KR20190093849A - Composition comprising extract of Gymnema sylvestre or compound isolated from thereof for preventing or treating of diabetes mellitus - Google Patents

Abstract

The present invention relates to a composition for preventing or treating diabetes, containing an extract of Gymnema sylvestre or a compound separated therefrom as an active component. By confirming that the extract of Gymnema sylvestre, a source thereof is Vietnam or the compound separated therefrom promotes absorption of sugar in fat cells and has an effect of alleviating glucose tolerance and insulin resistance, the extract of Gymnema sylvestre or the compound separated therefrom can be used for the development of medicines for preventing or treating diabetes or health functional foods for alleviating diabetes.

Description

Composition comprising extract of Gymnema sylvestre or compound isolated from conjugate for treating or treating of diabetes mellitus}

The present invention relates to a composition for preventing or treating diabetes comprising Gymnema sylvestre extract or a compound separated therefrom as an active ingredient.

Diabetes has problems with insulin secretion, such as decreased insulin secretion and resistance due to genetic and environmental causes, or malfunctions of insulin, causing glucose in the blood to not be transferred / stored to the cells, resulting in too much blood in the blood. It is a metabolic disease with symptoms of hyperglycemia that is much higher than normal. Diabetes ketoacidosis, hyperglycemic coma (Kitabachi, AE et al., 2009), cardiovascular disease, hypertension, stroke, chronic renal failure, diabetes It causes complications such as ulcers and diabetic retinopathy (WHO, 2013). This diabetes is known to be the fourth highest cause of death in Korea, including complications.

Diabetes is largely classified into Type 1 Diabetes (T1D), Type 2 Diabetes and Gestational Diabetes. Type 1 diabetes, which accounts for about 10% of all diabetes, occurs mainly in children, and is caused by the insufficiency of insulin by disruptive lesions of pancreatic β cells. Type 2 diabetes occurs mainly in adults, due to insulin resistance, in which the body does not secrete enough insulin to function properly or the cells do not respond to insulin. Gestational diabetes refers to a gradual increase in blood glucose levels because no insulin has been produced in the body during pregnancy, but has not been diagnosed before (WHO, 2013).

In 2014, there were about 380 million people with diabetes worldwide (International Diabetes Federation, 2015), and type 2 diabetes accounted for 90%, or 8.3% of the adult population (Shi, Y. et. al., 2014). From 2012 to 2014, it is estimated that between 1.5 million and 4.9 million people die of diabetes each year (World Health Organization, 2013; International Diabetes Federation, 2015). Diabetes is estimated to reach 590 million people by 2035, and the global economic cost of treating diabetes is estimated at US $ 612 billion (International Diabetes Federation, 2015). Indeed, in 2012 the cost of diabetes in the United States amounted to $ 245 billion (American Diabetes, Association, 2013).

The most basic of diabetes management is blood sugar control, exercise therapy or diet therapy to control blood sugar. If blood glucose is not controlled in this way, drug therapy is started. Type 1 diabetes is administered by insulin injection (WHO, 2013), and type 2 diabetes is administered by oral hypoglycemic agents or insulin (Kim, 2012). However, in addition to the positive aspects of continuous oral hypoglycemics, conventional oral hypoglycemic agents not only cause various side effects such as hypoglycemia, diarrhea, bloating, weight gain, lactic acid blood, cardiac toxicity, and liver toxicity. Since pancreatic β cells that function in insulin secretion are irreversibly damaged and destroyed, and insulin resistance occurs, the drug is finally ineffective and requires insulin injection. In addition, insulin, which is used most often as a diabetes treatment, has to be injected subcutaneously 2-3 times daily, causing discomfort and rejection for injections, and this also has a very high possibility of causing hypoglycemia. Therefore, there is a need for the development of more effective and safe drugs that have a pancreatic β-cell protective function even during long-term administration and can effectively lower blood glucose levels and significantly resolve insulin resistance without inducing hypoglycemia.

Gymnema sylvestre is a vine of perennial herb that grows widely in India, Southeast Asia and southern China. In Korea, it is called Kagaimo, and in Japan, sugar-weed, has been used for long time in diabetes treatment (Suttisri, R., et al., 1995). In Africa, milk from the roots is used to remove sweetness and bitterness. In China, it was used for the treatment of mammary fever, fever, premature and trauma. Recently, many countries, including Japan and the United States, have developed a dietary supplement in the form of extracts, powders, granules, jellies, and pastes, and their consumption is increasing (Shigematsu, N., et al., 2001).

Gimne sylvestre physiologically active substances include gymnemic acids, tartaric acid, gurmarin, triterpenoid saponins, stigmasterol and quercitol. (Nakamura, Y., et al., 1999), decreased glucose uptake in the small intestine (Shimizu, K., et al., 1997) and regulation of insulin concentrations after carbohydrate intake (Grover, JK, et al. , 2002). In addition, it has been reported that the alcoholic extract of Kimne Sylvestre improves blood cholesterol and triglyceride levels (Okazaki, M., et al., 2001), and Kimne Sylvestre improves body fat by improving lipids and controlling blood sugar. This is expected.

Accordingly, the present inventors, in the course of studying the Kimne sylvestre extract, the morphological and genetic difference between the Kimne sylvestre (GS-V), which is the origin of Vietnam, and the Kimne sylvestre (GS-I), which is native to India There was a difference in the chemical composition. In addition, by separating the compound from Kimne sylvestre extract, which is the origin of Vietnam, and confirming that the compound and the Kimne sylvestre extract containing the same to promote the absorption of glucose in adipocytes, and improve the glucose tolerance and insulin resistance The invention could be completed.

As a prior art, Japanese Laid-Open Patent Publication No. 2010-536806 discloses a diabetic treatment effect of an extract derived from Kimne sylvestre, from which India is a source, and a Kimnema acid compound contained therein, and Korean Patent No. 0625222 is a source of India. Although the diabetes treatment effect of the Jimnemic acid derivative isolated from phosphorus Kimne sylvestre has been described, the extract of Kimne sylvestre and the compound separated therefrom from which Vietnam of the present invention originates are not described, and the structure differs. In addition, U.S. Patent No. 6959261 describes the effect of diabetic treatment of Kimne sylvestre extract from India, but the effect of diabetic treatment of Kimne sylvestre extract of Vietnam from the present invention and the compound isolated therefrom is not described at all. And not mentioned.

Japanese Patent Publication No. 2010-536806, Herbal preparation for controlling blood sugar level in diabetic patients, published on December 12, 2010. Korean Registered Patent No. 0625222, Novel Jimnemic Acid Derivatives, Methods for Making the Same, and Pharmaceutical Use thereof, Registered Sep. 11, 2006. US Registered Patent No. 2264 261, Compositions for diabetes treatment and prophylaxis, registered on September 27, 2005.

International Diabetes Federation, Update 2014, 2015. Kim Dong-rim, Drug Treatment of Type 2 Diabetes, Konkuk University Hospital KRC, 2012. World Health Organization, The top 10 causes of death Fact sheet N ㅀ 310, 2013. American Diabetes, Association, Economic costs of diabetes in the U.S. in 2012, Diabetes Care, 36 (4), 1033-1046, 2013. Grover J.K. et al., Medicinal of plants of India with anti-diabetic potential. J. Ethnopharmacol., 81, 81-100, 2002. Kitabachi, A. E. et al., Hyperglycemic crises in adult patients with diabets, Diabetes care, 32 (7), 1335-1345, 2009. Nakamura, Y., et al., Fecal steroid excretion is increased in rats by oral administration of gymnemic acids contained in Gymnema sylvestre leaves, J. Nutr., 129, 1214-1222, 1999. Okazaki, M., et al., Effect of administration with the extract of Gymnema sylvestre R. Br leaves on lipid metabolism in rats, Biol. Pharma. Bull., 24, 713-717, 2001. Shi, Y. et al., The global implications of diabetes and cancer, The Lancet, 383 (9933), 1947-1948, 2014. Shigematsu, N., et al., Effect of long term-admintration with Gymnema sylvestre R. BR on plasma and liver lipid in rats, Biol. Pharm. Bull., 24, 643-649, 2001. Shimizu, K., et al., Suppression of glucose absorption by some fractions extracted from Gymnema sylvestre leaves, J. Vet. Med. Sci., 59, 245-251, 1997. Sutisri, R., et al., Plant-derived triterpenoid sweetness inhibitors, J. Ethnopharmacol., 47, 9-26, 1995. WHO, Diabetes Fact sheet N ㅀ 312, 2013.

