KR20210041353A - Composition comprising compound extracted from the root of Broussonetia papyrifera, and uses thereof - Google Patents

Abstract

The present invention relates to a composition containing a compound derived from Broussonetia papyrifera root, and uses thereof. The compound derived from Broussonetia papyrifera root according to an aspect activates AMPK in adipose tissue or liver tissue, thereby having effects of inhibiting the inflammatory response in the adipose tissue of mice fed a high-fat diet, and improving glucose tolerance induced by obesity by regulating lipid metabolism in liver tissue. Therefore, the composition can be usefully used in pharmaceuticals, foods, and the like for preventing, alleviating, or treating type 2 diabetes.

Description

Composition comprising compound extracted from the root of Broussonetia papyrifera, and uses thereof

The present invention relates to a composition comprising a compound derived from the root of Cucurbita, and to a use thereof.

Diabetes mellitus is a disease in which hyperglycemia occurs because the cells in the body cannot use sugar due to decreased insulin secretion or decreased insulin function. In the case of diabetes, due to an imbalance of hormones including insulin, it exhibits characteristic symptoms of hyperglycemia due to abnormal physiological metabolic control functions such as protein, lipid, and electrolyte metabolism, including carbohydrates. Diabetes is characterized by two types. Type 1 diabetes is caused by a lack of secretion of insulin, a hormone that regulates glucose in the blood, and is often called childhood diabetes because it occurs mainly in young age groups in their teens and 20s. Type 2 diabetes mainly occurs after the 40s and accounts for the majority of diabetic patients in Korea. As the etiology of type 2 diabetes, both impaired insulin secretion in pancreatic beta cells and defective insulin action (insulin resistance) in target cells are observed.

Recently, the number of obese and overweight populations is increasing rapidly, and the incidence of metabolic diseases related thereto is increasing. As a result of this, as research on adipose tissue that causes obesity increases rapidly, it is known that adipose tissue affects not only the regulation of body fat, but also insulin resistance, including inflammatory reactions, arteriosclerosis, and even the occurrence of certain cancers. As a result of many studies, adipose tissue, known as a simple storage of energy, has been identified as an endocrine organ that causes physiological and metabolic changes in the body. In addition, as evidence for the close relationship between adipose tissue, inflammatory response, and insulin resistance has been presented in various ways, pathological interest in adipose tissue is increasing.

Broussonetia papyrifera (Broussonetia papyrifera) is distributed in China, Japan, and Korea, and has been used for paper making since ancient times, and has also been used as a traditional Chinese medicine. Although it has been reported that Cudrania has an anti-inflammatory effect (Korean Patent Laid-Open Publication No. 10-2011-0064212), studies on its role as a diabetes treatment through improvement of insulin resistance are insignificant.

One aspect is for the prevention or treatment of diabetes comprising a compound represented by the following formula (1), a compound represented by the following formula (2), a solvate thereof, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient It is to provide a pharmaceutical composition.

[Formula 1]

[Formula 2]

Another aspect is for the prevention or improvement of diabetes comprising a compound represented by the following formula (1), a compound represented by the following formula (2), a solvate thereof, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient It is to provide a health functional food composition.

[Formula 1]

[Formula 2]

Another aspect is to provide a pharmaceutical composition for preventing or treating diabetes, including a root extract or a fraction thereof, as an active ingredient.

Another aspect is to provide a health functional food composition for preventing or improving diabetes, including a root extract or a fraction thereof, as an active ingredient.

One aspect is for the prevention or treatment of diabetes comprising a compound represented by the following formula (1), a compound represented by the following formula (2), a solvate thereof, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient Provides a pharmaceutical composition.

[Formula 1]

[Formula 2]

Brosoflavonol B (compound 1) represented by Chemical Formula 1 is a compound having a molecular formula of C ₂₆ H ₂₇ O ₇ and a molecular weight of 451 g/mol, from a root extract of Brosonetia papyrifera or a fraction thereof. It may be a separated natural compound, but the present invention is not limited thereto, and may be isolated from a plant related to the Japanese paper mulberry genus or other genus.

Kazinol J (compound 2) represented by Chemical Formula 2 is a compound having a molecular formula of C ₂₆ H ₃₃ O ₄ and a molecular weight of 409 g/mol, and is isolated from a root extract or a fraction thereof. Although it may be a natural compound, it is not limited thereto, and may be isolated from a plant related to the Japanese oak genus or other genus.

The "solvate" may mean a compound of the present invention or a salt thereof containing a stoichiometric or non-stoichiometric amount of a solvent bound by non-covalent intermolecular forces. Preferred solvents therefor may be volatile, non-toxic, and/or solvents suitable for administration to humans.

The "stereoisomer" refers to a molecule having the same molecular formula and a connection method of members, but different spatial arrangements between atoms. The stereoisomer may be an enantiomer or a diastereomer of the compound.

The "enantiomers" are compounds that have the same molecular formula as a specific compound and the same order of bonding of atoms, but differ in the three-dimensional configuration of atoms in the space, and are related to the relationship between a specific compound and its mirror image. It may mean a compound that is present.

The composition may be a racemic mixture in which two enantiomers are mixed in 1:1, that is, in the same ratio.

