KR20140100117A - Compositions comprising the extract of Alnus japonica or the compounds derived therefrom for inhibiting adipogenesis - Google Patents

Abstract

The present invention relates to a composition for inhibiting differentiation of adipocytes, which comprises Ascicus extract or a fraction thereof or a diarylheptanoyl compound as an active ingredient. Specifically, the present invention relates to a pharmaceutical composition for preventing and / or treating obesity, hyperlipidemia, dyslipidemia, arteriosclerosis, hepaticopathy, nonalcoholic fatty liver, type 2 diabetes, metabolic syndrome containing the above extract or fraction or compound as an active ingredient .

Description

TECHNICAL FIELD The present invention relates to a composition for inhibiting lipid differentiation, comprising an extract of Alnus japonica or a compound isolated therefrom,

The present invention relates to the medicinal or phytochemical uses of alder tree extract, fractions or compounds isolated therefrom. More specifically, the present invention relates to a methanol extract of Alnus japonica, a fraction thereof, and a composition for inhibiting lipid differentiation of a compound isolated therefrom.

Obesity is caused by abnormal hypertrophy of subcutaneous fat tissue caused by accumulation of excess energy in body into fat. It is important to control adipogenesis, which is the process of differentiation from adipocytes into adipocytes, in order to treat obesity. Diabetes is also a metabolic syndrome characterized by hyperglycemia resulting from insulin action, insulin secretion, or both. Diabetes is caused by autoimmune mechanism of beta cells in the pancreas is destroyed by insulin is absolutely insufficient to resistance to the action of insulin ranging from the insulin secretion disorder and insulin resistance in many cases There are many cases exist. Obesity is a risk factor for various adult diseases in that it causes complications such as cardiovascular disease, type 2 diabetes, metabolic diseases such as insulin resistance syndrome, respiratory diseases and osteoarthritis. Management and treatment of obesity are meaningful not only in obesity itself but also in the prevention or treatment of the above complications.

Currently, there are medications that affect the appetite and drugs that act on the gastrointestinal tract and inhibit absorption, acting on the central nervous system to treat obesity. Drugs acting on the central nervous system include drugs such as fenfluramine and dexfenfluramine, which inhibit the 5-hydroxytryptamine (5HT) nervous system, and drugs such as ephedrine and caffeine through the noradrenergic nervous system. In recent years, drugs such as sibutramine, which simultaneously acts on serotonin and noradrenergic nervous system to inhibit obesity, are on the market. In addition, the drugs that act on the gastrointestinal tract and inhibit obesity are typically Orlystate, which has recently been approved as a treatment for obesity by inhibiting the lipase produced by the pancreas to reduce fat absorption. However, pfuramilamine has been recently banned due to side effects such as primary pulmonary hypertension or heart valve lesion. Other drugs have also been reported to cause problems such as decreased blood pressure and acidosis. There is a problem that patients can not use. Therefore, in order to find a better treatment and prevention of obesity with fewer side effects, we have searched for obesity / diabetes treatment using direct obesity and diabetic animals.

Alnus sp . ) Plants belong to the Betulaceae, and there are about 30 species in North America and South America. Alnus japonica, Alnus hirasuta, Alnus borealis koidzumi, Alnus firm sieb. et zucc, and Alnus hirsuta var. sibirica).

At present, no component related to the activity of inhibiting adipocyte differentiation has been reported in Auspidia. The present inventors have identified candidates that can be developed as therapeutic agents for obesity, diabetes, etc. by isolating a substance having an activity of inhibiting adipocyte differentiation from a tree and identifying its chemical structure.

The present invention provides a composition for inhibiting differentiation of adipocytes, which contains Ascariasis extract, fraction thereof or diarylheptanoyl compound as an active ingredient.

The present invention provides a compound of formula (1). Such compounds include, for example, Alnus ≪ / RTI > Japonica ). The compound may be, for example, obtained from the fruit, leaf or stem skin of a mulberry tree.