An object of the present invention to provide a composition for preventing or treating diabetes comprising Gymnema sylvestre extract or a compound isolated therefrom as an active ingredient.

It is also an object of the present invention to provide a separation method and a novel compound of a compound separated from Kimne sylvestre extract.

The present invention relates to 3β, 16β, 28-trihydroxyolean-12-ene-29-oic acid 3-O-β-D-glucopyranosyl (1 → 3) -β-D-gluquin Lonofyranoside (Compound 1), 3β, 16β, 28-trihydroxyolean-12-ene-29-ioic acid 3-O-β-D-galactopyranosyl (1 → 3) -β-D -Glucuronopyranoside (compound 2), citakisogenin 3-O-β-D-glucopyranosyl (1 → 3) -β-D-glucuronopyranoside (compound 3), 3β, 16β-dihydroxyolean-12-ene 3-O-β-D-glucopyranosyl (1 → 3) -β-D-glucuronopyranoside (Compound 4), 29-O- (β- D-glucopyranosyl) Gimnegenol 3-O-β-D-glucuronopyranoside (Compound 5), citakisogenin 3-O-β-D-glucuronopyranoside (Compound 6) , Kimnemagenol 3-O-β-D-glucuronopyranoside (Compound 7), 28-benzoyl-22α-hydroxyrongispinogenin 3-O-β-D-glucuronopyranoside ( Compound 8), 3-O-β-D-glucuronopyranosyl 3β, 16β, 28-trihydroxyolean-12 -N-29,22β-oxide (Compound 9), 29-hydroxyrongispiginogenin 3-OD-glucopyranosyl (1 → 3) -D-glucuronopyranoside (Compound 10), rongisino Kimnema comprising at least one compound selected from the group consisting of genine 3-OD-glucopyranosyl (1 → 3) -D-glucuronopyranoside (Compound 11) and alternoside XII (Compound 12) The present invention relates to a pharmaceutical composition for preventing or treating diabetes or a dietary supplement for improvement, which contains an extract of Gymnema sylvestre as an active ingredient.

[Formula 1]

The Kimne sylvestre extract may be an extract of Kimne sylvestre extracted with one or more solvents selected from the group consisting of water, C1 to C4 lower alcohols, acetone, dichloromethane, ethyl acetate, n-hexane, and chloroform.

The Kimne Sylvestre may be originated from Vietnam.

In addition, the present invention is 3β, 16β, 28-trihydroxyolean-12-ene-29-oic acid 3-O-β-D- glucopyranosyl (1 → 3) -β-D- Glucuronopyranoside (Compound 1), 3β, 16β, 28-trihydroxyolean-12-ene-29-ioic acid 3-O-β-D-galactopyranosyl (1 → 3) -β -D-glucuronopyranoside (compound 2), citakisogenin 3-O-β-D-glucopyranosyl (1 → 3) -β-D-glucuronopyranoside (compound 3), 3β, 16β-dihydroxyolean-12-ene 3-O-β-D-glucopyranosyl (1 → 3) -β-D-glucuronopyranoside (Compound 4), 29-O- ( β-D-glucopyranosyl) Gimnegenol 3-O-β-D-glucuronopyranoside (Compound 5), citachisogenin 3-O-β-D-glucuronopyranoside (compound 6), Kimnemagenol 3-O-β-D-glucuronopyranoside (Compound 7), 28-benzoyl-22α-hydroxyrongispiginogenin 3-O-β-D-glucuronopyrano Seed (compound 8), 3-O-β-D-glucuronopyranosyl 3β, 16β, 28-trihydroxyol An-12-ene-29,22β-oxide (Compound 9), 29-hydroxyrongispiginogenin 3-OD-glucopyranosyl (1 → 3) -D-glucuronopyranoside (Compound 10) At least one compound selected from the group consisting of longispiginogen 3-OD-glucopyranosyl (1 → 3) -D-glucuronopyranoside (Compound 11) and alternoside XII (Compound 12) It relates to a pharmaceutical composition for preventing or treating diabetes or a functional food for improvement comprising the component.

[Formula 1]

The present invention also relates to a Kimne sylvestre extract, wherein the Kimne sylvestre is extracted using water, C1 to C4 lower alcohols, acetone, dichloromethane, ethyl acetate, n-hexane and chloroform as a solvent. Chromatographic analysis of 3β, 16β, 28-trihydroxyolean-12-ene-29-oic acid 3-O-β-D-glucopyranosyl (1 → 3) -β-D-gluquin Lonofyranoside (Compound 1), 3β, 16β, 28-trihydroxyolean-12-ene-29-ioic acid 3-O-β-D-galactopyranosyl (1 → 3) -β-D -Glucuronopyranoside (compound 2), citakisogenin 3-O-β-D-glucopyranosyl (1 → 3) -β-D-glucuronopyranoside (compound 3), 3β, 16β-dihydroxyolean-12-ene 3-O-β-D-glucopyranosyl (1 → 3) -β-D-glucuronopyranoside (Compound 4), 29-O- (β- D-glucopyranosyl) Gimnegenol 3-O-β-D-glucuronopyranoside (Compound 5) 3-O-β-D-glucuronopyranoside (Compound 6), Gimnegenol 3-O-β-D-glucuronopyranoside (Compound 7), 28-benzoyl-22α-hydroxylone Gispinogenin 3-O-β-D-glucuronopyranoside (Compound 8), 3-O-β-D-glucuronopyranosyl 3β, 16β, 28-trihydroxyolean-12- En-29,22β-oxide (compound 9), 29-hydroxyrongispiginogenin 3-OD-glucopyranosyl (1 → 3) -D-glucuronopyranoside (compound 10), rongispinogenin A method for separating at least one compound selected from the group consisting of 3-OD-glucopyranosyl (1 → 3) -D-glucuronopyranoside (Compound 11) and alternoside XII (Compound 12) will be.

In addition, the present invention is a novel compound having the chemical structure of formula 2 3β, 16β, 28-trihydroxyolean-12-ene-29-oic acid 3-O-β-D- glucopyranosyl (1 → 3 ) -β-D-glucuronopyranoside (Compound 1), 3β, 16β, 28-trihydroxyolean-12-ene-29-oic acid 3-O-β-D-galactopyranosyl ( 1 → 3) -β-D-glucuronopyranoside (Compound 2), citakisogenin 3-O-β-D-glucopyranosyl (1 → 3) -β-D-glucuronopyrano Seed (compound 3), 3β, 16β-dihydroxyolean-12-ene 3-O-β-D-glucopyranosyl (1 → 3) -β-D-glucuronopyranoside (compound 4) , 29-O- (β-D-glucopyranosyl) Gimnegenol 3-O-β-D-glucuronopyranoside (Compound 5), citakisogenin 3-O-β-D-gluco Lonofyranoside (Compound 6), Gimnegenol 3-O-β-D-Glucuronopyranoside (Compound 7), 28-benzoyl-22α-hydroxyrongispirogenin 3-O-β-D -Glucuronopyranoside (Compound 8) and 3-O-β-D-glucuronopy Nosil 3β, 16β, 28- trihydroxy oleic not -12- yen relates to come -29,22β- lead (Compound 9).

[Formula 2]

Hereinafter, the present invention will be described in detail.

The present invention relates to 3β, 16β, 28-trihydroxyolean-12-ene-29-oic acid 3-O-β-D-glucopyranosyl (1 → 3) -β-D-glucous Lonofyranoside (Compound 1), 3β, 16β, 28-trihydroxyolean-12-ene-29-ioic acid 3-O-β-D-galactopyranosyl (1 → 3) -β-D -Glucuronopyranoside (compound 2), citakisogenin 3-O-β-D-glucopyranosyl (1 → 3) -β-D-glucuronopyranoside (compound 3), 3β, 16β-dihydroxyolean-12-ene 3-O-β-D-glucopyranosyl (1 → 3) -β-D-glucuronopyranoside (Compound 4), 29-O- (β- D-glucopyranosyl) Gimnegenol 3-O-β-D-glucuronopyranoside (Compound 5), citakisogenin 3-O-β-D-glucuronopyranoside (Compound 6) , Kimnemagenol 3-O-β-D-glucuronopyranoside (Compound 7), 28-benzoyl-22α-hydroxyrongispinogenin 3-O-β-D-glucuronopyranoside ( Compound 8), 3-O-β-D-glucuronopyranosyl 3β, 16β, 28-trihydroxyolean-12 -N-29,22β-oxide (Compound 9), 29-hydroxyrongispiginogenin 3-OD-glucopyranosyl (1 → 3) -D-glucuronopyranoside (Compound 10), rongisino Kimnema comprising at least one compound selected from the group consisting of genine 3-OD-glucopyranosyl (1 → 3) -D-glucuronopyranoside (Compound 11) and alternoside XII (Compound 12) The present invention relates to a pharmaceutical composition for preventing or treating diabetes, which contains an extract of Gymnema sylvestre as an active ingredient.