The "diastereomers" may refer to all stereoisomers that are not enantiomers. It refers to a relationship between a cyclic compound without a stereocenter, a cyclic compound having a trans configuration, or a compound having a diastereoisomer relationship with an alkene, and a compound having two or more stereocenters, which have only partially opposite configurations. I can.

The "pharmaceutically acceptable" is suitable for use in contact with a subject, for example, human tissue, with a reasonable benefit/risk ratio without excessive toxicity, irritation, allergic reaction or other problems or complications, and is within the scope of sound medical judgment. It may mean a phosphorus compound or composition.

The "salt" may be an acid addition salt formed by a pharmaceutically acceptable free acid. The free acid may be an inorganic acid or an organic acid, and the inorganic acid may be hydrochloric acid, bromic acid, nitric acid, sulfuric acid, perchloric acid, phosphoric acid, etc., and the organic acid may be citric acid, acetic acid, lactic acid, maleic acid, fumaric acid, gluconic acid, methanesulfuric acid. Fonic acid, glycolic acid, succinic acid, tartaric acid, galacturonic acid, embonic acid, glutamic acid, aspartic acid, oxalic acid, (D) or (L) malic acid, maleic acid, methanesulfonic acid, ethanesulfonic acid, 4-toluenesulfonic acid, Salicylic acid, citric acid, benzoic acid or malonic acid, and the like. In addition, the salt may include an alkali metal salt (sodium salt, potassium salt, etc.) and an alkaline earth metal salt (calcium salt, magnesium salt, etc.). For example, the acid addition salt is acetate, aspartate, benzate, besylate, bicarbonate/carbonate, bisulfate/sulfate, borate, camsylate, citrate, edisylate, esylate, formate, fumarate, Gluceptate, Gluconate, Glucuronate, Hexafluorophosphate, Hybenzate, Hydrochloride/Chloride, Hydrobromide/Bromide, Hydroiodide/Iodide, Isethionate, Lactate, Malate, Male Eate, malonate, mesylate, methyl sulfate, naphthylate, 2-naphsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate/hydrogen phosphate/dihydrogen phosphate, saccha Rate, stearate, succinate, tartrate, tosylate, trifluoroacetate, aluminum, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, Potassium, sodium, tromethamine, zinc salts, and the like may be included.

The term “diabetes mellitus” refers to a group of metabolic disorders in which high blood sugar levels persist for a long period of time. The diabetes may occur because the pancreas does not make enough insulin or the cells of the body do not respond appropriately to the made insulin. The diabetes can be classified into insulin-dependent diabetes (type 1 diabetes) and insulin-independent diabetes (type 2 diabetes). The type 1 diabetes may be due to a deficiency of insulin due to a destructive lesion of pancreatic beta cells, and the type 2 diabetes may be due to the occurrence of insulin resistance in which cells do not respond appropriately to insulin. The insulin resistance may be caused by excess body fat accumulated in the abdomen. The pharmaceutical composition according to an aspect showed an effect of improving glucose tolerance in mice in which obesity and insulin resistance were induced by feeding a high fat diet (HFD), and the diabetes may be type 2 diabetes.

In one embodiment, the pharmaceutical composition may be to activate AMPK in adipose tissue or liver tissue.

In one embodiment, the pharmaceutical composition inhibits the activity of NF-κB induced by TNF-α in adipocytes or adipose tissues by activating AMPK, and thus pro-inflammatory genes, for example, For example, it may be to inhibit the expression of inflammatory cytokines such as IL-6 and MCP-1. In addition, the pharmaceutical composition is a gene related to lipid synthesis in adipose tissue (lipogenic genes), for example, Srebp-1c (sterol regulatory element-binding protein 1), Acc-1, and fatty acid synthase (Fasn). It may be to inhibit expression.

In one embodiment, the pharmaceutical composition is a gene related to lipid synthesis in liver tissue by activating AMPK, for example, Srebp-1c, Fasn, Scd-1 (stearoyl-CoA desaturase-1), Dgat-1 ( diglyceride acyltransferase-1) or inhibiting the expression of lipid oxidation-related genes (fatty acid oxidation-related genes), such as Acsl (acyl-CoA synthetase long-chain), Scad (short-chain acyl-CoA dehydrogenase) , Vlcad (very-long-chain acyl-CoA dehydrogenase) may be increased expression, and accordingly, fat in liver tissue may be reduced, thereby improving fatty liver.

The term “prophylaxis or treatment” may comprehensively mean improving the symptoms of diabetes by administering the composition in a pharmaceutically effective amount, and may mean improving one symptom or most of the symptoms caused by diabetes. The “pharmaceutically effective amount” refers to the type of disease, the patient's age, weight, health, sex, the patient's sensitivity to the drug, the route of administration, the method of administration, the number of administrations, the duration of treatment, the combination, or drugs used simultaneously. It can be easily determined by a person skilled in the art according to factors well known to. The “effective amount” may mean an amount capable of activating AMPK in adipose tissue or liver tissue when administered to an individual. The “individual” may be an animal, for example, an animal including a mammal, particularly a human, and may be a cell, tissue, organ, etc. derived from an animal.

The “pharmaceutical composition” can be administered in various oral and parenteral formulations at the time of clinical administration, and for human administration, the formulation has sterility, pyrogenicity, general stability and purity required by the FDA Bureau of Biological Standards. It may be conforming to the standard.