The present invention provides compounds of formula (2). The compound may be, for example, obtained from a mulberry tree. The compound may be, for example, obtained from the fruit, leaf or stem skin of a mulberry tree.

The present invention also relates to a method for the prevention of any one of the diseases selected from the group consisting of obesity, hyperlipidemia, dyslipidemia, arteriosclerosis, hepaticopathy, nonalcoholic fatty liver, type 2 diabetes and metabolic syndrome, Or a pharmaceutical composition for therapeutic use.

The present invention also relates to a pharmaceutical composition for the prevention and / or amelioration of any one of the symptoms selected from the group consisting of obesity, hyperlipidemia, dyslipidemia, arteriosclerosis, hepaticopenia, nonalcoholic fatty liver, type 2 diabetes and metabolic syndrome, ≪ / RTI >

The term "hyperlipidemia" refers to a state in which lipid components are excessively present in the blood because fat metabolism such as triglycerides and cholesterol is not smoothly performed. Generally, blood cholesterol is 240 mg / dL or more and triglycerides are 200 mg / dL or more. The hyperlipidemia broadly includes hypercholesterolemia, hypertriglyceridemia, hyperlipoproteinemia, and hypercholesterolemia, which occurs frequently in consultation.

The term "dyslipidemia" refers to a vascular disease caused by a decrease in total cholesterol, low density lipoprotein (LDL) cholesterol, triglyceride, free fatty acid, or high density lipoprotein (HDL) cholesterol in plasma such as hyperlipidemia and atherosclerosis do. When such anomalous lipidemia occurs, lipid deposits on the inner wall of the blood vessel, and as the inner surface of the blood vessel narrows, symptoms such as a circulatory disorder such as poor blood supply to various organs and extremities occur.

The term " arteriosclerosis "refers to a pathological condition in which cholesterol is deposited on the inner wall of the artery and the endothelium is proliferated, resulting in thickening of the blood vessels and reduction of elasticity. The arteriosclerosis includes arterial vascular diseases caused by excessive accumulation of cholesterol in blood vessel wall cells such as cerebral artery sclerosis, coronary artery sclerosis, and atherosclerosis.

The term " liver steatosis "refers to a state of abnormal accumulation of fat in hepatocytes. In particular, the term "nonaclcoric fatty liver disease" (NAFLD) refers to a fatty deposits in the liver that do not have enough alcohol consumption to cause hepatic damage and exclude clear causes such as viral hepatitis or autoimmune hepatitis .

The term " diabetes mellitus "refers to a group of diseases characterized by chronic hyperglycemia due to a deficiency of insulin action and accompanied by various characteristic metabolic abnormalities. "Type 2 diabetes" or "non-insulin dependent diabetes mellitus" as an object of treatment of the composition of the present invention is caused by insulin secretion deficiency or insulin resistance, and obesity is a key risk of type 2 diabetes or insulin resistance It is known as an argument.

The term "metabolic syndrome" means a syndrome in which a risk factor such as hyperlipidemia, glucose metabolism abnormality, obesity and hypertension mentioned above together.

In the present specification, the above-mentioned metabolic syndrome in which obesity, dyslipidemia, hyperlipidemia, atherosclerosis, fatty liver, type 2 diabetes and the above-mentioned diseases occur simultaneously is collectively referred to as "lipid metabolic disease".

The term "prophylactic " means any action that inhibits lipid metabolic disease by administration of the composition of the present invention. The terms " treatment "and" improvement "refer to all actions by which administration of the composition of the invention alleviates or alleviates a lipid metabolic disorder.

The present invention also relates to a pharmaceutical composition for treating a disease selected from the group consisting of obesity, hyperlipidemia, dyslipidemia, arteriosclerosis, hepaticopathy, nonalcoholic fatty liver, type 2 diabetes and metabolic syndrome Or a pharmaceutically acceptable salt thereof.

The pharmaceutical composition may be administered to mammals such as humans in various routes. The composition can be administered via conventional methods known in the art. For example, oral, intraarticular, intravenous, intramuscular, subcutaneous, transdermal, rectal, nasal, intraperitoneal or intracerebral administration, and the like are possible, but are not limited thereto.

