KR101472086B1 - Compositions for Preventing or Treating Diabetes Comprising Castanea crenata-Derived Compounds or Extract from Castanea crenata - Google Patents

Abstract

The present invention relates to a composition for preventing, treating, ameliorating or alleviating diabetes comprising a compound based on a natural compound contained in chestnut extract as an active ingredient. According to the present invention, the compound used as an active ingredient in the composition of the present invention and the chestnut extract can be effectively used for preventing, treating, ameliorating or alleviating diabetes.

Description

Compositions for Preventing or Treating Diabetes Containing Chestnut-Derived Compounds or Chestnut Extracts {Compositions for Preventing or Treating Diabetes Comprising Castanea crenata-Derived Compounds or Extracts from Castanea crenata}

The present invention relates to a composition for preventing or treating diabetes comprising a chestnut-derived compound or a chestnut extract.

Insulin activates the PI3K-Akt (Phosphoinositide 3-kinase-Akt), CbI and MAPK (Mitogen-Activated Protein Kinase) pathways and translocates GLUT4 (Glucose transporter 4) to the cell membrane to increase glucose uptake It is a hormone.

The problem of insulin secretion or resistance causes diabetes and can cause a number of complications. The worldwide prevalence of diabetes in adults (aged 20-79) is 6.4% (28.5 billion) and is projected to rise to 7.7% (409 million) by 2030. Therefore, it is necessary to develop anti-diabetic material which can mimic the insulin effect.

Meanwhile, the present inventors have developed a screening system for insulin-like action compounds using a fluorescent glucose probe (Jung DW, et al., Mol . Biosyst ., 7 (2): 346-58 A patent application was filed against it (Korean Patent Application No. 2010-0073670).

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors screened 999 Korean native plant extract libraries using a screening system based on NBDG uptake that can identify insulin mimic drugs developed previously. As a result, chestnut ( Castanea Crenata ) extract was selected as a 'heat' extract that increased NBDG intake by 25% or more as compared to untreated 3T3-L1 adipocytes. In addition, seven compounds (UP-2.2: Scopoletin, UP-3.2: Fraxidin, UP-3.3: 6,7,8- UP-5.2.1: 4-hydroxy-5-methoxycinnamic acid, UP-5.2.3: 3,4 , 5-methoxy-tree body acid (3,4,5-trimethoxycinnamic acid), UP -3.5.3: new plaque is A ₂ (Fragransins A ₂₎ and UP-3.11.1: Marsden acid (Maslinic acid)) is 3T3 Ll < / RTI > adipocytes. In particular, the above seven compounds were re-tested by bio-based assay using zebrafish, and it was confirmed that all seven compounds significantly increased glucose uptake.

Accordingly, an object of the present invention is to provide a composition for preventing, treating, ameliorating or alleviating diabetes.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, there is provided a composition for preventing, treating, ameliorating or alleviating diabetes comprising a compound represented by the following formula (1):

Formula 1

In Formula 1, R ₁ is hydrogen, hydroxyl, halo, amino or C _{1 -10} alkoxy; R ₂ is hydrogen, halo or C _{1 -10} alkyl; And R ₃ is hydrogen, halo or C _{1 -10} alkyl;

(2)

In Formula 2, R ₁ is hydrogen, hydroxyl, halo, amino or C _{1 -10} alkoxy; R ₂ is hydrogen, halo or C _{1 -10} alkyl; And R ₃ is hydrogen, halo or C _{1 -10} alkyl; And R ₄ is carboxyl, C _{3 -10} carboxyalkyl, C _{3 -10} carboxy alkenyl, C _{3 -10} alkyl, carboxyl, C _{3 -10} alkenyl carboxyl, C _{3 -10} alkyl, carboxy alkyl, C _{3- 10} carboxy alkyl alkenyl, C _{3 -10} alkenyl carboxyalkyl, C _{4 -10} alkenyl carboxy alkenyl, C _{1 -10} alcohol, C _{1 -10} Al kenol, C _{2 -10} acyl, C _{1 -10} aldehydes, C _{1 -10} amide, C _{1 -10} amine, C _{2 -10} ester or sulfate;

(3)

In Formula 3, R ₁ and R ₅ are independently of each other hydrogen, halo or C _{1 -10} alkyl; R ₂ and R ₆ are independently H, halo or C _{1 -10} alkyl and each other; And R ₃ And R ₄ are independently hydrogen, halo, C _{1 -10} alkyl or C _{2 -10} alkenyl each other;

Formula 4

The compound represented by Formula 4 is Maslinic acid.

Formula 5

The compound represented by Formula 5 is arctigenic acid.

The present inventors screened 999 Korean native plant extract libraries using a screening system based on NBDG uptake that can identify insulin mimic drugs developed previously. As a 'heat' extract that increased NBDG uptake by 25% compared to untreated 3T3-L1 adipocytes, chestnut ( Castanea Crenata ) extracts were selected. In addition, seven of the 14 pure compounds isolated from the chestnut extract (UP-2.2: Scopoletin, UP-3.2: Fraxidin, UP-3.3: UP-5.2.1: 4-hydroxy-5-methoxycinnamic acid, UP-5.2.3: 3,4, 5-methoxy-tree body acid (3,4,5-trimethoxycinnamic acid), UP -3.5.3: new plaque is A ₂ (Fragransins A ₂₎ and UP-3.11.1: Marsden acid (Maslinic acid)) is 3T3- Lt; RTI ID = 0.0 > L1 < / RTI > adipocytes. In particular, the above seven compounds were re-tested by bio-based assay using zebrafish, and it was confirmed that all eight compounds significantly increased glucose uptake.

The compound used as an active ingredient in the composition for preventing, treating, ameliorating or alleviating diabetes of the present invention is represented by any one of formulas (1) to (5).

As used herein, the term " halo " used in formula (1) defining an active ingredient refers to a halogen group element and includes, for example, fluoro, chloro, bromo and iodo.

The term " alkyl " means a straight or branched unsubstituted or substituted saturated hydrocarbon group including, for example, methyl, ethyl, propyl, isobutyl, pentyl, hexyl, heptyl, octyl, nonyl and decyl. C _{1 -10} alkyl means an alkyl group having an alkyl unit of 1 to 10 carbon atoms, and when C _{1 -10} alkyl is substituted, the number of carbon atoms of the substituent is not included.

The term " alkoxy " means an -O alkyl group. C _{1 -10} alkoxy is not included in the carbon number of a substituent when the alkyl group means a unit having an alkoxy group having 1 to 10, C _{1 -10} alkyl substituted.

As used herein, the term " alcohol " used in formula (2) defining the active ingredient means a compound in which the hydroxyl group is bonded to the carbon atom of the alkyl or substituted alkyl group, and includes compounds in which a plurality of hydroxyl groups are substituted . The C _{1 -10} alcohol means an alcohol compound having an alcohol unit of 1 to 10 carbon atoms, and when the C _{1 -10} alcohol is substituted, the carbon number of the substituent is not included.

The term " alkenol " means a compound wherein the hydroxyl group is bonded to the carbon atom of the alkyl or substituted alkenyl group. C _{1 -20} alkenol means an alkenyl compound having an alkenol unit having 1 to 10 carbon atoms, and when the C _{1 -10} alkenol is substituted, the number of carbon atoms of the substituent is not included.

The term " acyl " means a radical formed by removal of a hydroxyl group from a carboxylic acid. C _{2 -10} acyl means an acyl group having an acyl unit having 2 to 10 carbon atoms, and when C _{2 -10} acyl is substituted, the number of carbon atoms of the substituent is not included.

The term " amide " means a functional group consisting of an acyl group bonded to a nitrogen atom. C _1-10 amide means an amide group having an amide unit having 1 to 10 carbon atoms, and when the C _{1 -10} amide is substituted, the carbon number of the substituent is not included.