The Kimne Sylvestre is originated in Vietnam, and there are differences in morphological, genetic and chemical components from those originated in India.

The Kimne sylvestre extract may be obtained by extracting the Kimne sylvestre with at least one solvent selected from the group consisting of water, C1 to C4 lower alcohols, acetone, dichloromethane, ethyl acetate, n-hexane and chloroform, As the lower alcohol of C1 to C4, methanol, ethanol, propanol, isopropanol, butanol and the like can be used. It is preferably water and lower alcohols of C1 to C4, more preferably ethanol and most preferably 60% [v / v] ethanol.

The solvent may be added to the weight of 2 to 500 times the weight of Kimne sylvestre.

In addition, the present invention is 3β, 16β, 28-trihydroxyolean-12-ene-29-oic acid 3-O-β-D- glucopyranosyl (1 → 3) -β-D- Glucuronopyranoside (Compound 1), 3β, 16β, 28-trihydroxyolean-12-ene-29-ioic acid 3-O-β-D-galactopyranosyl (1 → 3) -β -D-glucuronopyranoside (compound 2), citakisogenin 3-O-β-D-glucopyranosyl (1 → 3) -β-D-glucuronopyranoside (compound 3), 3β, 16β-dihydroxyolean-12-ene 3-O-β-D-glucopyranosyl (1 → 3) -β-D-glucuronopyranoside (Compound 4), 29-O- ( β-D-glucopyranosyl) Gimnegenol 3-O-β-D-glucuronopyranoside (Compound 5), citachisogenin 3-O-β-D-glucuronopyranoside (compound 6), Kimnemagenol 3-O-β-D-glucuronopyranoside (Compound 7), 28-benzoyl-22α-hydroxyrongispiginogenin 3-O-β-D-glucuronopyrano Seed (compound 8), 3-O-β-D-glucuronopyranosyl 3β, 16β, 28-trihydroxyol An-12-ene-29,22β-oxide (Compound 9), 29-hydroxyrongispiginogenin 3-OD-glucopyranosyl (1 → 3) -D-glucuronopyranoside (Compound 10) At least one compound selected from the group consisting of longispiginogen 3-OD-glucopyranosyl (1 → 3) -D-glucuronopyranoside (Compound 11) and alternoside XII (Compound 12) It relates to a pharmaceutical composition for preventing or treating diabetes, including as an ingredient.

The compound is preferably 29-O- (β-D-glucopyranosyl) gimemmagenol 3-O-β-D-glucuronopyranoside (Compound 5), citakisogenin 3-O-β -D-Glucuronopyranoside (Compound 6), Gimnegenol 3-O-β-D-Glucuronopyranoside (Compound 7), 28-benzoyl-22α-hydroxyrongispinogenin 3- O-β-D-glucuronopyranoside (Compound 8), 3-O-β-D-glucuronopyranosyl 3β, 16β, 28-trihydroxyolean-12-ene-29,22β At least one member selected from the group consisting of -oxide (compound 9) and 29-hydroxyrongispiginogenin 3-OD-glucopyranosyl (1 → 3) -D-glucuronopyranoside (compound 10) Can be.

The compound can be separated from the Kimne sylvestre extract.

The present invention also relates to a method for separating the compound of Formula 1, wherein the Kimne sylvestre is selected from the group consisting of water, lower alcohols of C1 to C4, acetone, dichloromethane, ethyl acetate, n-hexane and chloroform One or more compounds selected from the group consisting of Compound 1 to Compound 12 of Chemical Formula 1 may be separated by chromatography of the Kimne sylvestre extract extracted with one or more solvents.

The separation method is to separate the Kimne sylvestre extract water with one or more selected from the group consisting of water, C1 to C4 lower alcohol, acetone, dichloromethane, ethyl acetate, n- hexane and chloroform as a solvent Stage 1; Adsorbing the Kimne sylvestre extract of step 1 on an ion exchange resin and eluting with ethanol to secure an eluate; A three step of securing a fraction obtained by fractionating the eluate of the second step by silica gel column chromatography; And four steps of separating the compounds 1 to 12 of Formula 1 by adding the fractions of step 3 to column chromatography.

The four steps of column chromatography include silica gel column chromatography, reverse-phase silica gel column chromatography, and diion HP-20 column chromatography. RP-18 column chromatography, Sephadex LH-20 column chromatography, preparative reversed-phase high performance chromatography, Medium pressure liquid chromatography, high-performance liquid chromatography, and the like can be selected and used.

Meanwhile, the compound of the present invention may be synthesized according to a conventional method in the art, and may be prepared as a pharmaceutically acceptable salt.

The pharmaceutical composition comprising the Kimne sylvestre extract or a compound isolated therefrom is oral formulation, external preparation, suppository, and sterilization such as powder, granule, tablet, capsule, suspension, emulsion, syrup, aerosol, etc. It can be formulated and used in the form of injectable solutions. Carriers, excipients and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose , Methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid preparations may include at least one excipient such as starch, starch sylvestre extract of the present invention, or a compound separated therefrom. It is prepared by mixing calcium carbonate, sucrose or lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The dosage of the pharmaceutical composition will vary depending on the age, sex, weight of the subject to be treated, the particular disease or pathology to be treated, the severity of the disease or pathology, the route of administration and the judgment of the prescriber. Dosage determination based on these factors is within the level of skill in the art and generally dosages range from 0.01 mg / kg / day to approximately 2000 mg / kg / day. More preferred dosage is 0.1 mg / kg / day to 500 mg / kg / day. Administration may be once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.

The pharmaceutical composition may be administered to mammals such as mice, livestock, humans, and the like by various routes. All modes of administration can be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine mucosal or cerebrovascular injections. Kimne sylvestre extract of the present invention or a compound isolated therefrom has little toxicity and side effects, so it is a drug that can be used with confidence even for long-term administration for the purpose of prevention.

The present invention also provides a dietary supplement for diabetic improvement comprising at least one compound selected from the group consisting of the Kimne sylvestre extract or the formula (I) isolated therefrom and a food supplement acceptable food supplement.

The Kimne sylvestre extract or a compound isolated therefrom may be included as 0.001 to 100% by weight based on the total weight of the health food. Preferably it is 0.001-50 weight%, More preferably, it is 0.001-30 weight%, Most preferably, it is 0.001-10 weight%.

The health functional food of the present invention includes the form of tablets, capsules, pills, or liquids, and the foods to which the Kimne sylvestre extract of the present invention or the compound separated therefrom can be added, for example, various foods , Drinks, gum, tea, vitamin complexes, and health functional foods.

The diabetes may be type 1 diabetes, type 2 diabetes or gestational diabetes. Preferably type 1 diabetes or type 2 diabetes, most preferably type 2 diabetes.

The present invention relates to a composition for preventing or treating diabetes comprising Gymnema sylvestre extract or a compound isolated therefrom as an active ingredient, wherein the Kimne sylvestre extract or a compound isolated therefrom is a fat cell It was confirmed that there is an effect of promoting the absorption of sugar in and improve the glucose tolerance and insulin resistance.

Through this, it is expected that the Kimne sylvestre extract of the present invention or the compound isolated therefrom can be usefully used in the development of diabetes preventive or therapeutic medicines or diabetes functional supplements.