The pharmaceutical composition may be formulated into conventional pharmaceutical formulations known in the art. The formulations may be formulated and administered in any formulation, including, but not limited to, oral dosage forms, injectables, suppositories, transdermal dosage forms, and expectorate dosage forms. But are not limited to, solid forms such as tablets, capsules, pills, powders, granules or suppositories, or in liquid form with solutions, suspensions, emulsions, syrups, elixirs or extensions . In addition, the composition may further comprise suitable carriers, excipients and diluents conventionally used in accordance with the formulation. Such carriers, excipients and diluents include, but are not limited to, fillers, extenders, binders, wetting agents, disintegrants, surfactants, and the like.

The present invention also relates to a pharmaceutical composition for treating a disease selected from the group consisting of obesity, hyperlipidemia, dyslipidemia, arteriosclerosis, hepaticopathy, nonalcoholic fatty liver, type 2 diabetes and metabolic syndrome There is provided a food composition for improvement.

The food composition includes all forms of functional foods, nutritional supplements, health foods and food additives. Food compositions of this type may be prepared according to conventional methods known in the art and may be formulated into conventional health functional food formulations known in the art. The composition may be prepared, for example, as a powder, a granule, a tablet, a capsule, a suspension, an emulsion, a syrup, a liquid, an extract, tea, jelly, or a drink. The composition may further comprise a pharmaceutically acceptable auxiliary additive. The composition may also contain various nutrients, vitamins, minerals (electrolytes), colorants and enhancers, pectic acid and salts thereof, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, A carbonating agent used in a carbonated beverage, and the like.

The extract can be obtained, for example, by successively extracting from an almond, leaf or stem skin extract of water, a C ₁ to C ₄ lower alcohol or a mixed solvent thereof with n-hexane, chloroform and n-butanol Chloroform fraction or n-butanol fraction. The chloroform fraction may contain a compound having a cyclic dianil heptanoid as a parent moiety. The compound having the cyclic dianil heptanoid as the parent nucleus may be, for example, a compound represented by the above formula (1) or (2).

The term "extract" includes not only extracts obtained by treating extracts of alder trees or extracts thereof, but also artificially formulated (e.g., pulverized) alcohols to be administered to animals. Also, the extract has the meaning of being used as a crude extract in the art, but broadly includes fractions obtained by further fractionating the extract and those obtained by additionally applying a purification process. The extracts include, for example, fractions obtained by passing through an ultrafiltration membrane having a constant molecular weight cut-off value or fractions isolated by various chromatographies (prepared for separation by size, charge, hydrophobicity or affinity) do. The terms "extract" and "fraction" are intended to encompass not only extracts and fractions obtained by extraction, but also the dried products obtained by drying diluents or concentrates, extracts and fractions of said extracts and fractions, And purified water.

The alder tree extract may be one obtained by extracting alder tree by a conventional extraction method using water, alcohol or a mixture thereof as a solvent and concentrating it under reduced pressure. The alcohol C ₁ to C ₄ alcohols are preferable and include those commonly used as extraction solvents in the art such as methanol, ethanol, propanol, n-propanol, isopropanol, butanol, n-butanol and the like. For example, the mulberry extract may be a methanol extract of mulberry. The extraction method includes, but is not limited to, a reflux cooling extraction method, a cold extraction extraction method, an ultrasonic extraction method, an onion extraction method, a supercritical extraction method, and an extraction method combining these methods. For example, the mushroom extract of the present invention may be prepared by repeating ultrasonic extraction 3-5 times with 2-10 kg (based on dry weight) of fruit of a mushroom in 80% methanol for 2-4 hours.

In addition to the above process, the method may further include, if necessary, concentrating and / or drying, and the concentration and / or drying may be carried out by a conventional method, for example, by concentration under reduced pressure and lyophilization or spray drying have. The alder extract used in the present invention may be in the form of a powder which is dried, for example, by concentration under reduced pressure and lyophilization.