The pharmaceutical composition may further include a pharmaceutically acceptable excipient or carrier. The carrier may be an excipient, a disintegrant, a binder, a lubricant, or a combination thereof. The excipient may be microcrystalline cellulose, lactose, low-substituted hydroxycellulose, or a combination thereof. The disintegrant may be sodium starch glycolate, anhydrous calcium monohydrogen phosphate, or a combination thereof. The binder may be polyvinylpyrrolidone, low-substituted hydroxypropylcellulose, hydroxypropylcellulose, or a combination thereof. The lubricant may be magnesium stearate, silicon dioxide, talc, or a combination thereof.

Another aspect is for the prevention or improvement of diabetes comprising a compound represented by the following formula (1), a compound represented by the following formula (2), a solvate thereof, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient It provides a health functional food composition. In the health functional food composition, the compound represented by Formula 1, the compound represented by Formula 2, solvate, stereoisomer, pharmaceutically acceptable salt, diabetes, and prevention are as described above.

[Formula 1]

[Formula 2]

The "health functional food" may be added to the health food composition as it is, or may be used together with other foods or food ingredients, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined depending on the intended use. In general, the amount of the composition in the health functional food may be added in an amount of 0.1 to 90 parts by weight of the total food weight. However, in the case of long-term intake for the purpose of maintaining health or for the purpose of health control, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range. These foods include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcohol, beverages, and vitamin complexes. It is not limited and may include all health foods in the usual sense.

The term “improvement” may refer to any action that at least reduces the severity of a parameter, eg, symptom, related to the alleviation or treatment of the condition.

Another aspect provides a pharmaceutical composition for preventing or treating diabetes, including a root extract or a fraction thereof, as an active ingredient. In the pharmaceutical composition, diabetes, prevention or treatment, the pharmaceutical composition is as described above.

The root extract or fractions thereof may contain the compound. For example, it may include one or more of the compound represented by Formula 1 or the compound represented by Formula 2. The description of the compound is as described above.

The root of the Giraffe tree may be the whole or part of the root. The roots of the Cudrania tree used for extraction may be crushed or shredded, or suitably dried, in whole or in part. The root of the Giraffe tree may be used only as a root bark for an efficient yield.

The extract may be extracted by a hydrophilic solvent, for example, alcohol, water, or a combination thereof. The alcohol may be a compound having one or more -OH groups of C1 to C10. The alcohol may be a C1 to C6 alcohol or a C3 to C6 polyhydric alcohol. The alcohol may be methanol, ethanol, n-propanol, isopropanol, n-butanol, sec-butanol, isobutanol, tert-butanol, n-pentanol, n-hexanol, or a mixture thereof. The solvent may be, for example, a mixture of water and alcohol, that is, an aqueous alcohol solution. The alcohol concentration of the aqueous alcohol solution is 1 to 100% (w/w), for example, 1 to 99.5% (w/w), 10 to 100% (w/w), 20 to 100% (w/w), 30 to 100% (w/w), 40 to 100% (w/w), 50 to 100% (w/w), 60 to 100% (w/w), 70 to 100% (w/w), 75 to 100% (w/w), 80 to 100% (w/w), 60 to 90% (w/w), 70 to 90% (w/w), 75 to 85% (w/w) days I can. The aqueous alcohol solution may be an aqueous methanol, ethanol, or butanol solution. In one embodiment, the extract may be extracted with 100% methanol.

The extract may be extracted by a conventional method in the art, such as warm extraction, pressurized extraction, ultrasonic extraction, hot water extraction, reflux cooling extraction, subcritical extraction, or supercritical extraction.

The extraction is 4 ℃ to 85 ℃, for example, 4 ℃ to 70 ℃, 4 ℃ to 60 ℃, 4 ℃ to 50 ℃, 4 ℃ to 40 ℃, 4 ℃ to 30 ℃, 10 ℃ to 85 ℃, 15 It may be performed at ℃ to 85 ℃, 20 ℃ to 85 ℃, 10 ℃ to 50 ℃, 10 ℃ to 40 ℃, 10 ℃ to 35 ℃, 10 ℃ to 30 ℃, 20 ℃ to 30 ℃, or at room temperature.

The extraction time may be appropriately selected according to the selected temperature and extraction method. For example, the extraction time is 1 hour to 3 days, 1 hour to 2 days, 1 hour to 1 day, 1 hour to 18 hours, 1 hour to 12 hours, 3 hours to 3 days, 3 hours to 2 days, 3 Time to 1 day, 3 hours to 18 hours, 3 hours to 12 hours, 6 hours to 3 days, 6 hours to 2 days, 6 hours to 1 day, 6 hours to 18 hours, 6 hours to 12 hours, or 6 hours To 9 hours.

The extraction may be one or more times, for example, 1 to 5 times, 1 to 4 times, 1 to 3 times, 2 to 5 times, or 2 to 4 times, and each extraction is performed by the same method, It can be done in different ways.

The extraction may be performed by separating plant residues and extracts by known methods such as filtration. The extraction may also include removing the solvent from the obtained extract by known methods such as concentration under reduced pressure. The extraction may also include preparing a dried extract by drying the obtained extract, such as lyophilization.