1 is the fragrance of (a) plant, (b) leaf shape of Gymnema sylvestre (GS-V) originated in Vietnam and Kimnema sylvestre originated in India (GS-I), (b) leaf shape, (c Morphological differences in fruit, (d) stem, and (e) leaf vein.
Figure 2 shows genetic schematics within or across species plants.
Figure 3 is a result of analyzing the difference in the chemical composition in the plant according to the Gimne sylvestre origin, (A) of the standard sample containing the gymnemagenin (B), Gimnema originated in India Of the Sylvestre (GS-I) extract, (C) shows the HPLC peak of the chemical composition of Kimne Sylvestre (GS-V) extract from Vietnam.
4 is a result confirming the effect of improving the glucose tolerance of gimne sylvestre (GS-V) extract originated in Vietnam, after the injection of glucose (sugar), (A) measures the amount of glucose in the blood according to the GS-V extract treatment As a result, (B) shows the results of analyzing the glucose-improving effect of the GS-V extract.
5 is a result confirming the effect of improving the insulin sensitivity of Kim Nem Sylvestre (GS-V) extract originated in Vietnam, after insulin injection, (A) is the amount of glucose (sugar) in the blood according to GS-V extract treatment As a result of the measurement, (B) shows the result of analyzing the insulin resistance reduction effect of the GS-V extract.
6 is a result of confirming the cytotoxicity of the compound isolated from the Kimne sylvestre (GS-V) extract of Vietnam origin, (A) cytotoxicity according to the treatment of 40uM compound, (B) cytotoxicity at 40uM The results of reconfirming the cytotoxicity according to the concentration of some compounds (compounds 4, 11, 12) showed.
Figure 7 shows the effect of promoting the absorption of glucose into adipocytes of the compound isolated from the Kimne sylvestre (GS-V) extract originated in Vietnam, (A) is 2-NBDG (glucose of Fluorescent Derivatives) The degree of absorption was analyzed by fluorescence intensity measurement.

Hereinafter, a preferred embodiment of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, the information provided herein is to be thorough and complete, and to fully convey the spirit of the present invention to those skilled in the art.

<Example 1. Kimne Sylvestre (Gymnema sylvestre, GS )  Gathering>

Kim Nee Sylvestre (GS-V) originated from Vietnam of the present invention and Kim Nee Sylvestre (GS-I) originated from India as a control group are Nam Dinh, 20 ° 09'26.2 "N 106 ° 19 ' 06.3 "E)) and cultivated under the same conditions in the Good Agriculture-Practice-certified farm (WHO GAP) in Thai Nguyen provinces (21 ° 52'47.5" N 105 ° 44'35.7 "E). Was used. In the case of Kimne Sylvestre, where Vietnam originated, two other regions of origin (GS-V1 and GS-V2) were used.

<Example 2. Comparison of Characteristics of Kimne Silvestre by Geographical Influence>

Example  2-1. Kimnema Sylvester  Morphological and Anatomical Comparison

We compared the morphological and anatomical characteristics of Kimne Sylvestre (GS-V) originated in Vietnam and Kimne Sylvestre (GS-I) originated in India.

Visual and EZ4 stereoscopic microscopes (Leica, Germany) were used to observe the morphology, stems, leaves, flowers, fruits and seeds of plants. Also, for anatomical analysis, fresh plant stems and leaves were sliced using a rotatory microtome, double stained with methylene blue and carmine red, and then anatomically analyzed using an optical microscope. The characteristic was observed and the result is shown in FIG.

There were no differences in morphological differences between the two samples (GS-V1 and GS-V2), the Kimne Sylvestre, originating in Vietnam. On the other hand, a similar aspect exists in the result of comparing the external morphological shape of Vietnam-origin Kimne Sylvestre (GS-V2) and India-origin Kimne Sylvestre (GS-I), as shown in FIG. It was found that there was a difference between GS-V2 and GS-I in the plant's fragrance (a), leaf form (b), fruit (c), stem (d), and leaf vein (e).

Example 2-2. Genetic Differences in Kimne Sylvestre

In order to confirm the genetic difference between the Kimne sylvestre of the present invention, the internal transcriped spacer (ITS) region of the Kimne sylvestre was used. ITS includes two unencrypted ITS1 and ITS2 separated by the highly conserved 5.8S rRNA gene.

From the leaves of two kinds of Kimnema Sylvestre (GS-V1 and GS-V2) originated in Example 1 for genetic analysis and Sylvestre (GS-I) originated in India, DNA was extracted from each plant using the DNeasy Plant Mini Kit. Using the extracted DNA as a template, PCR was performed using the primers of Table 1 to obtain a PCR product. After purifying the obtained PCR product, DNA sequencing was performed by Macrogen (Korea).

In addition, G. sylvestre , G. latifolium , G. inodorum , G. inodorum , and G. ema unnanse ( G ) are registered with the National Institutes of Health (GenBank). yusense and sequencing information of 18 species belonging to Marsdenia tenacissima were collected and used to analyze the differences between the nucleotide sequences and the nucleotide sequences between plants.

DNA sequencing was performed using Geneious DNA sequencing analysis software, version 8.1.8, Biomatters Ltd. New Zealand. Using the 18 nucleotide sequences collected from the GenBank and the nucleotide sequences of GS-V1, GS-V2 and GS-I confirmed through the analysis, the phylogenetic tree between the plants was confirmed, and the results are shown in FIG. It was.

Gene region Primer name Primer Sequence ITS1-5.8S-ITS2 ITS5 5′-GGAAGTAAAAGTCGTAACAAGG-3 ′ ITS4 5′-TCCTCCGCTTATTGATATGC-3 ′

As can be seen from Figure 2, it was confirmed that the distinction between the species Kimnema plant appears clearly. In addition, in the case of Kimne Sylvestre species level, it was divided into two groups, and it was confirmed that these groups are clearly distinguished by the source.

Example  2-3. Kimnema Sylvester  Identify chemical differences

The differences in the chemical composition of two kinds of Kimne sylvestre (GS-V, GS-I), which are the source of Vietnam and India collected in Example 1, were confirmed. At this time, in the case of GS-V, a mixture of two species (GS-V1 and GS-V2) originated in different regions was used.

50 ml of 60% [v / v] ethanol was added to 5 g of two finely powdered Gemmane Silvestres (GS-V and GS-I), respectively, and ultrasonically treated at 50 ° C. for 3 hours to extract plant components. It was. The plant extract obtained by repeating this process three times was cooled and passed through Whatman's filter paper to obtain a filtered plant extract. The filtered plant extract was evaporated under reduced pressure at 50 ° C. to obtain a plant extract in the form of a residue. 10 mg of the obtained plant extract in the form of a residue was dissolved in 1 ml of hydrolysis solution (5M HCl, 60% ethanol) and reacted at 90 ° C. for 24 hours. After the reaction was completed, the hydrolysis solution was removed using ethyl acetate and dried in vacuo. The dried product was dissolved in methanol to have a final concentration of 2 mg / ml, and then filtered with a 0.2 μm hydrophilic membrane filter manufactured by Whatman, to obtain a sample for plant extract component analysis.

High performance liquid chromatography (HPLC) was performed to analyze the chemical composition of each plant. HPLC was performed using an Agilent 1200 HPLC system (Agilent Technologies, Palo Alto, Calif., USA) at an acetonitrile: water (10 → 100% [v / v]) gradient at 30 ° C. After the extract concentration was 2 mg / ml, 10 μl of each was injected. The column used was INNO C18 (4.6 × 250mm, 5㎛ particle size, Young Jin Bio Chrom Co., Ltd), the flow rate was 0.6ml / min, UV detection was performed at 201nm. In this case, a sample containing gymnemagenin was used as a standard sample, and the results are shown in FIG. 3.

As shown in Figure 3, in the case of HPLC analysis of the standard sample containing the Kimnemaginin of (A), it was confirmed that the peak corresponding to the Kimnemaginin indicated by an asterisk, GS-I of (B) In the (C) GS-V of the (g), while the peak corresponding to the Kimnemagenin appears, the Kimnemaginin peak did not appear (X). Moreover, when comparing the peak pattern of GS-I and the peak pattern of GS-V, the peak pattern of both samples showed the difference and it turned out that the chemical composition of GS-V and GS-I differs.

 Thus, despite this, even though the genetically modified plants of Vietnam-based Kimne Sylvestre (GS-V) and India-derived Kimnema Sylvestre (GS-I) are homogeneous, there are morphological and genetic differences and they are included in each plant. It was found that there is a difference in the chemical composition.

<Example 3. Preparation of Kim Nema sylvestre (GS-V) extract originated from Vietnam>

Vietnam-based Kimnema Sylvestre (GS-V) extract is located in Nam Duoc, http: // namduoc, in the Dinh Vong Hau Ward, Cau Giay District, Hanoi, Vietnam. .vn /) The company used extracts used to produce dietary supplements.