The term " amine " refers to a functional group containing a basic nitrogen atom with a non-bonded pair (lone pair). C _{1 -10} amine means an amine group having an amine unit of 1 to 10 carbon atoms, and when C _{1 -10} amine is substituted, the number of carbon atoms of the substituent is not included.

The term " ester " means a functional group represented by RCOO- (R is alkyl or aryl). The C _{2 -10} ester means an ester group having an ester unit having 2 to 10 carbon atoms, and when the C _{2 -10} ester is substituted, the carbon number of the substituent is not included.

The term " carboxyl " means a functional group represented by -COOH.

The term " carboxyalkyl " means a carboxyl-bonded alkyl group. C _{3 -10} carboxyalkyl means a carboxyalkyl group having a carboxyalkyl unit having 3 to 10 carbon atoms, and when C _{3 -10} carboxyalkyl is substituted, the carbon number of the substituent is not included. C _{3 -10} carboxyalkyl means a carboxyalkyl group having a carboxyalkyl unit having 3 to 10 carbon atoms, and when C _{3 -10} carboxyalkyl is substituted, the carbon number of the substituent is not included.

The term " carboxyalkenyl " means a carboxyl-bonded alkenyl group. C _{3 -10} carboxyalkenyl means a carboxyalkenyl group having a carboxyalkenyl unit having 3 to 10 carbon atoms, and when the C _{3 -10} carboxyalkenyl is substituted, the number of carbon atoms of the substituent is not included.

The term " alkylcarboxy " means a carboxyl group to which alkyl is attached. C _{3 -10} carbon atoms, an alkyl carboxylic acid is in the case where alkyl refers to a carboxyl group with an alkyl carboxylic units having 3-10 carbon atoms, and substituted C _{3 -10} alkylcarboxylic Fifi substituent is not included.

The term " alkenylcarboxyl " means a carboxyl group to which an alkenyl is bonded. C _{3 -10} alkenylcarboxyl means an alkenylcarboxyl group having an alkenylcarboxyl unit having 3 to 10 carbon atoms, and when C _{3 -10} alkenylcarboxyl is substituted, the number of carbon atoms of the substituent is not included.

The term " alkylcarboxyalkyl " means an alkyl-C (O) -O-alkyl group. C _{3 -10} alkyl, carboxy alkyl means a carboxyalkyl group having an alkyl carboxyalkyl alkyl units having 3-10 carbon atoms, having a carbon number in the case where a C _{3 -10} alkyl, carboxy-substituted alkyl substituent is not included.

The term " alkylcarboxyalkenyl " refers to an alkyl-OC (O) -alkenyl group. C _{3 -10} alkyl, carboxy-alkenyl group is not included in the carbon number of a substituent when the alkyl means a group having a carboxy alkenyl alkyl carboxy alkenyl unit having 3 to 10 carbon atoms, the alkenyl C _{3 -10} alkyl substituted with carboxy.

The term " alkenylcarboxyalkyl " means an alkenyl-OC (O) -alkyl group. C _{3 -10} alkenylcarboxyalkyl means an alkenylcarboxyalkyl group having an alkenylcarboxyalkyl group having 3 to 10 carbon atoms, and when C _{3 -10} alkenylcarboxyalkyl is substituted, the number of carbon atoms of the substituent is not included.

The term " alkenylcarboxyalkenyl " means an alkenyl-OC (O) -alkenyl group. C _4-20 alkenylcarboxyalkenyl means an alkenylcarboxyalkenyl group having an alkenylcarboxyalkenyl unit having 4 to 10 carbon atoms, and when C _{4 -10} alkenylcarboxyalkenyl is substituted, the number of carbon atoms of the substituent is It is not.

According to a preferred embodiment, R ₁ is a hydrogen, hydroxy or C _{1 -10} alkyl, more preferably hydrogen, hydroxyl, halo, amino or C _{1 -10} alkyl, more preferably in formula (I) It is a hydrogen, hydroxy or C _{1 -5} alkoxycarbonyl, and most preferably hydrogen, hydroxy or C _{1 -5} alkoxycarbonyl.

According to a preferred embodiment, in the formula 1 R ₂ is hydrogen, halo or C _{1 -10} alkyl, more preferably hydrogen or C _{1 -10} alkyl, most preferably hydrogen or a C _{1 -5} alkyl .

According to a preferred embodiment of the present invention, R ₃ in the general formula (1) is hydrogen, halo or C _{1 -10} alkyl, more preferably hydrogen or C _{1 -10} alkyl, more preferably C _{1 -10} alkyl, preferably a C _{1 -5} alkyl.

According to a preferred embodiment of the present invention, R ₁ is hydrogen, hydroxyl, halo, amino or C _{1 -10} alkoxy, more preferably hydrogen or C _{1 -10} alkoxy, most preferably hydrogen or It is C _{1 -5} alkoxycarbonyl.

According to a preferred embodiment of the present invention, in the above formula 2, R ₂ is hydrogen, halo or C _{1 -10} alkyl, more preferably hydrogen or C _{1 -10} alkyl, still more preferably C _{1 -10} alkyl, most preferably a C _{1 -5} alkyl.

According to a preferred embodiment of the present invention, R ₃ is hydrogen, halo or C _{1 -10} alkyl, more preferably hydrogen or C _{1 -10} alkyl, most preferably hydrogen or C _{1 -5} alkyl to be.

According to a preferred embodiment, in Formula 2, R ₄ is carboxyl, C _3-10 carboxyalkyl, C _{3 -10} carboxy alkenyl, C _{3 -10} alkyl, carboxyl, C _{3 -10} alkenyl carboxylic acid, C _{3 -10} alkyl, carboxyalkyl, C _{3 -10} alkyl, carboxy-alkenyl, C _{3 -10} alkenyl carboxyalkyl, C _{4 -10} alkenyl carboxy alkenyl, C _{1 -10} alcohol, C _{1 -10} Al kenol , C _{2 -10} acyl, C _{1 -10} aldehyde, C _{1 -10} amide, C _{1 -10} amine, C _{2 -10} ester or sulfate. More preferably carboxyl, C _{3 -10} carboxyalkyl, C _{3 -10} carboxy alkenyl, C _{3 -10} alkyl, carboxyl, C _{3 -10} alkenyl carboxylic acid, C _3-10 alkyl, carboxyalkyl, C _{3 - 10} alkyl-carboxy alkenyl, C _3-10 alkenyl is carboxyalkyl, C _{4 -10} alkenyl carboxy alkenyl, C _{1 -10} alcohol or C _{1 -10} alkenyl play. Even more preferably C _{3 -10} carboxy alkenyl, C _{3 -10} alkenyl carboxyl, C _{3 -10} alkyl, carboxy-alkenyl, C _{3 -10} alkenyl carboxyalkyl, C _{4 -10} alkenyl carboxy alkenyl, C _{1 -10} alcohol. Even more preferably C _{3 -10} carboxyalkenyl or C _{1 -10} alcohol, and most preferably C _{3 -10} carboxyalkenyl.

According to a preferred embodiment, in Formula 3, R ₁ and R ₅ are independently hydrogen, halo or C _{1 -10} is alkyl, more preferably C _{1 -10} alkyl, most preferably C ₁ to each other _{- Lt; /} RTI >

According to a preferred embodiment of the present invention, R ₂ and R ₆ are independently of each other hydrogen, halo or C _{1 -10} alkyl, more preferably hydrogen or C _{1 -10} alkyl, most preferably hydrogen or a C _{1 -5} alkyl.