The extract is 0.001% to 80% by weight based on the total weight of the composition, for example, 0.01% to 60% by weight, 0.01% to 40% by weight, 0.01% to 30% by weight, 0.01% to 20% by weight %, 0.01% to 10%, 0.01% to 5%, 0.05% to 60%, 0.05% to 40%, 0.05% to 30%, 0.05% to 20%, 0.05% to 10% by weight, 0.05% to 5% by weight, 0.1% to 60% by weight, 0.1% to 40% by weight, 0.1% to 30% by weight, 0.1% to 20% by weight, 0.1% by weight % To 10% by weight, or 0.1% to 5% by weight.

The composition according to an embodiment may include a fraction of the root extract of Guji tree as an active ingredient.

The term "fraction" refers to a substance in which the extract of the roots of the Cudrania tree is divided into a part thereof, that is, a substance that has been fractionated. The fraction may be obtained by solvent fractionation. The solvent fractionation may be a mixture of the root extract of Cucurbita tree with a solvent and separating a substance present in the solvent. The fraction may be a butanol fraction, a methanol fraction, an acetonitrile fraction, or a water fraction obtained by sequentially fractionating the Guji tree root extract into butanol, methanol, acetonitrile, water, or a mixture thereof. In one embodiment, the fraction may be a methanol fraction, acetonitrile fraction, or a water fraction obtained by fractionating the root extract of Cudrania tree with methanol, acetonitrile, water, or a mixture thereof. Conditions for the fractionation, such as temperature conditions, pressure conditions, time, amount or concentration of the solvent used, and stirring, may be the same as those described for the extraction used to prepare the root extract of Cudrania tree. The fractionation may be repeated one or more times, for example, 1 to 5 times.

Separating the fraction may be performed by a known method such as filtration. The fractionation may also include removing the solvent from the obtained fraction by known methods such as concentration under reduced pressure. The fractionation may also comprise concentrating and/or drying the obtained fractions. The concentration may be concentrated under reduced pressure. The drying may include vacuum drying, boiling drying, spray drying, room temperature drying, or freeze drying.

The pharmaceutical composition may contain the extract or a fraction thereof in an effective amount or as an active ingredient. The effective amount can be appropriately selected according to the individual. The severity of the disease or condition, the age, weight, health, sex of the subject, the sensitivity to the extract of the subject, the time of administration, the route of administration and the rate of excretion, the period of administration, factors including other compositions used in combination or concurrent with the composition, and others. It can be determined according to factors well known in the field of physiology or medicine.

The pharmaceutical composition may further include a pharmaceutically acceptable excipient or carrier. The description of the pharmaceutically acceptable excipient or carrier is as described above.

Another aspect provides a health functional food composition for preventing or improving diabetes, including a root extract or a fraction thereof, as an active ingredient. In the health functional food composition, the root extract, fraction, diabetes, prevention or improvement, and health functional food are as described above.

According to one aspect, the compound derived from the roots of Cudrania chinensis activates AMPK in adipose tissue or liver tissue to inhibit inflammatory response in the adipose tissue of mice fed a high fat diet, and regulates lipid metabolism in liver tissue, thereby inhibiting glucose tolerance induced by obesity. As it has an effect of improving, it can be usefully used in medicines, foods, etc. for preventing, improving, or treating type 2 diabetes.

Figure 1 is a flow chart showing a process of preparing a fraction from the root extract of Guji tree and separating the compound.
Figure 2 is a result of confirming the effect on the NF-κB activity induced by TNF-α of the root bark extract of Guji tree.
3 is a result of confirming that PRE shows an inhibitory effect on pro-inflammatory gene expression and AMPK activation induced by TNF-α in 3T3-L1 adipocytes.
4 is a result of confirming that PRE shows an effect of improving glucose tolerance in obese mice induced by a high fat diet.
5 is a result of confirming that PRE exhibits an effect of improving inflammation in adipose tissue of obese mice induced by a high fat diet.
6 is a result of confirming that PRE shows the effect of improving fatty liver in liver tissue of obese mice induced by a high fat diet.
7 is a result of confirming that Brosoflavonol B and Kazinol J are active ingredients of PRE.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1. Preparation of Guji tree root extracts and fractions and separation of compounds 1 to 2

(1.1) Preparation of the roots of the Kuji tree

The root bark of the Broussonetia papyrifera was collected on June 23, 2015 at Mugori, Gongyang-myeon, Sacheon-si, Gyeongsangnam-do, and verified by Dr. Jinhyuk Paik. Samples of the collected raw materials (KRIBB 0059119) were deposited with the Korea Institute of Biotechnology (KRIBB) International Biological Sample Center (IBMRC). The collected roots were used only as root bark for efficient yield.

(1.2) Preparation of Guji tree root extract

After drying 13.36 kg of the root bark of Broussonetia papyrifera, 35 L of 100% methanol was added, followed by extraction at room temperature for 24 hours. After repeating the above process 3 times, the filtered extract was concentrated using a vacuum concentrator at 42°C, and all solvents were removed using a lyophilizer, and dried Guji tree root 100% methanol extract (total B. papyrifera extracts; TPRE). 1.33 kg was obtained.

(1.3) Preparation of Root Fractions of Cucurbita and Isolation of Compounds 1 to 2

Figure 1 is a flow chart showing a process of preparing a fraction from the root extract of Guji tree and separating the compound. As shown in FIG. 1, 1.33 kg of 100% methanol extract of Cudrania root prepared by the method of (1.1) was added to MeOH-H ₂ O (0-10 minutes 10% MeOH, 10-60 minutes 10-100% MeOH, SPOT-II MPLC (Gilson, Middleton, WI) using YMC-Pack ODS AQ HG (250 × 20 mm, 10 μm, Kyoto, Japan) eluted at 60-90 min 100% MeOH) and elution rate 30 mL/min. , USA), and fractions 1 to 8 were obtained (TPRE No. 1-8).