In detail describing the extract preparation method, 45 liters of 60% [v / v] ethanol was mixed in 10 kg of GS-V powdered with 0.4 mm of aerial parts (plant leaves and stems) of GS-V, and then mixed. Extracted three times with three high temperature continuous extraction systems. The solvent of the extract was evaporated in vacuo to make the water content 20%, and finally, the spray was dried to make the water content 3% to prepare an extract.

Example 4 Compound Separation from GS-V Extract

1 kg of the GS-V extract prepared in Example 3 was suspended in 30% [v / v] ethanol, and then adsorbed onto a diaion HP-20 resin, and 30% [v / v]. ] Elution was performed in order of ethanol, 50% [v / v] ethanol, 95% [v / v] ethanol, and acetone in order.

Among them, 95% [v / v] ethanol eluate (300 g) was converted into n-hexane: ethyl acetate (10: 1 → 0: 1 [v / v]), followed by ethyl Acetate: methanol (6: 1 → 0: 1 [v / v]) was subjected to silica gel column chromatography (column size: 15 × 45 cm, particle size: 63-200 μm) under elution conditions. 8 fractions were obtained (AH).

F fractions were subjected to reverse-phase silica gel column chromatography under a concentration gradient of methanol: water (2: 3 → 1: 0 [v / v]) to obtain 10 fractions (FI ~ FX).

FII fraction (10 g) was added to silica gel column chromatography with column size 5 × 20 cm and particle size 40-63 μm, followed by dichloromethane: methanol (6: 1 → 0: 1 [v / v]). Three fractions were obtained by elution under concentration gradient (FII.N1 to FII.N3).

FII.N2 fractions were subjected to Sephadex LH-20 column chromatography and acetonitrile: water (3: 7 [v / v]) under 100% [v / v] methanol isocratic conditions. Compound 1 (22.5 mg) and compound 2 (4.7 mg) were separated by continuous HPLC (column: Optima Pak C ₁₈ ) under isocratic elution, 2 ml / min flow rate.

The FII.N3 fractions were added to reverse phase silica gel column chromatography, and then eluted under a gradient of acetonitrile: water (1: 5 → 1: 0 [v / v]) to obtain four fractions (FII.N3.R1). ~ FII.N3.R4).

Compound 3 (12.0) by HPLC (column: Optima Pak C ₁₈ ) under isocratic conditions of acetonitrile: water (3: 7 [v / v]) with a flow rate of 2 mL / min as the FII.N3.R1 fraction Mg) and compound 10 (18.1 mg) were isolated.

FII.N3.R3 fractions were subjected to HPLC (column: Optima Pak C ₁₈ ) under isocratic conditions of acetonitrile: water (8:25 [v / v]) at a flow rate of 2 ml / min to give compound 5 (7.9 Mg) and compound 6 (14.0 mg) were isolated.

Separation of the FII.N3.R4 fractions with 100% [v / v] methanol isocratic conditions and isocratic conditions with acetonitrile: water (7:20 [v / v]), 2 mL / HPLC (column: Optima Pak C ₁₈ ) was performed successively at the flow rate of minutes to separate compound 7 (5.5 mg) and compound 9 (5.1 mg).

The FV fractions were subjected to reverse phase silica gel column chromatography under a gradient of acetonitrile: water (2: 5 → 1: 0 [v / v]) to obtain 10 fractions (FV.R1 to R10).

Compound 12 (9.1 mg) by HPLC (column: Optima Pak C ₁₈ ) under isocratic elution of acetonitrile: water (2: 5 [v / v]) with a flow rate of 2 ml / min as a FV.R10 fraction ) Was separated.

Sepadex LH-20 column chromatography with isotrope: water (7:20 [v / v]) isocratic elution conditions with 100% [v / v] methanol isocratic as FV.R6 fraction, 2 ml / min HPLC (column: Optima Pak C ₁₈ ) was carried out continuously at flow rate conditions of to separate compound 8 (6.1 mg) and compound 11 (17.0 mg).

FX 4 fractions were subjected to HPLC (column: Optima Pak C ₁₈ ) under isocratic elution conditions of acetonitrile: water (3: 5 [v / v]) at a flow rate of 2 ml / min to give compound 4 (5.0 mg). Separated.

Example 5 Confirmation of Physicochemical Structure of the Compound

Example 5-1. 3β, 16β, 28-trihydroxyolean-12-ene-29-ioic acid 3-O-β-D-glucopyranosyl (1 → 3) -β-D-glucuronopyranoside (Compound 1 )

3β, 16β, 28-trihydroxyolean-12-en-29-oic acid 3-O-β-D-glucopyranosyl (1 → 3) -β-D-glucuronopyranoside;

Gymnemoside ND1;

Amorphous powder;

-24.5°(c 0.2, 메탄올);[α]

-24.5 ° (c 0.2, methanol);

IR (KBr) V _max : 3399, 2943, 1706, 1371, 1051, 1030 cm ^-1 ;

¹ H NMR and ¹³ C NMR data see Tables 2 and 3 below;

HRESIMS m / z 825.4315 [M ⁻ H] ⁻ (calcd for C ₄₂ H ₆₅ O ₁₆ , 825.4278).

Example 5-2. 3β, 16β, 28-trihydroxyolean-12-ene-29-ioic acid 3-O-β-D-galactopyranosyl (1 → 3) -β-D-glucuronopyranoside (compound 2)

3β, 16β, 28-trihydroxyolean-12-en-29-oic acid 3-O-β-D-galactopyranosyl (1 → 3) -β-D-glucuronopyranoside;

Gymnemoside ND2;

Amorphous powder;

-23.3°(c 0.2, 메탄올);[α]

-23.3 ° (c 0.2, methanol);

IR (KBr) V _max : 3416, 2920, 1728, 1441, 1084, 1021 cm ^-1 ;

¹ H NMR and ¹³ C NMR data see Tables 2 and 3 below;

HRESIMS m / z 825.4297 [M ⁻ H] ⁻ (calcd for C ₄₂ H ₆₅ O ₁₆ , 825.4278).

Example 5-3. Citakisogenin 3-O-β-D-glucopyranosyl (1 → 3) -β-D-glucuronopyranoside (Compound 3)

Sitakisogenin 3-O-β-D-glucopyranosyl (1 → 3) -β-D-glucuronopyranoside;

Gymnemoside ND3;

Amorphous powder;

-5.9°(c 0.2, 메탄올);[α]

-5.9 ° (c 0.2, methanol);

IR (KBr) V _max : 3374, 2932, 1712, 1367, 1081, 1023 cm ^-1 ;

¹ H NMR and ¹³ C NMR data see Tables 2 and 3 below;

HRESIMS m / z 811.4521 [M ⁻ H] ⁻ (calcd for C ₄₂ H ₆₇ O ₁₅ , 811.4485).

Example 5-4. 3β, 16β-dihydroxyolean-12-ene 3-O-β-D-glucopyranosyl (1 → 3) -β-D-glucuronopyranoside (Compound 4)

3β, 16β-dihydroxyolean-12-en 3-O-β-D-glucopyranosyl (1 → 3) -β-D-glucuronopyranoside;

Gymnemoside ND4;

Amorphous powder;

-8.7°(c 0.2, 메탄올);[α]

-8.7 ° (c 0.2, methanol);

IR (KBr) V _max : 3399, 2926, 1720, 1459, 1352, 1164, 1083, 1022 cm ^-1 ;

¹ H NMR and ¹³ C NMR data see Tables 2 and 3 below;

HRESIMS m / z 779.4625 [M ⁻ H] ⁻ (calcd for C ₄₂ H ₆₇ O ₁₃ , 779.4582).

Example 5-5. 29-O- (β-D-glucopyranosyl) Gimnegenol 3-O-β-D-glucuronopyranoside (Compound 5)

29-O- (β-D-glucopyranosyl) gymnemagenol 3-O-β-D-glucuronopyranoside;

Gymnemoside ND5;

Amorphous powder;

-22.3°(c 0.2, 메탄올);[α]

-22.3 ° (c 0.2, methanol);

IR (KBr) V _max : 3389, 2913, 1731, 1435, 1085, 1025 cm ^-1 ;

¹ H NMR and ¹³ C NMR data see Tables 2 and 3 below;

HRESIMS m / z 811.4526 [M ⁻ H] ⁻ (calcd for C ₄₂ H ₆₅ O ₁₆ , 811.4485).