According to a preferred embodiment of the present invention, in Formula 3, R ₃ And R ₄ are independently of each other hydrogen, halo, C _{1 -10} alkyl or C _{2 -10} alkenyl, more preferably C _{1 -10} alkyl or C _{2 -10} alkenyl, even more preferably C ₁ and _-10 alkyl, most preferably C _{1 -5} alkyl.

As used herein, the term " alkenyl " as used in formula (3) for defining an active ingredient refers to a straight or branched chain unsubstituted or substituted unsaturated hydrocarbon group having a designated number of carbon atoms such as ethenyl, Butenyl, isobutenyl, t -butenyl, n -pentenyl and n -hexenyl. C _{2 -10} alkenyl means an alkenyl group having an alkenyl unit having 1 to 10 carbon atoms, and when C _{2 -10} alkenyl is substituted, the number of carbon atoms of the substituent is not included.

According to a preferred embodiment of the present invention, the compound represented by Formula 1 is represented by Formula 6, Formula 7, Formula 8 or Formula 9; The compound represented by Formula 2 is represented by Formula 10, Formula 11, Formula 13 or Formula 14; The compound represented by Formula 3 is a compound represented by Formula 16 or 18, and more preferably, a compound represented by Formula 1 is represented by Formula 6, 7, 8 or 9; The compound represented by Formula 2 is represented by Formula 10, Formula 12, Formula 13 or Formula 15; The compound represented by Formula 3 is represented by Formula 17 or Formula 19, and most preferably, the compound represented by Formula 1 is represented by Formula 7, Formula 8 or Formula 9; The compound represented by Formula 2 is represented by Formula 13 below. The compound represented by Formula 3 is a compound represented by Formula 16:

6

The compound represented by Formula 6 is Scoparone.

Formula 7

The compound represented by the general formula (7) is scopoletin.

8

The compound represented by Formula 8 is Fraxidin.

Formula 9

The compound represented by the above formula (9) is 6,7,8-trimethoxycoumarin.

10

The compound represented by the formula (10) is 4-hydroxy-5-methoxycinnamic acid.

Formula 11

Formula 12

The compound represented by the above formula (12) is (7S, 8S) -4-O-methoxysilane salicylcerol) [(7S, 8S) -4-O-methylsyringoylglycerol].

Formula 13

The compound represented by the above formula (13) is 3,4,5-trimethoxycinnamic acid.

Formula 14

Formula 15

The compound represented by the above-mentioned formula (15) can be obtained by reacting (1S, 2S) -1- (4-hydroxy-3-methoxyphenyl) -hydroxy-3-methoxyphenyl) -1,2,3-propanetriol].

Formula 16

Formula 17

Compound of the formula 17 is a new plaque is A ₂ (Fragransins A _2).

18

Formula 19

The compound represented by the above formula (19) is Galgravin.

According to a preferred embodiment of the present invention, the active ingredients used in the present invention include not only the compounds represented by the formulas (1) to (5) but also their pharmaceutical compositions.

When the composition of the present invention is manufactured from a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. The pharmaceutically acceptable carriers to be contained in the pharmaceutical composition of the present invention are those conventionally used in the present invention and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, But are not limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. It is not. The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention can be administered orally or parenterally, and in the case of parenteral administration, it can be administered by intravenous injection, subcutaneous injection, muscle injection, intraperitoneal injection, transdermal administration or the like.

The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate, . According to a preferred embodiment of the present invention, the daily dosage of the pharmaceutical composition of the present invention is 0.0001-100 mg / kg.

The pharmaceutical composition of the present invention may be formulated into a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. The formulations may be in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media, or in the form of excipients, powders, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

According to a preferred embodiment of the present invention, the active ingredient used in the present invention includes not only the compounds represented by the general formulas (1) to (5) but also pharmaceutically acceptable salts, hydrates, solvates or prodrugs thereof.

The term " pharmaceutically acceptable salts " refers to salts of the compounds of Formulas 1 to 5 having desired pharmacological effects, i. E., Diabetes prophylactic or therapeutic activity. Such salts include, but are not limited to, inorganic acids such as hydrochloride, hydrobromide and hydroiodide, organic acids such as acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, p- toluenesulfonate, bisulfate, sulfamate, Naphthylate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentane propionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, Is formed using an organic acid such as sodium carbonate, sodium carbonate, sodium carbonate, sodium carbonate, sodium carbonate, sodium carbonate, sodium carbonate, sodium carbonate, sodium carbonate, sodium carbonate, sodium carbonate, do.

The term " pharmaceutically acceptable hydrates " refers to the hydrates of the compounds of formulas (1) to (5) having the desired pharmacological effect. The term " pharmaceutically acceptable solvates " refers to solvates of the compounds of formulas (1) to (5) having the desired pharmacological effect. The hydrates and solvates may also be prepared using the acids described above.

The term " pharmaceutically acceptable prodrugs " refers to derivatives of the compounds of formula (1) which must undergo bioconversion prior to exerting the pharmacological effects of the compounds of formulas (1) to (5). Such prodrugs are prepared for prolonged duration of action and for the reduction of side effects, in order to improve the chemical stability, patient acceptability, bioavailability, organ selectivity or convenience of formulation. The preparation of the prodrug of the present invention can be carried out by using the compound of the above formula (1) according to a conventional method in the art, for example, Burger's Medicinal Chemistry and Drug Chemistry , 5 ^th ed . , 1: 172-178 and 949-982 (1995).

According to a preferred embodiment of the present invention, the active ingredients used in the present invention include not only the compounds represented by the formulas (1) to (5) but also food compositions for preventing, improving or alleviating diabetes.

When the composition of the present invention is prepared with a food composition, it includes not only chestnut extract as an active ingredient but also components that are ordinarily added in the manufacture of food, such as protein, carbohydrate, fat, nutrients, seasoning or flavoring . The above-mentioned carbohydrates can be used in combination with monosaccharides (such as glucose and fructose), disaccharides (such as maltose, sucrose and oligosaccharides) or polysaccharides (such as common sugars such as dextrins and cyclodextrins) , Sorbitol and erythritol)]. Natural flavorings such as tau martin and stevia extract (e.g., rebaudioside A and glycyrrhizin) or synthetic flavors (saccharin, aspartame, etc.) can be used as flavorings.

When the food composition of the present invention is prepared as a drink, it may further contain citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, juice, mulberry extract, jujube extract or licorice extract in addition to the chestnut extract of the present invention.

According to another aspect of the present invention, there is provided a composition for preventing, treating, ameliorating or alleviating diabetes comprising chestnut extract as an active ingredient.

According to a preferred embodiment of the present invention, the chestnut extract is a tubercle extract.

As used herein, the term "chestnut extract" is a chestnut (Castanea crenata ), and the term " tubercle extract " means chestnut twigs, chestnut bark or chestnut extract of chestnut fruit.

(B) an anhydrous or hydro-lower alcohol having 1-4 carbons such as methanol, ethanol, propanol, butanol, n-propanol, iso Propanol and n-butanol), (c) a mixed solvent of the lower alcohol and water, (d) acetone, (e) ethyl acetate, (f) chloroform, (g) 1,3-butylene glycol h) hexane, (i) diethyl ether, or (j) butyl acetate.

The extract of the present invention includes not only those obtained by using the above-mentioned solvents, but also those obtained by additionally applying a purification process thereto. For example, a fraction obtained by passing the above extract through an ultrafiltration membrane having a constant molecular weight cut-off value, and a separation by various chromatography (manufactured for separation according to size, charge, hydrophobicity or affinity) The fraction obtained through the purification method also includes the extract of the present invention.

The extract of the present invention can be prepared in powder form by an additional process such as vacuum distillation and freeze-drying or spray-drying.

According to a preferred embodiment of the present invention, the composition comprising the chestnut extract as an active ingredient comprises a pharmaceutical composition.

