branches, leaves, roots, outposts, or mixtures thereof to be extracted, water, lower alcohols having 1 to 4 carbon atoms (methanol, ethanol, butanol, etc.), methylene chloride, ethylene, Acetone, hexane, ether, chloroform, ethyl acetate, butyl acetate, N, N-dimethylformamide (DMF), dimethylsulfoxide (DMSO), 1,3-butylene glycol, propylene glycol or a mixed solvent thereof. An extract obtained by leaching (ie, an extract soluble in the extraction solvent), an extract obtained using a supercritical extraction solvent such as carbon dioxide or pentane, or a fraction obtained by fractionating the extract, wherein the extraction method is the polarity of the active substance, the degree of extraction , Considering the degree of preservation, any method such as cold acupuncture, reflux, warming, ultrasonic radiation, and supercritical extraction can be applied. In the case of fractionated extracts, the extract is suspended in a specific solvent and then mixed and politicized with a solvent having a different polarity, and the crude extract is adsorbed on a column filled with a polar or non-polar high-phase material such as silica gel, followed by hydrophobic solvent, hydrophilic solvent, or It means that the fraction obtained by using these mixed solvents as a mobile phase is included. In addition, the meaning of the extract includes a concentrated liquid extract or a solid extract in which the extraction solvent is removed by a method such as freeze drying, vacuum drying, hot air drying and 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 an extract obtained by removing the extract solvent from the extract It means a fraction of each layer obtained when a mixed solvent extract of water and ethanol in the form is suspended in water and sequentially fractionated with normal hexane, chloroform, ethyl acetate and normal butanol. In particular, among the fractions, as shown in Examples and Experimental Examples below, a fraction of a normal hexane layer having excellent AMPK activation and ACC inactivation is preferable. Here, 'sequential fractionation' means that after fractionation, the residual water layer is continuously used to fractionate with a fractional solvent in the order listed above.

In addition, "anti-diabetes" in the present specification refers to a decrease in blood sugar and prevention, treatment, improvement or delay of onset of diabetes, specifically hyperglycemia caused by lack of insulin or lack of insulin, and sugar detected in urine, etc. It means the prevention, treatment, improvement of pathological symptoms or delayed development of these pathological symptoms.

In addition, in the present specification, "diabetes" refers to insulin-dependent diabetes and insulin-independent diabetes as described above, and furthermore, diabetes that occurs due to damage to the pancreas due to other diseases such as hyperthyroidism, adrenal cortical hyperactivity , Growth hormone hypercholesterolemia or catecholamine hyperglycemia, gestational diabetes. Preferably, it means insulin-independent type 2 diabetes.

In addition, in the present specification, "active ingredient" refers to a component that can exhibit the desired activity alone or itself can exhibit activity with an inactive carrier.

The active ingredient in the composition of the present invention can be included in any amount (effective amount) according to the use, formulation, etc., as long as it can exhibit anti-diabetic effect, etc., the typical effective amount is 0.001% by weight based on the total weight of the composition To 15% by weight. Here, the "effective amount" refers to the intended medical and pharmacological effect, such as an anti-diabetic effect, when the composition of the present invention is administered to a mammal, preferably a person, to which the object is applied, according to the recommendation of a medical expert or the like. , Refers to the amount of active ingredient contained in the composition of the present invention. Such an effective amount can be determined empirically within the 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 can be produced in any form, for example, beverages such as tea, juice, carbonated beverages, ionic beverages, processed oils such as milk, yogurt, gums, rice cakes, Chinese medicine, bread, It can be made of foods such as confectionery, noodles, tablets, capsules, pills, granules, liquids, powders, flakes, pastes, syrups, gels, jelly, bars, etc. In addition, the food composition of the present invention can be classified into any product as long as it complies with the enforcement regulations at the time of manufacturing and distribution in terms of legal and functional classification. For example, it is a health functional food in accordance with the Korea "Act on Health Functional Food", or a confectionary, soybean, or tea product according to each food type in the Korea Food and Drug Administration's Food Fair (which is the "Standard and Specification of Food" by the Ministry of Food and Drug Safety). Beverages, special-purpose foods, and the like.

The food composition of the present invention may include a food additive in addition to the active ingredient. Food additives can be understood as substances that are added to foods and mixed or infiltrated in general in the manufacture, processing, or preservation of foods, and their safety must be ensured because they are consumed daily and for a long time with foods. Food additives in accordance with national laws governing the manufacture and distribution of food ("Food Sanitation Law" in Korea) are limited in terms of ingredients or functions in terms of food additives with guaranteed safety. In the Korea Food Additives Fair (Korea Food and Drug Administration's notice and standards for food additives), food additives are classified into chemical synthetic products, natural additives, and mixed preparations in terms of ingredients, and these food additives are sweeteners and flavors in terms of function. , Preservatives, emulsifiers, acidulants, thickeners, etc.