Example 5-6. Citakisogenin 3-O-β-D-glucuronopyranoside (Compound 6)

Sitakisogenin 3-O-β-D-glucuronopyranoside;

Gymnemoside ND6;

Amorphous powder;

-15.7°(c 0.2, 메탄올);[α]

-15.7 ° (c 0.2, methanol);

IR (KBr) V _max : 3399, 2926, 1720, 1455, 1083, 1032 cm ^-1 ;

¹ H NMR and ¹³ C NMR data see Tables 4 and 5 below;

HRESIMS m / z 649.3967 [M ⁻ H] ⁻ (calcd for C ₃₆ H ₅₇ O ₁₀ , 649.3957).

Example 5-7. Gimnegenol 3-O-β-D-glucuronopyranoside (Compound 7)

Gymnemagenol 3-O-β-D-glucuronopyranoside;

Gymnemoside ND7;

Amorphous powder;

-12.4°(c 0.2, 메탄올);[α]

-12.4 ° (c 0.2, methanol);

IR (KBr) V _max : 3409, 2946, 1736, 1465, 1362, 1067, 1032 cm ^-1 ;

¹ H NMR and ¹³ C NMR data see Tables 4 and 5 below;

HRESIMS m / z 649.3985 [M ⁻ H] ⁻ (calcd for C ₃₆ H ₅₇ O ₁₀ , 649.3957).

Example 5-8. 28-benzoyl-22α-hydroxyrongispinogenin 3-O-β-D-glucuronopyranoside (Compound 8)

28-benzoyl-22α-hydroxylongispinogenin 3-O-β-D-glucuronopyranoside;

Gymnemoside ND8;

Amorphous powder;

-2.4°(c 0.2, 메탄올);[α]

-2.4 ° (c 0.2, methanol);

IR (KBr) V _max : 3374, 2933, 1716, 1446, 1380, 1095, 1020 cm ^-1 ;

¹ H NMR and ¹³ C NMR data see Tables 4 and 5 below;

HRESIMS m / z 769.4210 [M ⁻ H] ⁻ (calcd for C ₄₃ H ₆₁ O ₁₂ , 769.4169).

Example 5-9. 3-O-β-D-glucuronopyranosyl 3β, 16β, 28-trihydroxyolean-12-ene-29,22β-oxide (Compound 9)

3-O-β-D-glucuronopyranosyl 3β, 16β, 28-trihydroxyolean-12-en-29,22β-olide;

Gymnemoside ND9;

Amorphous powder;

-22.5°(c 0.2, 메탄올);[α]

-22.5 ° (c 0.2, methanol);

IR (KBr) V _max : 3414, 2953, 1706, 1350, 1051, 1005 cm ^-1 ;

¹ H NMR and ¹³ C NMR data see Tables 4 and 5 below;

HRESIMS m / z 661.3616 [M ⁻ H] ⁻ (calcd for C ₃₆ H ₅₃ O ₁₁ , 661.3593).

Example 5-10. 29-hydroxyrongispiginogenin 3-O-D-glucopyranosyl (1 → 3) -D-glucuronopyranoside (Compound 10)

29-hydroxylongispinogenin 3-O-D-glucopyranosyl (1 → 3) -D-glucuronopyranoside;

Amorphous powder;

+10.3°(c 0.12, 메탄올);[α]

+ 10.3 ° (c 0.12, methanol);

IR (KBr) V _max : 3422, 2928, 1618, 1430, 1028 cm ^-1 ;

¹ H NMR and ¹³ C NMR data see Tables 6 and 7 below;

ESIMS m / z 811.4 [M ⁻ H] ⁻ .

Example 5-11. Rongispiginogenin 3-O-D-glucopyranosyl (1 → 3) -D-glucuronopyranoside (Compound 11)

Longispinogenin 3-O-D-glucopyranosyl (1 → 3) -D-glucuronopyranoside;

Amorphous powder;

+18.15°(c 0.12, 메탄올);[α]

+ 18.15 ° (c 0.12, methanol);

IR (KBr) V _max : 3440, 2948, 1636, 1420, 1078, 1028 cm ^-1 ;

¹ H NMR and ¹³ C NMR data see Tables 6 and 7 below;

ESIMS m / z 795.4 [M ⁻ H] ⁻ .

Example 5-12. Alternoside XII (Compound 12)

Alternoside XII;

Amorphous powder;

+15.9°(c 0.12, 메탄올);[α]

+ 15.9 ° (c 0.12, methanol);

IR (KBr) V _max : 3400, 2900, 1706, 1730, 1665, 1240, 1160 cm ^-1 ;

¹ H NMR and ¹³ C NMR data see Tables 6 and 7 below;

ESIMS m / z 893.5 [M ⁻ H] ⁻ .

< Example  6. GS -V extract extracts for diabetic treatment

Example 6-1. Confirmation of improvement in glucose tolerance

8-week-old male C57BL / 6J mice (5 per experimental group) were ingested freely for 9 weeks in a high-fat diet providing 60% kcal of fat to induce a diabetic model. Once, orally administered to 300 mg / kg, mice treated with metformin (300 mg / kg) as a positive control group, the mice treated with nothing was used as a control.

To confirm the effect of improving glucose tolerance, nine weeks after GS-V administration, all mice in the group were fasted for 16 hours, and then glucose (sugar) was administered intraperitoneally at a concentration of 2 g / kg, and 0, 30, 60, 90 and After 120 minutes, a small amount of blood at the tail end is collected, and the blood glucose level is directly measured using a glucometer. Then, the area between the line graph and the Y axis (AUC) is calculated by group. The degree of glucose tolerance improvement was compared and analyzed, and the results are shown in FIG. 4.

As shown in Figure 4, the result of confirming the amount of glucose in the blood (A), in the case of the group treated with GS-V extract, it was confirmed that the amount of glucose is reduced compared to the control group treated with nothing. In addition, in the results of glucose tolerance analysis (B), the group treated with the GS-V extract is weaker than the positive control group treated with metformin, but the effect of improving glucose tolerance is weak compared to the control group without any treatment, it was found that Confirmed.

Example 6-2. Identify the effects of improving insulin resistance

To confirm the effect on improving insulin resistance, 8-week-old male C57BL / 6J mice (5 per experimental group) were freely ingested for 10 weeks in a high-fat diet providing 60% kcal of fat in the same manner as in Example 6-1. In the meantime, the GS-V extract of the present invention was orally administered to 300 mg / kg once a day, and mice treated with metformin 300 mg / kg as a positive control group were used as controls, and mice without any treatment were used. .

After 10 weeks of administration, mice in each group were fasted for 4 hours, and insulin was administered at a concentration of 1.0 IU for each mouse, and a small amount of blood at the tail end was collected at 0, 30, 60, 90, and 120 minutes. Measure the blood glucose level directly using a glucometer, and then calculate the area under curve (AUC) between the line graph and the Y-axis for each group, and compare and analyze the degree of insulin resistance. 5 is shown.

As shown in Figure 5, the result of confirming the amount of glucose in the blood (A), in the group treated with the GS-V extract, the amount of glucose in the blood compared to the control group treated nothing and metformin treated positive In the insulin resistance analysis (B), the group treated with GS-V extract was found to have reduced insulin resistance compared to the metformin-treated positive control group.

Through this, it can be seen that the Kimne sylvestre (GS-V) extract of Vietnam of the present invention has a therapeutic effect on diabetes by improving glucose tolerance and insulin resistance.

<Example 7. Confirmation of Diabetes Treatment Effects of Compounds Isolated from GS-V Extracts>

Example 7-1. Induce differentiation of 3T3-L1 preadipocyte

Adiose tissue, composed of adipocytes, plays an important role in the overall lipid and energy homeostasis of the human body. Excess sugar in the body absorbs sugar into adipose and muscle tissue for removal of excess sugar. In this case, diabetes may occur when a problem occurs in the absorption of sugar into adipose tissue.

Thus, 3T2-L1 adipocytes differentiated from 3T3-L1 fibroblasts were used to confirm the diabetes treatment effect of the compound isolated from the GS-V extract of the present invention.