The extracts and insulin mimetic compounds of the present invention are chestnut extract and chestnut extract-derived pure compounds which are likely to be studied as medicines and foods that can directly treat insulin deficiency of diabetes.

The features and advantages of the present invention are summarized as follows:

(a) The compound used as an active ingredient in the composition of the present invention can be effectively used for preventing, treating, ameliorating or alleviating diabetes.

(b) Chestnut extract used as an active ingredient in the composition of the present invention can be effectively used for prevention, treatment, improvement or alleviation of diabetes.

Figure 1 shows the results of screening the native plant extract library using a 3T3-L1 adipocyte-based screening system based on 2-NBDG uptake. * = P <0.05 compared to untreated, error = standard deviation; 27-53 is the number of the native plant extract library sample. This sample is the chestnut ( Castanea) collected on June 16, 2005 crenata ) flowers. And the Y axis represents the intensity value of the light read by the fluorescent microplate reader.
FIG. 2 shows the results of screening of single compounds isolated from the uri extract. Screening methods used a 3T3-L1 adipocyte-based screening system based on 2-NBDG uptake, * = P <0.05 compared to untreated, error = standard deviation. Insulin was used as a positive control to promote glucose uptake of cells. Each sample was treated at a concentration of 10, and the Y axis represents the intensity value of the light read from the fluorescence microplate reader.
FIG. 3 shows the result of screening a single compound isolated from the tubercle bark extract. As a screening method, a zebrafish-based screening system based on 2-NBDG uptake was used, and four single compounds (UP-2.2, UP-3.11.1, UP-3.5.3 and UP-5.2.1) The result of accelerating glucose uptake in zebrafish was obtained. Insulin, rosiglitazone, berberine, and PC5 were used as positive controls to stimulate glucose uptake, and the Y-axis represents the intensity value of the light read from the fluorescence microplate reader.
Fig. 4 shows a representative image of the experimental result shown in Fig. The photographs were taken at about 76 hpf (hours post fertilization) and were taken using DIC and FITC fluorescence filters at 50X magnification.
FIG. 5 shows the result of screening a single compound isolated from the tubercle bark extract. A zebrafish-based screening system based on 2-NBDG uptake was used as a screening method and the absorption of glucose in zebrafish treated with 3 single compounds (UP-3.2, UP-3.3 and UP-5.2.3) . Insulin was used as a positive control to accelerate the absorption of glucose, and the Y axis represents the intensity value of light read from a fluorescence microplate reader.
6 shows a representative image of the experimental result shown in Fig. The photographs were taken at about 76 hpf (hours post fertilization) and were taken using DIC and FITC fluorescence filters at 50X magnification.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Experimental material

2- (N- (7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino) -2-deoxyglucose (2-NBDG) was purchased from Invitrogen (OR, USA). Insulin from bovine pancreas was purchased from Sigma-Aldrich (SL, USA). Rosiglitazone was purchased from Cayman Chemicals (MI, USA). Berberine and PC5 (emodin), which were used as positive controls to increase intracellular glucose uptake, were purchased from Professor Ohwon Oh (College of Pharmacy, Chosun University).

Cell culture

3T3-L1 mouse embryonic fibroblast (ATCC) was cultured in DMEM (Dulbecco &apos; s Modified) supplemented with 10% fetal calf serum (Calf Serum; Invitrogen, OR, USA), 50 U / mL penicillin and 50 ug / mL streptomycin Eagle's Medium; Invitrogen, OR, USA). The following steps were performed to differentiate 3T3-L1 fibroblasts into adipocytes: 48h post-confluent cells (designated on day 0) were incubated with 10% FBS, 0.5 mM 3-isobutyl- -Methyl-xanthine, 2 / mL dexamethasone (Sigma-Aldrich, SL, USA), 1 / mL insulin, 1 rosiglitazone, 50 / mL penicillin and 50 / mL streptomycin. Then, the cells were cultured for 2 days in DMEM supplemented with 10% FBS, 1 / mL insulin, and 1 logjistitazone. 5 days after induction of differentiation, 3T3-L1 adipocytes were used in the experiment. The incubator used for cell culture was maintained at a temperature of 37 ° C and a CO ₂ concentration of 5%.

Cell-based screening system based on 2-NBDG uptake

To test whether 2-NBDG can be used to detect insulin-stimulated glucose uptake in a 96-well plate cell culture system that is well suited for cell-based screening already developed to identify novel insulin mimetic compounds, 3T3- L1 mouse embryonic fibroblasts were used. 3T3-L1 is a lipid precursor cell that is representative of liver and fat, the major forms sensitive to insulin activity.

Cells were seeded at a concentration of 5000 cells / well in a 96-well tissue culture plate (BD Falcon, NJ, USA), and the differentiation into adipocytes was induced by the above method. Cells differentiated into adipocytes were changed to DMEM medium, and 10 wells of UBP extract were treated for 30 minutes in each well. Berberine, which increases intracellular glucose uptake, was used as a positive control. Each well was treated with DMEM supplemented with 50 2-NBDG for 10 minutes, and then each well was washed three times with PBS. Subsequently, fluorescence microplate reader (SpectraMAX GeminiXS and SoftMax Pro V5 software, Molecular Devices, CA, USA) was used for quantification of intracellular 2-NBDG uptake. Cells were lysed in each well using 70 cell lysis buffer (0.1 M Potassium Phosphate, 1% Triton X-100, pH 10 in PBS), followed by addition of 30 dimethylsulfoxide (DMSO, Generay Biotech, Canada) After 2 to 3 times of peptetting, the intensity of light was measured using 466 nm excitation light and 540 nm emission light, which can detect 2-NBDG.

Compound separation

The UV spectrum in methanol was measured using a spectrometer (Shimadzu) and the optical rotation in methanol was measured using a Rudolph Autopol IV Polarimeter (Rudolph Research Analytical, Inc., Autopol IV). NMR spectra were measured by JEOL FT NMR (300 MHz) and Varian Inova (500 MHz) using TMS (Tetramethylsilane), an internal standard of Korea Basic Science Institute (KBSI, Gwangju Center, Korea). All mass experiments were performed with a Micromass QTOF2 (Micromass, Wythenshawe, UK) mass spectrometer. The column chromatography was performed using HP-20, silica gel (Merck, 63-200 mm particle size) and RP-C18 (Merck, 75 mm particle size), and the thin plate was silica gel 60 F254 and RP-18 F254 Plate (Merck) was used. HPLC analysis was performed using a UV detector and an Optima Pak C18 column (10x250 mm, 10 mm particle size, RS Tech, Korea). HPLC solvents were purchased from Burdick & Jackson (USA).

Zebrafish Danio rerio ), Preservation and scattering

The zebrafish family was purchased from Lotte Mart (Gimpo Branch, Gwangju). 15-20 zebrafishes were preserved in a 10 L glass water bath. The setting of the water tank was maintained at a light / dark cycle of 28 hours at a temperature of 14/10 hours. Meal times were 1 in the morning, 1 in the afternoon, 2 in total, and 6 meals a week were allocated. Feeding flakes (Amazon Flake, Foosung, Taiwan) and Brine Shirin (Artemia salina, Inve Aquaculture Nutrition, USA) were given periodically in parallel. Health status was checked every day except the weekend, and it was examined based on observations such as fish mobility, disease symptoms and death.

Prior to spawning, male and female zebrafish were separated at a ratio of 2: 1 and placed in a spawn bath and kept in the incubator at 9 pm. There is a scattering tray with a mesh that can separate the zebrafish and zebrafish from the egg reservoir. After egg laying, the egg sinks on the bottom of the tank. Light was illuminated at 9 am and spawning was initiated by removing the barrier that separated the magnetic and male zebrafish. Spawning was continued for about 1 hour.