Fraction 6 (TPRE No.6 (hereinafter referred to as PRE) (55.7 g) was subjected to high performance liquid chromatography using a reverse phase column (YMC-Pack ODS-AQ-HG, 10 μm), and a sample (2 g/mL Methanol diluent) was repeatedly injected 25 times _{and eluted with an ACN-H 2} O concentration gradient system (55% → 100% MeOH, 70 minutes) to obtain four fractions 6-1 to 6-4 (PRE No. 6- 1-6-4) After that, preparative HPLC was repeatedly performed under the following conditions.

Of these, fraction 6-3 (14.4 g) was subjected to preparative-HPLC using a reversed-phase column (Hypersil GOLD), and a sample (10 mg/mL methanol dilution) was injected into the MeOH-H ₂ O concentration gradient system (70%). → 100% MeOH) to give 7 fractions 6-3-1 to 6-3-7.

Of these fractions 6-3-5 (345.1 mg) were eluted with Hypersil GOLD with a MeOH-H ₂ O concentration gradient system (70% → 100% MeOH) to obtain the purity of 6-3-5-4-2 in 7 fractions. To increase, additional column chromatography was performed by preparative HPLC (PLC 2020 personal purification system, Gilson, Middleton, MA, USA) using a YMC-Pack pro C8 column using a 55% ACN (acetonitrile) isocratic system. , Compound 1 (44.9 mg) was obtained.

In addition, fraction 6-3-7 (337.5 mg) was eluted with 55% ACN with a J'sphere to increase the purity of 6-3-7-2-3 among the 8 fractions. MeOH-H ₂ O (75% → 90% MeOH) using a YMC-Pack pro C8 column with a gradient system was subjected to additional column chromatography by preparative HPLC (PLC 2020 ) to give compound 2 (48.7 mg).

(1.4) NMR analysis and HRMS analysis of compounds 1 to 2

The structure was analyzed by performing ^{HRMS, 1} H, and ¹³ C NMR on the compounds 1 and 2 above. HRMS analysis is an ultra-performance liquid chromatography quadrupole time of flight mass spectrometry equipped with an electrospray ionization (ESI) interface (Waters Q-TOF PremierTM, Waters Corporation). ; UPLC-QTOF-MS) was used. 1D NMR spectrum was obtained with a JEOL ECZ500R ( ¹ H NMR at 500 MHz, ¹³ C NMR at 125 MHz, Tokyo, JP) spectrometer _{using acetone-d 6 as a solvent.}

Compound 1: brosoflavonol B

The molecular formula of Compound 1 was determined to have a _{molecular formula of C 26} H ₂₇ O ₇ by HRMS spectrum (m/z 451.1778 [MH]-calcd. for 451.1757). Compound 2 was confirmed to be broussoflavonol B according to the following NMR analysis results.

¹ H-NMR (500 MHz, acetone-d ₆ ) δ: 3.60 (2H, d, J = 6.5 Hz, H-11), 5.20 (1H, m, H-12), 1.65 (3H, s, H- 14), 1.78 (3H, s, H-15), 3.42 (2H, d, J = 6.9 Hz, H-16), 5.23 (1H, m, H-17), 1.66 (3H, s, H-19 ), 1.79 (3H, s, H-20), 7.74 (1H, s, H-2'), 7.00 (1H, d, J = 8.0 Hz, H-5'), 7.59 (1H, d, J = 8.1 Hz, H-6'), 3.86 (3H, s, 3-OCH ₃ ).

¹³ C-NMR (125 MHz, acetone-d ₆ ) δ: 156.6 (C-2), 139.2 (C-3), 179.8 (C-4), 153.1 (C-5), 107.0 (C-6), 159.6 (C-7), 111.8 (C-8), 157.5 (C-9), 106.1 (C-10), 22.6 (C-11), 123.4 (C-12), 132.9 (C-13), 25.9 (C-14), 18.1 (C-15), 22.2 (C-16), 123.0 (C-17), 132.6 (C-18), 26.0 (C-19), 18.3 (C-20), 123.5 ( C-1'), 116.5 (C-2'), 145.9 (C-3'), 149.0 (C-4'), 116.3 (C-5'), 122.1 (C-6'), 60.2 (3- OCH ₃ ).

Compound 2: Kazinol J

The molecular formula of Compound 2 was determined to have a _{molecular formula of C 26} H ₃₃ O ₄ by HRMS spectrum (m/z 409.2410 [MH]-calcd. for 409.2379). Compound 2 was confirmed to be Kazinol J according to the following NMR analysis results.

¹ H-NMR (500 MHz, acetone-d ₆ ) δ: 2.54 (2H, t, J = 7.5 Hz, H-1), 1.71 (2H, m, H-2), 2.45 (2H, t, J = 8.0 Hz, H-3), 6.43 (1H, d, J = 2.3 Hz, H-3'), 6.33 (1H, dd, J = 2.3, 8.1 Hz, H-5'), 6.90 (1H, d, J = 8.1 Hz, H-6'), 6.55 (1H, s, H-6''), 3.21 (2H, d, J = 6.1 Hz, H-7''), 4.95 (1H, t, J = 6.1 Hz, H-8``), 1.65 (3H, s, H-10``), 1.70 (3H, s, H-11''), 3.35 (2H, d, J = 6.6 Hz, H-12 ''), 5.09 (1H, t, J = 6.6 Hz, H-13``), 1.64 (3H, s, H-15''), 1.72 (3H, s, H-16''), 3.75 ( 3H, s, 2-OCH ₃ ).