Sweeteners are used to impart a moderate sweetness to food, and both natural and synthetic 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 aroma, and both natural and synthetic ones can be used. Preferably it is a case of using a natural thing. In addition to flavor, when using natural ones, the purpose of enhancing nutrition can also be combined. The natural flavoring agent may be obtained from apples, lemons, tangerines, grapes, strawberries, peaches, etc., or may be obtained from green tea leaves, perilla, large leaves, cinnamon, chrysanthemum leaves, jasmine, and the like. In addition, those obtained from ginseng (red ginseng), bamboo shoots, aloe vera, and ginkgo can be used. Natural flavors may be liquid concentrates or solid extracts. In some cases, synthetic flavoring agents may be used. As the synthetic flavoring agent, 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. can be used, and as 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 a thickener, a suspending agent, a sedimentation agent, a gel-forming agent, a swelling agent, etc. can be used.

The food composition of the present invention, in addition to the food additives as described above, may include bioactive substances or minerals known in the art for the purpose of supplementing and reinforcing functional and nutritional properties and guaranteed stability as food additives.

Examples of such bioactive substances include catechins contained in green tea, vitamins such as vitamin B1, vitamin C, vitamin E, and vitamin B12, tocopherols, dibenzoyl thiamine, and the like, and calcium preparations such as calcium citrate and magnesium stearate as minerals. Magnesium preparations such as iron, iron preparations such as iron citrate, chromium chloride, potassium iodine, selenium, germanium, vanadium, zinc, and the like.

The food composition of the present invention may include the food additives as described above 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 food or food additives according to laws of each country.

The composition of the present invention may be identified 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 the route of administration by a conventional method known in the art, including a pharmaceutically acceptable carrier in addition to the active ingredient. Here, "pharmaceutically acceptable" means that the target of application (prescription) has no toxicity beyond adaptability 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, liquids, gels, syrups, suspensions, wafers according to methods known in the art with suitable carriers And the like. At this time, examples of suitable pharmaceutically acceptable carriers include sugars such as lactose, glucose, sucrose, dextrose, sorbitol, mannitol, xylitol, starch such as corn starch, potato starch, wheat starch, cellulose, methylcellulose, ethyl cellulose, Celluloses such as sodium carboxymethylcellulose and hydroxypropylmethylcellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, mineral oil, malt, gelatin, talc, polyol, vegetable And the like. In the case of formulation, if necessary, it can be formulated by including diluents and / or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, 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 dosage forms, nasal inhalants and suppositories according to methods known in the art with a suitable carrier. As a suitable carrier in the case of formulation as an eye drop, an isotonic solution such as sterile water, saline, or 5% dextrose can be used, and if necessary, benzalkonium chloride, mefilparaben, ethylparaben, etc. can be added for preservative purposes. When formulated as an injection, suitable carriers may be sterile water, ethanol, polyols such as glycerol or propylene glycol, or mixtures thereof. Preferably, Ringer's solution, PBS (phosphate buffered saline) containing triethanol amine or sterilized for injection Isotonic solutions such as water and 5% dextrose can be used. When formulated as a transdermal dosage form, it can be formulated in the form of ointments, creams, lotions, gels, external solutions, pasta, linen agents, aerosols, and the like. For nasal inhalants, dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, etc. can be formulated in the form of an aerosol spray using a suitable propellant, and when formulated as a suppository, Witepsol ( witepsol), tween 61, polyethylene glycols, cacao butter, laurin, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearate, sorbitan fatty acid esters, and the like.

Regarding the specific formulation of the pharmaceutical composition, it is known in the art, and for example, see Remington's Pharmaceutical Sciences (19th ed., 1995) and the like. The above documents are regarded as part of this specification.

Preferred dosages of the pharmaceutical compositions of the present invention range from 0.001 mg / kg to 10 g / kg per day, preferably 0.001 mg / kg to 1 g per day, depending on the patient's condition, weight, sex, age, patient severity, and route of administration. / kg range. Administration can be done once a day or divided into several times. Such doses 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 anti-diabetic composition using an unrepaired extract. The composition of the present invention may be commercialized as a food such as a health functional food or a medicine such as a natural medicine.