Zebrafish-based toxicity testing

The scattered zebrafish eggs were placed on a 96-well tissue culture plate at a distribution of 1 well / well, and the number of eggs was set at three times per sample. To each well was added 200 E3 water (E3 water) and each sample was treated by concentration on a 5/4 hpf (Hours Post Fertilization) of 8 embryo cell lines. The concentrations used in this experiment were 20, 10, and 5, and were replaced with E3 water treated at each concentration every 24 hours. The total treatment time was 72 hours in the incubator, and each health condition was examined at 24 hpf, 48 hpf and 72 hpf. The test items included the development of truncation, development of the tail, development of the isoform and the eardrum, development of the eye, heartbeat, flow of the circulatory system, delay of the incubation time, deformity of the skeleton, body position and ability to swim A total of 10 items were identified through observation to identify the health status of the fish. In addition, 50 embryos were placed on 96-well tissue culture plates containing 200 E3 water and cultured in the same environment as the experimental group. The initial mortality rate (IMR), which can affect the outcome, was measured and the reference point of the incubation time required for the delayed incubation time item was set through the incubation time of 50 embryos. After each item was examined at 72 hpf, each larva was anesthetized with a solution of 0.02% tricane (3-aminobenzoic acid ethylester, Sigma, USA) for about 10 minutes. The larvae were placed in E3 water with 3% methylcellulose dissolved and then analyzed using a fluorescence microscope (DM 2500, Leica, Wetzlar, Germany) equipped with a digital camera (DFC425 C, Leica, Wetzlar, Germany) and the Lieca Application Suite Microsystems, Wetzlar, Germany) software. The magnification was 50X, and the image was corrected in Photoshop CS4 (Adobe Systems Incorporated, CA, USA).

2- NBDG  Ingestion-based Zebra fish - based screening system

The scattered zebrafish eggs were stored in an incubator in E3 water (5 mM NaCl, 0.17 mM KCl, 033 mM CaCl ₂ and 0.33 mM MgSO _{4 in} distilled water) containing 0.2 mM PTU (1-phenyl-2-thiourea) . The larvae removed after 48 hours were kept in the dishes for up to 72 hours. The medium was replaced with fresh medium once a day. At 72 hours after the spawning, the larvae were placed in a 96-well tissue culture plate (BD Falcon, NJ, USA) at a distribution of 4 wells / well. The control group (control group) did not receive any treatment, and 2 / mL insulin, 1 rosiglitazone, 10 berberine, 10 / mL PC5, 10 UP-2.2, 10 UP- 3.11.1, 10 UP- 3.5.3, 10 UP-4.2.3 and 10 UP-5.2.1 were each treated with larvae for 30 minutes. Each well was replaced with E3 water containing 600 2-NBDG. After holding for 90 minutes, 2-NBDG in vitro was removed by immersing in E3 water. After anesthesia with 0.02% tricane (3-aminobenzoic acid ethyl ester, Sigma, USA) solution for about 10 minutes, one animal was placed in E3 water with 3% methyl cellulose dissolved therein. Using a fluorescence microscope (DM 2500, Leica, Wetzlar, Germany) equipped with a digital camera (DFC425 C, Leica, Wetzlar, Germany) and Lieca Application Suite software (Leica Microsystems, Wetzlar, Germany) Respectively. The magnification was 50X, and DIC and FITC filters were used. Images were corrected in Photoshop CS4 (Adobe Systems Incorporated, CA, USA). The remaining three anesthetized pieces were placed in a laboratory tweezers and placed in a tube containing 120 μL of CelLytic M cell disruption reagent (Sigma-Aldrich, SL, USA) and disrupted using a sonicator. The cell disruption proceeded at 4 &lt; 0 &gt; C and was carried out for 10 minutes at a 10 &quot; / 5 &quot; cycle. The cell lysate was centrifuged at 4O &lt; 0 &gt; C at 10,000 rpm for 10 minutes, and then the supernatant 100 was placed in a 96-well plate to measure the intensity of fluorescence. A fluorescence microplate reader (SpectraMAX-Gemini XS (Molecular Devices, Calif., USA) and SoftMax Pro V5 software was used and excitation was performed at 466 nm and emission wavelengths in the 540 nm region, The intensity of light was quantified.

Experiment result

999 native plant extract libraries were screened using a 3T3-L1 adipocyte-based screening system based on 2-NBDG uptake. This experimental technique makes it possible to detect plant extract-stimulated glucose uptake in cells cultured in 96-well plates, and the difference in intensity values of light read from a microplate reader, one of the major human tissues sensitive to insulin activity, 2-NBDG in the 3T3-L1 adipocyte cell line, which can represent the endometrial cancer cells. The chest extracts from the native plant library were selected as heat extracts through this technique (Fig. 1).

 During the analysis of the chestnut extract, common substances were found in extracts from various parts of chestnut trees. Therefore, it was used in the experiment for long time storage and easy collection and purchase than chestnut flower. The purchased ufi was fractionated by polar solvent fractionation method using an organic solvent and separated into methanol, hexane, ethylacetate and water fractions.

The four fractions were tested in a 3T3-L1 adipocyte-based screening system based on 2-NBDG uptake, and high glucose uptake was observed in adipocytes of the wells treated with the ethyl acetate fraction. 14 compounds were then separated from the ethyl acetate fraction after separation and structure determination using high performance liquid chromatography (HPLC) and nuclear magnetic resonance (NMR) (Tables 1 and 2 ).

number Name of compound UP-1.1 Scoparone UP-2.2 Scopoletin UP-3.2 Fraxidin UP-3.3 6,7,8-Trimethoxycoumarin (6,7,8-trimethoxycoumarin) UP-5.2.1 4-hydroxy-5-methoxycinnamic acid (4-hydroxy-5-methoxycinnamic acid) UP-5.2.2 (7S, 8S) -4-O-methoxysiline &lt; / RTI &gt;
[(7S, 8S) -4-O-methylsyringoylglycerol] UP-5.2.3 3,4,5-trimethoxycinnamic acid (3,4,5-trimethoxycinnamic acid) UP-4.2.1 (1S, 2S) -1- (4-hydroxy-3-methoxyphenyl) -1,2,3-propanetriol
[(1S, 2S) -1- (4-hydroxy-3-methoxyphenyl) -1,2,3-propanetriol] UP-3.5.1 Saucernetindiol &lt; / RTI &gt; UP-3.5.2 Nectandrin B UP-3.5.3 Plaque is the new A ₂ (Fragransins A ₂₎ UP-3.6.1 Galgravin UP-3.11.1 Maslinic acid UP-4.2.2 Arctigenic acid

High Performance Liquid Chromatography ( HPLC ) result

(2.4 kg) was extracted with methanol (5 L x 3 times) using ultrasound (5 hr each step) at 50 캜. The methanol-soluble extract was filtered and completely dried to give concentrated methanol extract (490 g). The methanol extract was suspended in H ₂ O (1 L) and then loaded on an HP-20 column (10 x 60 cm) and eluted with MeOH-H ₂ O (0: 100, 25:75, 50:50, 75:25, 0, 2 L each), and finally washed with acetone (2 L) to separate into six fractions. The above 100% methanol fraction (UP-MeOH fr, eluted with 100% MeOH, 2 L) was passed through a silica gel column (6 x 60 cm; 63200 μm particle size) with a gradient of n-hexane / acetone Size), and six fractions (UP.1UP.6) were obtained according to the TLC profile.

Chromatography of fraction 1 (UP. 1) on a reverse phase ODS-A column (5.0 x 60 cm; 12 nm, S150 μm particle size) was repeated with MeOH-H2O (1: 2, 1: 1.5 and 1: L) to separate scopolone (UP-1.1, 85 mg).