¹³ C-NMR (125 MHz, acetone-d ₆ ) δ: 30.5 (C-1), 33.1 (C-2), 33.7 (C-3), 122.2 (C-1'), 159.4 (C-2' ), 99.8 (C-3'), 157.7 (C-4'), 107.4 (C-5'), 130.9 (C-6'), 132.7 (C-1''), 130.5 (C-2'' ), 127.8 (C-3``), 142.3 (C-4''), 143.1 (C-5''), 114.7 (C-6''), 28.2 (C-7''), 125.9 (C -8''), 130.8 (C-9''), 25.9 (C-10''), 18.2 (C-11''), 26.3 (C-12''), 125.0 (C-13'') , 130.8 (C-14''), 25.9 (C-15''), 18.1 (C-16''), 55.6 (2-OCH ₃ ).

Experimental Example 1. Confirmation of the effect of inhibiting the activity of NF-κB induced by TNF-α of the root fraction (PRE)

In order to confirm the effective fraction of the methanol extract from the root of Cucurbita on the anti-inflammatory action, the transcriptional activity of NF-κB, a key regulator of the inflammatory response, was measured.

Specifically, HEK293 cells were obtained from the American Tissue Culture Collection (ATCC, USA) and in Dulbecco's modified Eagle's medium (DMEM, Life Technologies, NY, USA) in 10% fetal bovine serum (Atlas, CO, USA). Cultured. All chemicals for cell culture were obtained from Sigma-Aldrich (St. Louis, Missouri, USA). HEK-293 cells were transfected with NF-κB-reactive luciferase reporter and pRL-renillia. Thereafter, the cells were treated with each concentration for 24 hours with methanol extract (TPRE), Fraction 1 to Fraction 8, Fraction 6 (PRE), respectively, and then additionally treated with TNF-α (10 ng/ml) to NF -κB activation was induced, and the cells were harvested to measure luciferase activity. Figure 2 is a result of confirming the effect on the NF-κB activity induced by TNF-α of the root bark extract of Guji tree.

As a result, as shown in FIG. 2A, it was confirmed that TPRE inhibited NF-κB activity induced by TNF-a treatment in a concentration-dependent manner. In order to separate the effective fraction for the anti-inflammatory action of TPRE, TPRE was separated into a total of 8 fractions, and then the activity of NF-κB for each fraction was measured. As a result, as shown in FIG. 2B, 4 out of fractions 1 to 8 Significantly inhibited the activity of NF-κB induced by TNF-α. Particularly, fraction 6 (PRE) showed the highest inhibitory effect, and as shown in FIG. 2C, it was confirmed that fraction 6 (PRE) inhibited NF-κB activity in a concentration-dependent manner.

Experimental Example 2. Confirmation of the inhibitory effect of gene expression related to the inflammatory response induced by TNF-α in adipocytes of the root fraction (PRE)

To further confirm the effect of PRE on the inflammatory response in vitro, 3T3-L1 adipocytes are treated with TNF-α to induce the expression of inflammatory response-related genes, and PRE-treated to increase the expression of inflammatory response-related genes. It was confirmed that it was suppressed.

Specifically, 3T3-L1 cells were obtained from the American Tissue Culture Collection (ATCC, USA) and obtained from Dulbecco's modified Eagle's medium (DMEM, Life Technologies, NY, USA) in 10% bovine calf serum (Invitrogen, CA, USA). ) Was incubated. Adipocyte differentiation was induced by treating the cells in DMEM containing 10% FBS, 0.5 mM isobutylmethylxanthine, 1 μM dexamethasone, and 850 nM insulin. After 48 hours, the medium was replaced every other day with DMEM containing 10% FBS and 850 nM insulin. All chemicals for cell culture were obtained from Sigma-Aldrich (St. Louis, Missouri, USA). 3T3-L1 adipocytes were incubated with 3, 10, 20, 40 μg/ml PRE for 24 hours, respectively, and treated with 20 ng/ml TNF-α for 5 hours, followed by real-time-PCR to perform inflammatory cytokines. (IL-6, MCP-1), iNOS, and cox2 mRNA expression levels were analyzed. In addition, 3T3-L1 adipocytes were treated with 5, 10, 20, 40 μg/ml of PRE for 24 hours, treated with 20 ng/ml TNF-α for 30 minutes, and then subjected to western blot to phosphorylation-I. The expression levels of -κB, I-κB, phosphorylation-NF-κB, and NF-κB were analyzed. In addition, in order to confirm the mechanism of inhibiting PRE's NF-κB signaling pathway, we investigated whether PRE activates AMPK, which is known as an important inhibitor of inflammatory response. To this end, 3T3-L1 adipocytes were incubated for 1 hour in the presence or absence of AMPK-specific inhibitor Compound C (10 μM), treated with PRE or AICAR (500 μM) for 2 hours, and phosphorylated by performing Western blot- The expression levels of AMPK, AMPK, phosphorylation-ACC, and ACC were analyzed. 3 is a result of confirming that PRE shows an inhibitory effect on the expression of inflammatory response-related genes and AMPK activation induced by TNF-α in 3T3-L1 adipocytes.