The total extract of Duckwood obtained from the above step is suspended in distilled water, and fractions of the extract can be sequentially obtained therefrom according to the solvent polarity. For example, the fraction may be n-hexane, chloroform, and n-butanol soluble fractions obtained by successive fractionation with n-hexane, chloroform, and n-butanol. The fraction used in the composition for inhibiting lipid differentiation of the present invention may be, for example, a chloroform fraction or an n-butanol fraction.

The composition according to the present invention contains an extract of Alnus japonica, a fraction or a compound as an active ingredient, and exhibits an effect of inhibiting adipocyte differentiation. Also, it has no toxicity to cells, and is composed of natural or natural-derived components, so that it is preferable because there are no side effects on the human body. Accordingly, the composition according to the present invention is useful for the prevention, amelioration and treatment of diseases caused by excessive fat cells, such as obesity, hyperlipidemia, dyslipidemia, arteriosclerosis, hepaticopathy, nonalcoholic fatty liver, type 2 diabetes, metabolic syndrome May be used for the manufacture of a medicinal product or health functional food which can be used for the purpose.

1 shows a process for obtaining a methanol extract and a n-hexane, chloroform and n-butanol fraction thereof from a mulberry fruit.
Figure 2 shows the process of isolating the active compound from the chloroform fraction of the Ascariasis extract.
Fig. 3 is a graph showing the effect of the n -butanol Lt; RTI ID = 0.0 > fractions. ≪ / RTI >
FIG. 4A shows the structures of Compounds 1 to 6, which were identified by separation from the mulberry extract. 4B to 4G show the ¹ H-NMR spectrum and the ¹³ C-NMR spectrum of the compounds 1 to 6, respectively.
FIG. 5 shows the results of confirming the activity of inhibiting adipocyte differentiation by treating the 3T3-L1 cell line with the extracts of Oriental fruit, leaf and stem skin.
6A is a graph showing the activity of inhibiting adipocyte differentiation by treating methanol extract (T), n-hexane fraction (H), chloroform fraction (C) and n-butanol fraction (B) It shows the result of confirmation. FIG. 6B is a microphotograph showing the activity of inhibiting adipocyte differentiation by treating methanol extract of Oriole fruit with various concentrations of 3T3-L1 cell line by oil red staining.
FIG. 7 shows the results of confirming the activity of inhibiting adipocyte differentiation by treating the 3-1 / 3-L1 cell line with the compounds 1 to 6 derived from Oriole.
FIG. 8 shows the results of real-time PCR analysis of the effect of Compound 2 on gene expression involved in adipocyte formation of 3T3-L1 cell lines.

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

Example  1. Extracts, fruit extracts, Fraction  Produce

The fruit, leaf, and stem skin (10 g dry weight) of the three herbs of the duckwood were crushed and sonicated with 80% MeOH for 3 hours each. The extract was collected, filtered, and concentrated under reduced pressure to obtain a total methanol extract.

Duckwood (dry weight 1.5 kg) was pulverized and ultrasonically extracted 5 times for 3 hours with addition of 80% MeOH. The extract was collected, filtered, and concentrated under reduced pressure to obtain about 419.1 g of a total methanol extract. After the total methanol extract was suspended in distilled water, n -hexane fraction (29.5 g), CHCl ₃ fraction (47.6 g) and n- BuOH fraction (251.6 g) were obtained according to the polarity of the solvent 1).

Example  2. Isolation and Structure Identification of Active Compounds

2-1. Duck CHCl3 ₃ From the fraction  Separation of compounds

Alder silica gel column targeting CHCl ₃ fraction chromatography of the fruit of our _{(CHCl 3: MeOH: Water =} 100: 4: 1 → 100% MeOH) sequentially into 7 small fraction (C1 to C7) according to the polarity to conduct . Among them, fraction C4 having strong adipocyte differentiation inhibiting activity was subjected to normal phase chromatography or recrystallization to finally obtain four pure compounds (compounds 1, 2, 3 and 4) (FIG. 2). The result of identifying the structure is shown in FIG. 4A.