In a similar manner, MeOH-H ₂ O (40:60, 45:55, 50:50, 70:30, and 2.5 L each, using a reverse phase ODS-A column (5.0 x 60 cm; 12 nm, S150 μm particle size) ), And scopollen (UP-2.2, 75 mg) was isolated from fraction 2 (UP.2).

Fraction 3 (UP.3) was also eluted with MeOH-H ₂ O (50:50, 55:45, 60:40 and 100: 1) using a reverse phase ODS-A column (6.0 x 60 cm; 12 nm, S150 μm particle size) (UP-3.2, 50 mg), 6,7,8-trimethoxycoumarin (UP-3.3, 10 mg) and four sub-fractions (UP.3.4, UP .3.5, UP.3.6, and UP.3.7).

Semi-preparative Gilson HPLC systems (RSTech OptimaPak_C18 column (10 x 250 mm, 10 μm particle size); Mobile phase MeOH / H ₂ O (48:52) isocratic; Flow rate 2 mL / min; Sub-fraction 4 (UP.3.4) was purified by UV-detection at 205 and 254 nm to give (lS, 2S) -l- (4-hydroxy-3- methoxyphenyl) Propanetriol (UP-4.2.1, 5.7 mg, tR 24.5 min) and actinic acid (UP-4.2.2, 8.7 mg, tR 28.5 min) were separated.

Purification of sub-fraction 5 with an apreparative Gilson HPLC system equipped with RSTech OptimaPak_C18 column (10 x 250 mm, 10 μm particle size) and 205 and 254 nm UV / Vis-155 detectors was performed with H ₂ O + 0.1% formic acid (UP-3.5.1, 12.6 mg, tR 27.1 min), nectandrin B (UP-3.5.2, 6.5 mg, tR 30.0 min) was eluted with a flow rate of 2 mL / And pragramin A ₂ (UP-3.5.3, 6.0 mg, tR 33.5 min), respectively.

H ₂ O &lt; + &gt; at a flow rate of 2 mL / min for 70 min under the same conditions as above (Gilson HPLC equipped with RSTech OptimaPak C18 column (10 x 250 mm, 10 μm particle size) and 205 and 254 nm UV / Vis- (UP-3.6.1, 3.6 mg, tR 37.1 min) and masulinic acid (UP-3.11.1, 36.0 mg, tR 53.5 min) were eluted from fraction 6 by elution with 0.1% formic acid.

Fraction 5 (UP.5), which was further fractionated by RP-C18 column chromatography (4.0 x 60 cm, 150 μm particle size) was also eluted with MeOH / H ₂ O (1: 2, 1: 1.5, 1: To obtain five sub-fractions (UP.5.1UP.5.5). A semi-aqueous solution of isomers of 42% MeCN in H ₂ O + 0.1% formic acid for 60 minutes with UV-detection at 205 and 254 nm, flow rate of 2 mL / min, OptimaPak_C18 column (10 x 250 mm, Sub-fraction 2 (UP.5.2) was purified by preparative Gilson HPLC system to give 4-hydroxy-5-methoxycinnamic acid (UP-5.2.1, 10.9 mg, tR 22.0 min), (7S, (UP-5.2.2, 17.7 mg, tR 39.0 min) and 3,4,5-trimethoxycinnamic acid (UP-5.2.3, 12.0 mg, tR 45.1 min) .

Nuclear magnetic resonance NMR ) result

number NMR  result UP-1.1 Yellow powder;
¹ HNMR (300 MHz, acetone-d6)? (Ppm):
(1H, d, J = 9.3, H-4), 7.18 (1H, s, H-5), 6.98 ), 3.92 (3H, s, 7-OCH 3), 3.84 (3H, s, 6-OCH 3);
¹³ CNMR (75 MHz, acetone-d6)? (Ppm):
113.2 (C-2), 154.0 (C-9), 150.9 (C-7), 147.4 , 109.8 (C-5), 100.7 (C-8), 56.5 (7-OCH 3), 56.4 (6-OCH 3) UP-2.2 Yellow crystals;
¹ HNMR (300 MHz, acetone-d6)? (Ppm):
(1H, s, H-8), 6.11 (1H, s) d, J = 9.3, H- 3), 3.85 (3H, s, 6-OCH 3);
¹³ CNMR (75 MHz, acetone-d6)? (Ppm):
161.3 (C-2), 152.0 (C-9), 150.5 (C-7), 146.1 (C-6), 144.6 (C-5), 109.8 ( C-8), 56.6 (6-OCH 3) UP-3.2 Yellow powder;
¹ HNMR (300 MHz, acetone-d6)? (Ppm):
(1H, d, J = 9.6, H-3), 3.93 (3H, s, 7-OCH _{3), 3.88 (3H, s} , 6-OCH 3);
¹³ CNMR (75 MHz, acetone-d6)? (Ppm):
144.9 (C-7), 144.1 (C-4), 135.7 (C-9), 113.3 (C-3), 111.6 (C-10), 105.0 ( C-5), 56.7 (6-OCH 3), 61.3 (7-OCH 3) UP-3.3 Yellow amorphous powder;
¹ H NMR (300 MHz, acetone-d6)? (Ppm): 7.87 (1H, d, J = 9.6, H-4), 7.16 , H-3), 3.93 ( 3H, s, 7-OCH 3), 3.85 (3H, s, 6-OCH 3), 3.81 (3H, s, 8-OCH 3);
¹³ C-NMR (75 MHz, acetone-d6)? (Ppm): 160.5 (C-2), 151.2 (C-6), 145.9 -8), 114.8 (C-3 ), 113.2 (C-10), 105.8 (C-5), 56.3 (7-OCH 3), 61.4 (6-OCH 3), 61.8 (8-OCH 3) UP-5.2.1 Amorphous powder;
¹ HNMR (300 MHz, acetone-d6)? (Ppm):
(1H, d, J = 8.1, 1.8, H-5), 6.86 (1H, d, J = , d, J = 8.1, H -6), 6.36 (1H, d, J = 15.8, H-7), 3.90 (3H, s, 2-OCH 3);
¹³ CNMR (75 MHz, acetone-d6)? (Ppm):
(C-2), 148.0 (C-7), 144.9 (C-1), 128.8 (C-4), 119.7 (C-5), 116.8 -8), 111.8 (C-3 ), 56.3 (2-OCH 3) UP-5.2.2 Amorphous powder;
¹ HNMR (300 MHz, acetone-d6)? (Ppm):
(1H, d, J = 6.0, 7.8, H-9a), 3.80 (1H, d, J = (1H, dd, J = 4.2,8.1 , H-9b), 3.81 (9H, br, s, 1/2/6-OCH 3), 3.06 (1H, m, H-6);
¹³ CNMR (75 MHz, acetone-d6)? (Ppm):
105.9 (C-3/5), 87.7 (C-7), 73.5 (C-9), 56.5 (1- OCH ₃ ), 56.2 (2/6-OCH ₃ ), 56.2 (1/2/6-OCH ₃ ), 55.3 UP-5.2.3 Amorphous powder;
¹ HNMR (300 MHz, acetone-d6)? (Ppm):
D, J = 15.9, H-7), 6.76 (2H, br, s, H-3 / H-5), 6.34 , br, s, 1/2 /6-OCH 3);
¹³ CNMR (75 MHz, acetone-d6)? (Ppm):
(C-4), 115.6 (C-8), 106.5 (C-3/5), 171.2 (COOH), 150.4 _{, 56.3 (1-OCH 3)} , 56.2 (2/6-OCH 3) UP-4.2.1 Amorphous powder;
¹ HNMR (300 MHz, acetone-d6)? (Ppm):
D, J = 8.1, 1.8, H-5), 6.78 (1H, d, J = 8.1, H-6), 4.66 (d, J = 4.5, H-7), 4.20 (1H, dd, J = 6.5, 7.8, H-9a), 3.80 (1H, dd, J = 4.0, 9.0, _{s, 2-OCH 3),} 3.80 (1H, dd, J = 4.5,6.0, H-8);
¹³ CNMR (75 MHz, acetone-d6)? (Ppm):
146.4 (C-4), 119.7 (C-5), 115.6 (C-6), 110.8 (C-3), 86.7 (C-9), 56.3 ( 2-OCH 3), 55.3 (C-8) UP-3.5.1 Colorless oil;
[a] +10.0 (c1.38, CHCl 3);
HREIMS m / z 344.1636 (calcd for C 20 H 24 O 5, 344.1626);
¹ H- and ¹³ C-NMR, see Table 4 UP-3.5.2 Colorless oil;
[a] O (c0.3, MeOH);
HREIMS m / z 344.1610 (calcd for C 20 H 24 O 5, 344.1622);
¹ H- and ¹³ C-NMR, see Table 4 UP-3.5.3 Colorless oil;
[a] +69.0 (c1.24, CHCl 3);
HREIMS m / z 344.1633 (calcd for C 20 H 24 O 5, 344.1622);
¹ H- and ¹³ C-NMR, see Table 5 UP-3.6.1 Colorless oil;
[a] O (c0.3, MeOH);
HREIMS m / z 372.1910 (calcd for C 22 H 28 O 5, 372.1928);
¹ H- and ¹³ C-NMR, see Table 5 UP-3.11.1 White amorphous powder;
LRIMS m / z 432 [M &lt; + &gt;];
¹ HNMR (500 MHz, acetone-d6)? (Ppm):
D, J = 3.0, 4.5, 10.5, H-2), 2.91 (1H, d, J = 10.0 (1H, m, H-3), 0.87 (1H, m, H-5), 1.54 (1H, m, H-9), 1.91-1.93 (2H, m, H-11), 5.25 (1H, m, H-16a), 2.00-2.03 (1H, m, H-16b), 2.88 (1H, m, H-22b), 1.21 (1H, m, H-21b), 1.21 (3H, s, H3-23), 0.81 (3H, s, H3-24), 0.99 (3H, s, H3-25), 0.80 -27), 0.94 (3H, s, H3-29), 0.91 (3H, s, H3-30);
^{13 C} NMR (125 MHz, acetone-d6)? (Ppm):
(C-12), 84.0 (C-3), 68.9 (C-2), 56.2 (C-5), 48.6 (C-9), 47.7 (C-1), 46.9 (C-19), 46.8 (C-17), 42.6 (C-14), 42.3 C-10), 34.5 (C-21), 33.6 (C-30), 33.4 (C-22), 31.4 (C-26), 17.7 (C-24), 17.1 (C-16), 23.9 25) UP-4.2.2 White amorphous powder;
¹ HNMR (500 MHz, acetone-d6)? (Ppm):
(1H, d, J = 8.0, 2.0, H-6), 6.69 (1H, d, J = d, J = 2.0, H-2 '), 6.72 (1H, d, J = 7.5, H-5'), 6.58 dd, J = 7.5, 9.0, H-9'a), 3.89 (1H, dd, J = 8.0, 9.0, H-9'b), 2.93 ), 2.83 (1H, d, J = 7.0,14.0, H-7b), 2.66 (1H, m, H- -8 '), 2.53 (1H, m, H-7'b), 3.81 (3H, s, 3'-OCH 3), 3.79 (3H, s, 3-OCH 3);
^{13 C} NMR (125 MHz, acetone-d6)? (Ppm):
(C-3), 148.3 (C-3), 146.2 (C-4), 146.1 (C-4 '), 131.1 113.0 (C-2), 71.7 (C-6), 115.1 (C-5) '), 47.1 (C-8 ), 42.3 (C-8'), 38.5 (C-7 '), 35.2 (C-7), 56.3 (3'-OCH 3), 56.2 (3-OCH 3)