3T3-L1 fibroblasts were treated with 10% fetal bovine serum (FBS), 1 uM dexamethasone, 520 uM 3-isobutyl-1-methyl-xanthine and 1 μg / ml insulin. DMEM (Dulbecco's modified eagle's medium) was included and incubated for 4-6 days at 37 ℃, CO ₂ cell incubator. During the incubation process, every two days, the cells were replaced with DMEM medium containing 10% FBS, 1 μg / ml insulin, 100 U / ml penicillin, and 100 μg / ml streptomycin to induce differentiation into adipocytes.

Example 7-2. Confirmation of Cytotoxicity of Compounds Isolated from GS-V Extracts

MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) assay was performed to confirm the cytotoxicity of the compounds of the present invention.

 The 3T2-L1 adipocytes obtained in Example 7-1 were dispensed into 96-well plates, incubated for 24 hours in DMEM medium containing 10% FBS, and the compound isolated in Example 5 had a concentration of 40 uM. It was replaced with DMEM medium contained as and incubated for 24 hours. At this time, cells treated with nothing were used as a control. After 24 hours, 20 μl of 2 mg / ml MTT solution was added to each well, followed by 4 hours of reaction at 37 ° C., the culture medium was removed, and the formed formazan precipitate was dissolved in 100 μl of DMSO (dimethyl sulfoxide). Absorbance was measured at nm and the results are shown in FIG. 6.

As can be seen from FIG. 6, when Compound 1 to Compound 12 isolated in Example 5 were treated with 40 uM (A), the other compounds except Compound 4, Compound 11, and Compound 12 were not cytotoxic. . In order to further examine the effects of Compound 4, Compound 11 and Compound 12 on cytotoxicity, when varying concentrations of Compound (B), all of Compound 4, Compound 11 and Compound 12 showed cytotoxicity in a concentration dependent manner. B, it was confirmed that the toxicity does not appear when the concentration is less than 2.5uM.

Example 7-3. Confirmation of Sugar Absorption Promoting Effect of Compounds from GS-V Extract

In order to confirm the diabetes treatment effect of the compound of the present invention, it was confirmed whether the glucose uptake of adipocytes (glucose uptake).

The 3T3-L1 adipocytes obtained in Example 7-1 were dispensed into 96-well plates and incubated for 24 hours in sugar-free DMEM medium containing 10% FBS. 50 uM of 2-NBDG (2- (N- (7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino) -2-deoxyglucose) (Invtrogen, USA), a fluorescent derivative of glucose, was added to all wells. After treatment, insulin 1 used as a compound 1 to compound 12 or a positive control isolated in Example 5 was treated in each well and incubated for 1 hour. In this case, Compound 4, Compound 11 and Compound 12 of the present invention was treated with a concentration of 2uM, the remaining compounds were treated at a concentration of 20uM, insulin was treated to 100nM, only 2-NBDG was used as a control. After 1 hour, the cells were washed with cold PBS (phosphate buffer saline) and then the excitation / emission wavelengths were reduced using a fluorescence microplate reader (Spectra Max GEMINI XPS, Molecular Devices, USA). Fluorescence intensity was measured under conditions of 450/535 nm. In addition, fluorescence images were taken using a fluorescence microscope (Olympus ix70 Fluorescence Microscope, Olympus Corporation, Japan), and the results are shown in FIG. 7.

As can be seen from Figure 7, the results of measuring the intracellular fluorescence intensity by the absorption of adipocytes of 2-NBDG (A), most of the compounds isolated from the GS-V extract promotes the glucose uptake of adipocytes It was confirmed. In addition, as a result of confirming the fluorescence image in the adipocytes (B), it was confirmed that the cells expressing the fluorescence in the adipocytes treated with the compound isolated from the GS-V extract significantly increased compared to the control (vehicle).

Through this, it can be seen that the compound isolated from the Kimne sylvestre (GS-V) extract, which is the origin of Vietnam of the present invention, has a therapeutic effect on diabetes by promoting glucose uptake of fat cells.

Preparation Example 1. Pharmaceutical Formulations

Formulation Example 1-1. Manufacture of tablets

20 g of Kimne sylvestre (GS-V) extract or 200 mg of Compound 7, which is the source of Vietnam of the present invention, was mixed with 175.9 g of lactose, 180 g of potato starch, and 32 g of colloidal silicic acid, respectively. 10% gelatin solution was added to the mixture, which was then ground and passed through a 14 mesh sieve. It was dried and the mixture obtained by adding 160 g of potato starch, 50 g of talc and 5 g of magnesium stearate was made into a tablet.

Formulation Example 1-2. Preparation of Injection

100 mg of Compound 7 of the present invention, 0.6 g of sodium chloride and 0.1 g of ascorbic acid were dissolved in distilled water to make 100 ml. The solution was bottled and sterilized by heating at 20 ° C. for 30 minutes.

Preparation Example 2 Preparation of Health Functional Food

Formulation Example 2-1. Preparation of Health Functional Food

Kimne Sylvestre (GS-V) extract of the present invention 20g, vitamin mixture proper amount, vitamin A acetate 70 ㎍, vitamin E 1.0 mg, vitamin B1 0.13 mg, vitamin B2 0.15 mg, vitamin B6 0.5 mg, vitamin B12 0.2 ㎍, vitamin C 10 mg, biotin 10 µg, nicotinic acid amide 1.7 mg, folic acid 50 µg, calcium pantothenate 0.5 mg, mineral mixture appropriate amount, ferrous sulfate 1.75 mg, zinc oxide 0.82 mg, magnesium carbonate 25.3 mg, potassium monophosphate 15 mg, 55 mg of dibasic calcium phosphate, 90 mg of potassium citrate, 100 mg of calcium carbonate, and 24.8 mg of magnesium chloride were mixed to prepare granules. In addition, the composition ratio of the above-mentioned vitamin and mineral mixture may be arbitrarily modified, and it may be prepared by mixing the above components according to a conventional health functional food manufacturing method.

Formulation Example 2-2. Preparation of Health Functional Drink

Kimne sylvestre (GS-V) extract 1 of the present invention 1g, citric acid 0.1g, fructooligosaccharide 100g, purified water 900g was mixed, stirred, heated, filtered, sterilized, refrigerated according to the conventional beverage production method to prepare a beverage. .

Claims (10)

3β, 16β, 28-trihydroxyolean-12-ene-29-ioic acid 3-O-β-D-glucopyranosyl (1 → 3) -β-D-glucuronopyrano of Formula 1 Seed (compound 1), 3β, 16β, 28-trihydroxyolean-12-ene-29-oic acid 3-O-β-D-galactopyranosyl (1 → 3) -β-D-gluco Lonofyranoside (Compound 2), Citakisogenin 3-O-β-D-glucopyranosyl (1 → 3) -β-D-glucuronopyranoside (Compound 3), 3β, 16β-di Hydroxyolean-12-ene 3-O-β-D-glucopyranosyl (1 → 3) -β-D-glucuronopyranoside (Compound 4), 29-O- (β-D-glucose Pyranosyl) Kimnemagenol 3-O-β-D-glucuronopyranoside (Compound 5), citakisogenin 3-O-β-D-glucuronopyranoside (Compound 6), Kimnemager Nol 3-O-β-D-glucuronopyranoside (Compound 7), 28-benzoyl-22α-hydroxyrongispinogenin 3-O-β-D-glucuronopyranoside (Compound 8) , 3-O-β-D-glucuronopyranosyl 3β, 16β, 28-trihydroxyolean-12-ene-29,22β- Reed (Compound 9), 29-hydroxyrongispiginogenin 3-OD-glucopyranosyl (1 → 3) -D-glucuronopyranoside (Compound 10), longispiginogenin 3-OD-glucopyranosyl An effective extract of Gymnema sylvestre comprising at least one compound selected from the group consisting of (1 → 3) -D-glucuronopyranoside (Compound 11) and alternoside XII (Compound 12) A pharmaceutical composition for preventing or treating diabetes, comprising as an ingredient.
[Formula 1]