2-2. Duck n - BuOH ₃ From the fraction  Separation of compounds

The n- BuOH ₃ fraction of nuts was subjected to HP-20 column chromatography using a mixed solvent of MeOH and H ₂ O (0% → 100% MeOH) to obtain six small fractions (B1 to B6) . The third and fourth small fractions were separated and purified by HPLC to obtain two pure compounds (compounds 5 and 6) (FIG. 3), and the structure thereof was identified and shown in FIG. 4A.

2-3. Structure identification of compounds

The physical properties, molecular weight, mass spectrometry, ¹ H-NMR spectrum and ¹³ C-NMR spectrum of the compounds 1 to 6 were measured, and their physicochemical properties and chemical structures were as follows. ¹ H-NMR spectrum and ¹³ C-NMR spectrum of each compound are shown in FIGS. 4B to 4G. Of the six compounds in total, compounds 1 and 2 are the first reported substances to be isolated from nature.

Compound 1: (5 R) -5,5'- dihydroxy-4'-methoxy-3 ', 4''- oxo-1,7-diphenyl-1,3-heptene [(5 R) -5,5'-dihydroxy-4'-methoxy-3 ', 4'-oxo-1,7-diphenyl-1,3-diheptene]

Compound 1 was estimated to have a molecular formula of C ₂₀ H ₂₀ O ₄ based on a peak at 324.1358 [M] ⁺ and a ¹³ C-NMR spectrum in HRFABMS (m / z). Strong absorption was observed at the wavelength of 287 nm of the UV spectrum, and it was presumed that this compound was a biphenyl ether type cyclic diaryl heptanoid. ¹ H NMR spectrum at _{δ H 6.36 (1H, d,} J = 1.9 Hz, H-6 '), 5.49 (1H, d, J = 1.9 Hz, H-2' 1 of 1,3,4 through), It was confirmed with a 5-substituted aromatic _{ring, δ H 7.32 (1H, dd} , J = 8.2, 2.1 Hz, H-2 ''), 7.25 (1H, dd, J = 8.2, 2.1 Hz, H-5 ), 7.14 (1H, dd, J = 8.2, 2.5 Hz, H-3 ''), 6.98 (1H, dd, J = 8.2, 2.5 Hz, H- Substituted aromatic rings. In _{addition, δ H 6.10 (1H, d} , J = 11.4 Hz, H-1), 5.91 (1H, dd, J = 11.4, 9.2 Hz, H-2), 5.64 (1H, m, H-3), 5.63 (1H, m, H-4), and two conjugated double bonds were confirmed through δ _H 4.11 (3H, s) and δ _H 4.09 (1H, And a secondary hydroxy moiety. ^{The 13} C NMR spectrum supported this, and the position of each substituent was confirmed by 2D NMR spectrum. In addition, the stereostructure of the hydroxyl group at the 5-position was confirmed as the R- type by the MTPA method. Above results in the compound 1 (5 R) -5,5'- dihydroxy-4'-methoxy-3 ', 4''- oxo-1,7-diphenyl-1,3-determined heptene Respectively. This is the first reported material to be isolated from nature.

Compound 2 : 4-hydroxy-alnus-3,5-dione [

Carbonyl group (1700 cm ^-1 ), and aromatic rings (1588 and 1430 cm ^-1 ) were identified in the IR spectrum. ¹ H NMR δ _H 7.13 in the spectrum (1H, d, J = 2.0 Hz, H-2 '), 6.98 (1H, dd, J = 8.1, 2.0 Hz, H-6'), 6.83 (1H, d, J = 8.1 Hz, H-5 ' ) to the one 1, 3,4-position was confirmed by a substituted aromatic _{ring, δ H 2.77 (2H, m} , H-1b, H-7b) , and 2.46 (2H, m, H-2b, H-6b). One hydroxy-bearing methane was identified through δ _H 5.88 (1H, s, H-4) and the position of each substituent was confirmed via 2D NMR spectrum. Based on the above data, Compound 2 was estimated to be an arylbutanoid series. Based on the peak of 326.1152 [M] ⁺ in HRFABMS (high resolution fast-atom bombardment mass spectroscopy) (m / z), the molecular formula was C ₂₀ H ₂₀ O ₄ It was confirmed that the compound was a cyclic diaryl heptanoid which was symmetric with the compound. As a result, compound 2 was determined to be 4-hydroxy-alk-3, 5-dione, which is the first report to be reported in nature.