position UP -3.5.1 UP -3.5.2 d _C d _H  ( J inHz ) d _C d _H  ( J inHz ) One 2 84.8 4.65, d, 9.6 87.2 4.53, d, 6.6 3 43.4 2.33, m 44.1 2.35, m 4 47.6 2.33, m 44.1 2.35, m 5 85.8 5.46, d, 4.2 87.2 4.53, d, 6.6 One 132.6 133.9 2 108.3 6.92, d, 2.5 114.1 6.99, d, 2.0 3 146.6 146.4 4 146.3 144.9 5 113.9 6.77, d, 8.4 109.2 6.92, d, 8.4 6 119.3 6.80, dd, 2.4, 8.4 119.1 6.98, dd, 1.8, 7.8 One 135.0 133.9 2 108.7 6.92, d, 2.4 114.1 6.99, d, 1.8 3 145.0 146.4 4 144.3 144.9 5 114.0 6.77, d, 8.4 109.2 6.91, d, 7.8 6 118.8 6.80, dd, 2.4, 8.4 119.1 6.93, dd, 1.8, 7.8 3- Me 9.4 1.01, d, 6.6 12.7 1.05, d, 6.6 4- Me 11.8 0.63, d, 6.6 12.7 1.05, d, 6.6 3- OMe 56.0 3.90, s 55.6 3.85, s 4- OMe 4 / 4- OH 5.65, br, s 5.96, br, s 3- OMe 55.8 3.92, s 55.6 3.85, s 4- OMe

position UP -3.5.3 UP -3.6.1 d _C d _H  ( J inHz ) d _C d _H  ( J inHz ) One 2 88.9 4.44, d, 6.6 84.4 5.44, d, 6.0 3 45.6 2.30, m 44.6 2.28, m 4 45.6 2.30, m 44.6 2.28, m 5 88.9 4.44, d, 6.6 84.4 5.44, d, 6.0 One 134.6 135.6 2 115.9 7.00, d, 1.8 111.5 6.93, d, 2.0 3 148.9 150.1 4 147.2 149.4 5 111.2 6.80, d, 8.1 112.5 6.92, d, 8.5 6 120.3 6.87, dd, 1.8, 8.1 119.4 6.85, dd, 2.0, 8.0 One 134.6 135.6 2 115.9 7.00, d, 1.8 111.5 6.93, d, 2.0 3 148.9 150.1 4 147.2 149.4 5 111.2 6.80, d, 8.1 112.5 6.92, d, 8.5 6 120.3 6.87, dd, 1.8, 8.1 119.4 6.85, dd, 2.0, 8.0 3- Me 13.1 0.99, d, 6.6 15.0 0.66, d, 6.5 4- Me 13.1 0.99, d, 6.6 15.0 0.66, d, 6.5 3- OMe 56.3 3.82, s 56.1 3.81, s 4- OMe 56.2 3.79, s 4 / 4- OH 5.60, br, s 3- OMe 56.3 3.83, s 56.1 3.81, s 4- OMe 56.2 3.79, s

2- NBDG  A cell-based screening system based on ingestion

Up-2.2, UP-3.11.1, UP-3.2, UP-3.3, UP-3.5.3, and UP-3.3 were tested using the 3T3-L1 adipocyte-based screening system based on 2-NBDG uptake. A total of seven pure compounds, including UP-5.2.1 and UP-5.2.3, were shown to exhibit high glucose uptake in adipocytes of wells treated at a concentration of 10 (Fig. 2). 20 insulin was used as a positive control and glucose intensities of the same or higher intensity were measured in the group treated with the above seven pure compounds. From this, it was found that the insulin - sensitivity of the seven pure compounds affected the intracellular glucose uptake, and the effect was similar to or higher than that of a certain concentration of insulin.