The method of claim 1,
The Kimne sylvestre extract is characterized in that the Kimne sylvestre extract is extracted with one or more solvents selected from the group consisting of water, C1 to C4 lower alcohol, acetone, dichloromethane, ethyl acetate, n-hexane and chloroform Pharmaceutical composition for preventing or treating diabetes. The method according to claim 1 or 2,
The Kimne sylvestre is a diabetic preventive or therapeutic pharmaceutical composition, characterized in that the origin. 3β, 16β, 28-trihydroxyolean-12-ene-29-ioic acid 3-O-β-D-glucopyranosyl (1 → 3) -β-D-glucuronopyrano of Formula 1 Seed (compound 1), 3β, 16β, 28-trihydroxyolean-12-ene-29-oic acid 3-O-β-D-galactopyranosyl (1 → 3) -β-D-gluco Lonofyranoside (Compound 2), Citakisogenin 3-O-β-D-glucopyranosyl (1 → 3) -β-D-glucuronopyranoside (Compound 3), 3β, 16β-di Hydroxyolean-12-ene 3-O-β-D-glucopyranosyl (1 → 3) -β-D-glucuronopyranoside (Compound 4), 29-O- (β-D-glucose Pyranosyl) Kimnemagenol 3-O-β-D-glucuronopyranoside (Compound 5), citakisogenin 3-O-β-D-glucuronopyranoside (Compound 6), Kimnemager Nol 3-O-β-D-glucuronopyranoside (Compound 7), 28-benzoyl-22α-hydroxyrongispinogenin 3-O-β-D-glucuronopyranoside (Compound 8) , 3-O-β-D-glucuronopyranosyl 3β, 16β, 28-trihydroxyolean-12-ene-29,22β- Reed (Compound 9), 29-hydroxyrongispiginogenin 3-OD-glucopyranosyl (1 → 3) -D-glucuronopyranoside (Compound 10), longispiginogenin 3-OD-glucopyranosyl (1 → 3) -D-glucuronopyranoside (compound 11) and alternoside XII (compound 12), the prevention of diabetes, characterized in that it comprises at least one compound selected from the group consisting of Therapeutic pharmaceutical composition.
[Formula 1]

3β, 16β, 28-trihydroxyolean-12-ene-29-oic acid 3-O-β-D-glucopyranosyl (1 → 3) -β-D-glucuronopyrano of Formula 1 Seed (compound 1), 3β, 16β, 28-trihydroxyolean-12-ene-29-oic acid 3-O-β-D-galactopyranosyl (1 → 3) -β-D-gluco Lonofyranoside (Compound 2), Citakisogenin 3-O-β-D-glucopyranosyl (1 → 3) -β-D-glucuronopyranoside (Compound 3), 3β, 16β-di Hydroxyolean-12-ene 3-O-β-D-glucopyranosyl (1 → 3) -β-D-glucuronopyranoside (Compound 4), 29-O- (β-D-glucose Pyranosyl) Kimnemagenol 3-O-β-D-glucuronopyranoside (Compound 5), citakisogenin 3-O-β-D-glucuronopyranoside (Compound 6), Kimnemager Nol 3-O-β-D-glucuronopyranoside (Compound 7), 28-benzoyl-22α-hydroxyrongispinogenin 3-O-β-D-glucuronopyranoside (Compound 8) , 3-O-β-D-glucuronopyranosyl 3β, 16β, 28-trihydroxyolean-12-ene-29,22β- Reed (Compound 9), 29-hydroxyrongispiginogenin 3-OD-glucopyranosyl (1 → 3) -D-glucuronopyranoside (Compound 10), longispiginogenin 3-OD-glucopyranosyl (1 → 3) -D-glucuronopyranoside (Compound 11) and alternoside XII (Compound 12) containing a Kimne sylvestre extract comprising at least one compound selected from the group consisting of an active ingredient Dietary supplement for diabetes improvement. The method of claim 5,
The Kimne sylvestre extract is characterized in that the Kimne sylvestre extract is extracted with one or more solvents selected from the group consisting of water, C1 to C4 lower alcohol, acetone, dichloromethane, ethyl acetate, n-hexane and chloroform Dietary supplement for diabetes improvement. The method according to claim 5 or 6,
The Kimne Sylvestre is a dietary supplement for diabetes improvement, characterized in that the origin of Vietnam. 3β, 16β, 28-trihydroxyolean-12-ene-29-oic acid 3-O-β-D-glucopyranosyl (1 → 3) -β-D-glucuronopyrano of Formula 1 Seed (compound 1), 3β, 16β, 28-trihydroxyolean-12-ene-29-oic acid 3-O-β-D-galactopyranosyl (1 → 3) -β-D-gluco Lonofyranoside (Compound 2), Citakisogenin 3-O-β-D-glucopyranosyl (1 → 3) -β-D-glucuronopyranoside (Compound 3), 3β, 16β-di Hydroxyolean-12-ene 3-O-β-D-glucopyranosyl (1 → 3) -β-D-glucuronopyranoside (Compound 4), 29-O- (β-D-glucose Pyranosyl) Kimnemagenol 3-O-β-D-glucuronopyranoside (Compound 5), citakisogenin 3-O-β-D-glucuronopyranoside (Compound 6), Kimnemager Nol 3-O-β-D-glucuronopyranoside (Compound 7), 28-benzoyl-22α-hydroxyrongispinogenin 3-O-β-D-glucuronopyranoside (Compound 8) , 3-O-β-D-glucuronopyranosyl 3β, 16β, 28-trihydroxyolean-12-ene-29,22β- Reed (Compound 9), 29-hydroxyrongispiginogenin 3-OD-glucopyranosyl (1 → 3) -D-glucuronopyranoside (Compound 10), longispiginogenin 3-OD-glucopyranosyl (1 → 3) -D-glucuronopyranoside (compound 11) and alternoside XII (compound 12) for improving diabetes, comprising at least one compound selected from the group consisting of Health functional food. Kimne sylvestre extracted with water, C1 to C4 lower alcohol, acetone, dichloromethane, ethyl acetate, n-hexane and chloroform as a solvent at least one selected from the group consisting of chromatographic extract of the Kimne sylvestre 1 3β, 16β, 28-trihydroxyolean-12-ene-29-ioic acid 3-O-β-D-glucopyranosyl (1 → 3) -β-D-glucuronopyranoside ( Compound 1), 3β, 16β, 28-trihydroxyolean-12-ene-29-ioic acid 3-O-β-D-galactopyranosyl (1 → 3) -β-D-glucuronopy Lanoside (Compound 2), Citakisogenin 3-O-β-D-glucopyranosyl (1 → 3) -β-D-glucuronopyranoside (Compound 3), 3β, 16β-dihydroxy Olean-12-ene 3-O-β-D-glucopyranosyl (1 → 3) -β-D-glucuronopyranoside (Compound 4), 29-O- (β-D-glucopyranosyl ) Kimnemagenol 3-O-β-D-glucuronopyranoside (Compound 5), citakisogenin 3-O-β-D-glucu Nopyranoside (Compound 6), Kimnemagenol 3-O-β-D-Glucuronopyranoside (Compound 7), 28-benzoyl-22α-hydroxyrongispinogenin 3-O-β-D- Glucuronopyranoside (compound 8), 3-O-β-D-glucuronopyranosyl 3β, 16β, 28-trihydroxyolean-12-ene-29,22β-oxide (compound 9 ), 29-hydroxyrongispiginogenin 3-OD-glucopyranosyl (1 → 3) -D-glucuronopyranoside (Compound 10), longgispinogenin 3-OD-glucopyranosyl (1 → 3) A method for separating at least one compound selected from the group consisting of) -D-glucuronopyranoside (Compound 11) and alternoside XII (Compound 12). Novel Compounds 3β, 16β, 28-trihydroxyolean-12-ene-29-ioic acid 3-O-β-D-glucopyranosyl (1 → 3) -β-D Glucuronopyranoside (Compound 1), 3β, 16β, 28-trihydroxyolean-12-ene-29-oic acid 3-O-β-D-galactopyranosyl (1 → 3)- β-D-glucuronopyranoside (Compound 2), citakisogenin 3-O-β-D-glucopyranosyl (1 → 3) -β-D-glucuronopyranoside (Compound 3) , 3β, 16β-dihydroxyolean-12-ene 3-O-β-D-glucopyranosyl (1 → 3) -β-D-glucuronopyranoside (Compound 4), 29-O- (β-D-glucopyranosyl) Gimnegenol 3-O-β-D-glucuronopyranoside (Compound 5), citachisogenin 3-O-β-D-glucuronopyranoside ( Compound 6), Kimnemagenol 3-O-β-D-glucuronopyranoside (Compound 7), 28-benzoyl-22α-hydroxyrongispinogenin 3-O-β-D-glucuronopy Lanoside (Compound 8) and 3-O-β-D-glucuronopyranosyl 3β, 16β, 28- Li-hydroxy oleic not -12- yen -29,22β- all lead (Compound 9).
[Formula 2]

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