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

500 ml of 70% ethanol was added to 500 g of dry powder to the vespedeza cuneata stem and leaf mixture and extracted at room temperature for 24 hours, followed by ultrasonic irradiation for 1 hour. Centrifugation at 10,000 × g for 20 minutes to take the supernatant, extract the remaining residue three times in the same manner as above, mix these extracts, filter with filter paper (Whatman No 2), and concentrate the filtrate under reduced pressure. Lyophilization afforded 220 g of solid extract. Then, the extract was suspended in water, and then fractionated sequentially with the same amount of n-hexane, chloroform, ethyl acetate, and n-butanol, 32.77 g of hexane fraction (LC-H), 4.05 g of chloroform fraction (LC-C) , Ethyl acetate fraction (LC-E) 8.50g, butanol fraction (LC-B) 30.80g and residual water layer fraction (LC-W) 167.51g water was obtained. These non-repair extract manufacturing process is schematically shown in FIG. 1.

<Experimental Example> Anti-diabetic activity of non-repair extract

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

The sample was treated with L6 muscle cells to measure the degree of AMPK and ACC enzyme activation by Western blot. Western blot was performed by applying 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, the non-repair extract was treated for 2h according to concentration, and then the cells were added to the RIPA buffer solution (10) to which the 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 then the protein concentration was measured using a BCA protein kit. To the protein extracted from the cells, a buffer for electrophoresis (sample buffer) was added and heated at 100 ° C. for 5 min, followed by SDS-PAGE (sodium dodecyl sulfatepolyacrylamide gel electrophoresis). After electrophoresis, proteins separated by molecular weight were transferred from a gel to a 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 the primary antibody was diluted as follows. Used. Antibody dilution was diluted by reacting with 5% Skim milk solution at room temperature for 1 h, after reaction, washed 3-4 times for 5 min with washing buffer (0.1% Tween-20, PBS) to 1: 5000. The diluted secondary antibody (HRP-conjugated anti-goat IgG antibody) solution was reacted at room temperature for 1 h. After the secondary antibody reaction, the protein band to be analyzed was developed and quantified using ECL (enhanced chemiluminescence) reagent.

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

 <Experiment 2> Evaluation of glucose absorption capacity

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

Specifically, L6 muscle cells were cultured in a DMEM culture medium containing 10% FBS using a 37 ° C CO ₂ incubator, and then cultured in an appropriate amount of cell number (5 × 10 ⁴ cells) 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, before processing the extract, starvation was performed by replacing with FMEM-free DMEM culture (4-10 hours). After washing three times with PBS, it was pretreated with KRPH / 2% BSA buffer (100 μL / well) for 40 minutes. After treating the extract by concentration (Total volume 100 μL), 10 mM 2-deoxyglucse (2-DG) was treated in 10 μL and reacted for 20 minutes. After washing with PBS three times to remove 2-DG, 80 μL of Extraction buffer was added and the cells were decomposed by reacting at 85 ° C. for 40 minutes. After cooling on ice for 5 minutes, Neutralization buffer is neutralized by adding 10 μL of each. After centrifugation at 500 rpm for 2 minutes, 5 μL of supernatant was dispensed into a new 96 well plate to which assay buffer was added at 45 μL / well. Reaction mix A was added at 10 μL / well and reacted at 37 ° C. for 1 hour. After the reaction, extraction buffer was added in increments of 90 μL and reacted at 90 ° C. for 40 minutes. After cooling on ice for 5 minutes, neutralization buffer is added and neutralized by adding 12 μL. After neutralization, 38 μL of Reaction mix B was added. The mixture was thoroughly mixed and measured at OD 412 nm using a microplate reader. The activity was measured by comparing the band size of the control and the extract compared to the control.

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

Claims (6)

Anti-diabetic composition comprising a non-repair extract as an active ingredient.
According to claim 1,
The anti-diabetic composition is characterized in that the prevention or treatment activity of insulin-independent type 2 diabetes.
According to claim 1,
When the extract is (i) water, ethanol or a mixed solvent extract of the unrepaired leaf, and (ii) the mixed solvent extract is suspended in water as a solid, and sequentially fractionated with normal hexane, chloroform, ethyl acetate and butanol. The obtained composition, characterized in that each fraction.
According to claim 3,
The fraction is a composition characterized in that the normal hexane fraction.
The method according to any one of claims 1 to 4,
The composition is characterized in that the food composition.
The method according to any one of claims 1 to 4,
The composition is characterized in that the pharmaceutical composition.

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