Zebra fish -Based toxicity test results

Zebrafish was used as a model animal to test the toxicity of the seven active ingredients, particularly teratogenicity / developmental toxicity and the glucose absorption enhancement in the animal body. Zebrafish has 75% genetic similarity with humans and has been known as an animal suitable for testing toxicity or efficacy as a vertebrate animal whose myocardial system, nervous system, and digestive organs are very similar in function, development, and structure to mammals. (Chakraborty, C., et al. 2009). In a recent report, a method for testing toxicity of compounds using zebrafish has been published (Selderslaghs, IW et al. 2011), and the present inventors have tested the toxicity test of the above seven efficacious individuals according to the standard toxicity test method presented in this paper (Table 6).

                                                      <+: Toxic -: None> - UP -2.2 UP -3.11.1 UP -3.5.3 UP -5.2.1 UP -3.2 UP -3.3 UP -5.2.3 20 μM +++ + + - ++ - - 10 μM - - - - ++ - - 5 [mu] M - - - - ++ - -

The degree of teratogenicity to the zebrafish embryos was checked by treating the seven tubal extract compounds selected with a zebrafish-based screening system based on 2-NBDG uptake for 3 days. If there is toxicity, it is indicated as "+". If there is no toxicity, it is indicated as "". The number of "+" increases according to the intensity of toxicity. Concentrations of 20, 10, and 5 were used. Of these, 6 pure compounds were found not to be teratogenic for zebrafish at concentrations below 10. As shown in Table 2 above, two out of three zebrafish groups in the group treated with 20 UP-2.2 stopped their heartbeat at 24 and 48 hours after compound treatment and stopped the development. The other one found edema in the yolk sac, lacked fin development, and had no normal appearance at the developmental stage, such as the vertebrae. In the UP-3.11.1-treated zebrafish group of 20, all three out of three showed abnormal development of the minor caudal fin. In the UP-3.5.3 treated zebrafish group of 20, one out of three showed edema in the vicinity of the heart. Zebrafish in the group treated with other concentrations and other pure compounds showed normal health and development status. As a result of analysis of 50 untreated groups, the infant mortality rate (IMR) was 0%, and all 50 animals showed normal health status and development status. In summary, UP-2.2, UP-3.11.1 and UP-3.5.3 showed stability to zebrafish in the range of 0-10, while UP-5.2.1 showed the stability in the range of 0-20 It was confirmed that zebrafish was not teratogenic. After 20 μM UP-3.2 treatment, delayed incubation occurred in one of three embryos. In the former test concentration group, it was judged that there was no abnormality in all the items including the heart and blood circulation, but it showed that it was not able to swim. As a result, it can be determined that the UP-3.2 is toxic to ++. UP-3.3 and UP-5.2.3 did not show teratogenicity and showed normal developmental status. Overall, six of the seven active compounds listed above, except UP-3.2, did not exhibit teratogenicity at 10 concentrations. Since teratogenic induction in zebrafish does not necessarily imply the possibility of teratogenic induction in humans, the teratogenic induction of UP-3.2 may need to be retested at the mammalian level.

2- NBDG  Ingestion-based Zebra fish - based screening system results

Zebrafish is a well-suited model for screening compounds for the treatment of diabetes or for in vivo validation. As mentioned above, the following advantages can be gained, in addition to being 75% genetic information similar to humans, the myocardium, the nervous system, and digestive organs being very similar to mammals in function and structure. First, many (200-300) embryos are produced, and spawning is not affected by the season. Second, the development of the embryo is rapid, and at 96 hours after spawning, the internal organs such as heart, liver, kidney, and organs are all developed. Third, it has a transparent body that allows real-time observation and visualization. Recent studies have examined the mRNA transcription levels of AMP-activated protein kinase (AMPKα), cAMP response element binding protein 3-like 3 (CREB3l3) and mammalian target of rapamycin (mTOR) in zebrafish liver Studies have shown that zebrafish is an animal model that is well suited to study human metabolic diseases including diabetes ( Craig, PM and TW Moon 2011 ). ( Jensen, PJ et al., 2006; Jensen, PJ, 2011 ) . In these papers, a fluorescent glucose analogue is used as a probe Respectively. Therefore, the present inventors measured the absorption of 2-NBDG, a fluorescent glucose-like analogue, into zebrafish embryos after compound treatment to evaluate the in vivo activity (enhancement of glucose uptake) of the above seven active ingredients (Figs. 3 to 6). Insulin that promotes glucose absorption in the body, rosiglitazone as an insulin-sensitive compound, and berberine and PC5, which exhibit a high glucose uptake effect among plant extracts, were used as positive control. As with the cell-based experiments, the positive control results in the zebrafish-based 2-NBDG absorption screening system showed a significant difference in glucose uptake as compared to the negative control, which could be seen in both graphs and images. UP-2.2, UP-3.2, UP-3.3, UP-5.2.1, UP-5.2.3, UP-3.5.3 and UP-3.11.1 showing high glucose uptake Seven compounds were selected, and the glucose uptake in the body of each group was imaged using a fluorescence photographic technique. As shown in FIG. 4 and FIG. 6, acceleration of glucose uptake of each treated compound was able to be compared according to the intensity of fluorescence.

Additional discussion

The data presented herein demonstrate that a novel insulin mimetic compound candidate can be rapidly identified using a cell-based screening system using 2-NBDG, which is an existing study. In addition, a new zebrafish-based screening system using 2-NBDG was proposed, and a known glucose uptake inducing substance was identified using the system and a screening test of the extract was carried out. Similar to the cell-based screening system, glucose uptake was quantified using the intensity of light generated in the wavelength range of light capable of detecting 2-NBDG, fluorescence pictures were presented and the difference in fluorescence signal was verified And succeeded in visualizing the extent of glucose uptake in the treated group. This screening technique adopts a method of absorption of the plant extract through the oral cavity, skin and gill, rather than injection, so that the practical use of the screened candidate substance can be applied to many fields such as medicines, food and beverage, . In addition, the use of native plant extracts minimized adverse effects and provided applicable concentrations based on the results of zebrafish-based toxicity tests to confirm safety and bioactivity effects on toxicity.

references

1. Selderslaghs, I. W., R. Blust, and H.E. Witters, Feasibility study of the zebrafish assay as an alternative method to screen for developmental toxicity and embryotoxicity using a training set of 27 compounds. Reprod Toxicol, 2011. 33 (2): p. 142-54.

2. Chakraborty, C., et al., Zebrafish: a complete animal model for in vivo drug discovery and development. Curr Drug Metab, 2009. 10 (2): p. 116-24.

3. Craig, P.M. and T.W. Moon, Fasted zebrafish mimic genetic and physiological responses in mammals: a model for obesity and diabetes? Zebrafish, 2011. 8 (3): p. 109-17.

4. Jensen, P.J., J.D. Gitlin, and M.O. Carayannopoulos, GLUT1 deficiencies, and nutrient availability during apoptosis during embryonic development. J Biol Chem, 2006. 281 (19): p. 13382-7.

5. Jensen, PJ, LB Gunter, and MO Carayannopoulos, Akt2 modulates glucose availability and downstream apoptotic pathways during development. J Biol Chem, 2011. 285 (23): p. 17673-80.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (18)

A composition for preventing, treating, ameliorating or alleviating diabetes comprising a compound represented by the following formula (9) or (13) as an active ingredient:
Formula 9

Formula 13

delete delete delete delete delete delete delete delete delete delete delete The composition of claim 1, wherein the composition is a pharmaceutical composition.
The composition according to claim 1, wherein the composition is a food composition for preventing, ameliorating or alleviating diabetes.
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