As a result, as shown in FIG. 3A, the expression of inflammatory cytokines (IL-6, MCP-1) and iNOS, cox2 induced by TNF-α in 3T3-L1 adipocytes decreased in a concentration-dependent manner by PRE treatment. Was confirmed. In addition, as shown in FIG. 3B, it was confirmed that phosphorylation of I-κB and NF-κB induced by TNF-α in 3T3-L1 adipocytes was reduced by PRE treatment. In addition, as shown in Figure 3C, phosphorylation of acetyl-CoA carboxylase (ACC), which is a substrate of AMPK and AMPK, in 3T3-L1 adipocytes is increased by PRE treatment, and is an AMPK-specific inhibitor. It was confirmed that phosphorylation was blocked when co-treated with Compound C. This means that by activating AMPK, PRE inhibits the activity of NF-κB induced by TNF-α, thereby reducing the inflammatory response.

Experimental Example 3. Confirmation of the effect of improving glucose tolerance of the root fraction (PRE)

To confirm whether PRE exhibits anti-diabetic effects in vivo, wild-type C57BL/6 mice were fed a high fat diet (HFD) to induce obesity and insulin resistance, and then pre-administered glucose tolerance test (glucose tolerance). test; GTT) was performed.

Specifically, 7-week-old male C57BL/6 mice were fed a high-fat diet (60% kcal fat, D12492, Research Diets Inc., New Jersey, USA) for 10 weeks, and then 40 mg/kg of PRE or vehicle was administered intraperitoneally for 1 week. My injection, and I fasted overnight before D-glucose injection (2g/kg body weight). Thereafter, glucose tolerance test was performed, and body weight and fasting insulin (Crystal Chem., IL, USA) level were measured. All animal experiments were performed according to procedures approved by the Animal Management Committee of Ulsan Institute of Science and Technology. 4 is a result of confirming that PRE shows an effect of improving glucose tolerance in obese mice induced by a high fat diet.

As a result, as shown in FIG. 4B, there was no change in body weight due to PRE administration, but as shown in FIG. 4A, it was confirmed that glucose tolerance was significantly improved in the PRE-treated group. In addition, as shown in Figure 4C, it was confirmed that PRE significantly reduced the insulin level. This means that PRE improves glucose tolerance and hyperinsulinemia due to obesity.

Experimental Example 4. Inhibition of the expression of genes involved in the inflammatory response and lipid synthesis in adipose tissue of the root fraction (PRE)

PRE activated AMPK in vitro and inhibited the inflammatory response by inhibiting the activity of NF-κB, and in obese adipose tissues in vivo, PRE inhibited AMPK activation, NF-κB activity, inhibited inflammatory response-related gene expression, and It was confirmed whether there is an effect of suppressing gene expression related to lipid synthesis.

Specifically, adipose tissue was isolated from obese mice induced by a high fat diet, and Western blot was performed to analyze the expression levels of phosphorylation-NF-κB, NF-κB, phosphorylation-AMPK, and AMPK. In addition, quantitative real-time-PCR was performed to analyze the mRNA levels of proinflammatory genes and adipogenic genes. 5 is a result of confirming that PRE exhibits an effect of improving inflammation in adipose tissue of obese mice induced by a high fat diet.

As a result, as shown in FIG. 5A, it was confirmed that NF-κB phosphorylation was significantly decreased by PRE administration in adipose tissue, while AMPK phosphorylation was significantly increased. In addition, as shown in FIG. 5B, it was confirmed that the expression of inflammatory response-related genes such as IL-1β, TNF-α, and iNos was significantly reduced by PRE administration in adipose tissue. In addition, as shown in FIG. 5C, it was confirmed that the expression of genes involved in lipid synthesis was significantly reduced by the administration of PRE in adipose tissue. This means that PRE inhibits obesity-mediated adipose tissue inflammation and fat formation.

Experimental Example 5. Confirmation of the effect of improving the fatty liver of Cuji tree root fraction (PRE)

Since PRE activated AMPK and suppressed the expression of genes involved in lipid synthesis, it was confirmed that PRE has an effect of improving fatty liver, which is increased by obesity in liver tissue.

Specifically, liver tissue was isolated from obese mice induced by a high fat diet, and H&E staining was performed. In addition, since AMPK plays an important role in the pathogenesis of fatty liver and PRE activated AMPK in adipose tissue, AMPK signaling was investigated in the liver. For this, Western blot was performed to analyze the expression levels of phosphorylation-AMPK and AMPK. In addition, quantitative real-time-PCR was performed to analyze the mRNA levels of adipose-forming genes and fatty acid oxidation-related genes. 6 is a result of confirming that PRE shows the effect of improving fatty liver in liver tissue of obese mice induced by a high fat diet.

As a result, as shown in FIG. 6A, it was confirmed that the fat in the liver tissue was reduced by administering PRE in obese mice induced by a high fat diet. In addition, as shown in Figure 6B, PRE administration increased AMPK phosphorylation in the liver. In addition, as shown in Figs. 6C and 6D, it was confirmed that the expression of genes involved in lipid synthesis in the PRE-treated group was decreased, while the expression of genes related to lipid oxidation was significantly increased.

Summarizing the above results, PRE means that by activating AMPK, inhibiting the inflammatory response in the adipose tissue of mice fed a high fat diet, and improving the glucose tolerance induced by obesity (insulin resistance) by regulating lipid metabolism in the liver tissue. do.

Experimental Example 6. Confirming that Broussoflavonol B and Kazinol J are active ingredients of PRE

In order to confirm the active ingredient of PRE that activates AMPK, Broussoflavonol B (Compound 1) and Kazinol J (Compound 2) that remarkably activate AMPK in 3T3-L1 adipocytes among 20 compounds isolated from methanol extracts from the roots of Koji tree. The following experiment was performed for.

Specifically, 3T3-L1 adipocytes were incubated in the presence or absence of Compound C (10 μM) for 1 hour, and the cells were treated with BF or KJ for 2 hours, and then subjected to Western blot phosphorylation-AMPK, AMPK, phosphorylation. -ACC, the expression of ACC was analyzed. In addition, HEK-293 cells were transfected with NF-κB-reactive luciferase reporter and pRL-renillia. Cells were treated with 5, 10, 20 μM concentration of BF or KJ for 24 hours, then treated with TNF-α (10 ng/ml) for 6 hours, and the cells were harvested to measure luciferase activity. In addition, after pretreatment of 3T3-L1 adipocytes in the presence or absence of compound C, the cells were treated with BF or KJ for 24 hours, and then treated with TNF-α (20 ng/ml) for 30 minutes. Then, Western blot was performed to analyze the expression of phosphorylation-I-κB, I-κB, phosphorylation-NF-κB, and NF-κB. In addition, 3T3-L1 adipocytes were incubated with BF or KJ at 1, 5, 10, 20 μM concentration for 24 hours, then treated with 20 ng/ml TNF-α for 5 hours, and quantitative real-time-PCR was performed. The mRNA expression level of each gene was analyzed. 7 is a result of confirming that Broussoflavonol B (BF) and Kazinol J (KJ) are active ingredients of PRE.

As a result, as shown in FIG. 7B, in 3T3-L1 adipocytes, both BF and KJ significantly increased AMPK and ACC phosphorylation, which was confirmed to be blocked by co-treatment with Compound C. In addition, as shown in Fig. 7C, BF and KJ significantly inhibited the transcriptional activity of NF-κB induced by TNF-α. In addition, as shown in FIG. 7D, it was confirmed that BF and KJ block the NF-κB signaling pathway induced by TNF-α in 3T3-L1 adipocytes. In addition, as shown in Fig. 7E, it was confirmed that both BF and KJ inhibited the expression of pro-inflammatory genes in 3T3-L1 adipocytes stimulated with TNF-α. The above results indicate that BF and KJ are active ingredients of PRE that activates AMPK, and can block the inflammatory response by blocking the NF-κB signaling pathway through AMPK activation.

Claims (19)

A pharmaceutical composition for preventing or treating diabetes comprising a compound represented by the following formula (1), a compound represented by the following formula (2), a solvate thereof, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient .
[Formula 1]

[Formula 2]

The pharmaceutical composition according to claim 1, wherein the diabetes is type 2 diabetes. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition activates AMPK in adipose tissue or liver tissue. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition inhibits the activity of NF-κB in adipose tissue. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition inhibits the expression of a gene related to an inflammatory response in adipose tissue. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition inhibits the expression of a gene related to lipid synthesis in adipose tissue. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition inhibits the expression of a gene related to lipid synthesis in liver tissue or increases the expression of a gene related to lipid oxidation. Health functional food for preventing or improving diabetes comprising a compound represented by the following formula (1), a compound represented by the following formula (2), a solvate thereof, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient Composition.
[Formula 1]

[Formula 2]

A pharmaceutical composition for preventing or treating diabetes, comprising a root extract or a fraction thereof, as an active ingredient. The composition of claim 9, wherein the extract is an extract of C1-C6 alcohol, water, or a mixture thereof. The composition of claim 9, wherein the C1-C6 alcohol is ethanol, methanol, butanol, or a mixture thereof. The composition of claim 9, wherein the fraction is a methanol fraction, acetonitrile fraction, or a water fraction obtained by fractionating the root extract of Cudrania tree with methanol, acetonitrile, water, or a mixture thereof. The pharmaceutical composition according to claim 9, wherein the diabetes is type 2 diabetes. The pharmaceutical composition of claim 9, wherein the pharmaceutical composition activates AMPK in adipose tissue or liver tissue. The pharmaceutical composition of claim 9, wherein the pharmaceutical composition inhibits the activity of NF-κB in adipose tissue. The pharmaceutical composition of claim 9, wherein the pharmaceutical composition inhibits the expression of a gene related to an inflammatory response in adipose tissue. The pharmaceutical composition of claim 9, wherein the pharmaceutical composition inhibits the expression of a gene related to lipid synthesis in adipose tissue. The pharmaceutical composition of claim 9, wherein the pharmaceutical composition inhibits the expression of a gene related to lipid synthesis in liver tissue or increases the expression of a gene related to lipid oxidation. A health functional food composition for preventing or improving diabetes, comprising the root extract of Broussonetia papyrifera or a fraction thereof as an active ingredient.

Images