Compound 3 : dihydroalnusone < RTI ID = 0.0 >

Compound 3 was estimated to be a compound of molecular formula C ₁₉ H ₂₀ O ₃ based on the peak of m / z 295.23 [MH] ^- in electrospray ionisation mass spectrometry (ESIMS). The hydroxyl group (3341 cm ^-1 ), the carbonyl group (1702 cm ^-1 ), and the aromatic rings (1608 and 1430 cm ^-1 ) were identified through the IR spectrum. In ¹ H and ¹³ C NMR spectrum _{[δ H 6.98 (1H, dd} , J = 8.2, 2.2 Hz, H-6 '), 6.78 (1H, d, J = 8.2 Hz, H-5'), and 6.58 ( (1H, dd, J = 2.2 Hz, H-2 ') and [delta _H 7.13 H-6 ") and 6.83 (1H, d, J = 8.2 Hz, H-5"). δ _H 2.86, 2.83, 2.71, 2.67, 1.83, and 1.63 confirmed 6 methylenes and δ _C 212.9 confirmed the carbonyl group. As a result, Compound 3 was determined to be dihydroalanosone (Nomura et al., 1981, Phytochemistry, 20: 1097-1104).

Compound 4 : alnusol

Compound 4 estimated the formula as C ₁₉ H ₂₀ O ₄ through a peak at m / z 311.10 [MH] ^- in ESIMS. The IR spectrum showed an absorption line corresponding to the hydroxyl group (3335 cm ^-1 ) and the carbonyl group (1735 cm ^-1 ). The ¹ H and ¹³ C NMR spectra of Compound 4 were similar to the spectra of Compound 3 except for the substitution of the hydroxyl group at C-5 [隆_H 3.61 (1H, m, H-5), 隆_C 68.0]. The absolute arrangement of hydroxyl groups at C-5 was confirmed by the modified Mosher's method in the S-configuration (Hoye et al, 2007, Nat Protoc, 2: 2451-2458). As a result, Compound 4 was determined with an aniline (Nomura et al., 1981).

Compound 5 : alnusone < RTI ID = 0.0 >

Compound 5 was estimated to be C ₁₉ H ₁₈ O ₃ via ESIMS (negative mode, 293.15 [MH] ^- ). The IR spectrum showed an absorption line corresponding to the hydroxyl group (3349 cm ^-1 ) and the carbonyl group (1735 cm ^-1 ). The ¹ H and ¹³ C NMR spectra of the compound 5 showed that the trans arrangement at < RTI ID = 0.0 ># _H 6.93 (1 H, dt, J = 15.5, 7.6 Hz, H- Lt; RTI ID = 0.0 > 3 < / RTI > As a result, Compound 5 was determined to be Alunosin (Nomura et al., 1981).

Compound 6 : betulatetraol < RTI ID = 0.0 >

Compound 6 confirmed the presence of a hydroxyl group through an absorption line at 3373 cm ^-1 in the IR spectrum, but no absorption line corresponding to a carbonyl group was observed. ¹ H in NMR spectrum _{[δ H 7.00 (1H, d} , J = 8.0 Hz, H-5 '), 6.79 (1H, d, J = 8.0 Hz, H-6'), and 6.68 (1H, br s, H-2 ')], and the methylene was identified through δ _H 2.79. Two adjacent hydroxy-containing methanes were identified through delta _H 4.15 and 4.40, and carbonyl groups were identified via delta _C 193.7. Ten carbons were identified through ¹³ C NMR spectra. Based on the above data, compound 6 was estimated to be an arylbutanoid series, but ESIMS was presumed to be a compound whose molecular formula is C ₁₉ H ₂₂ O ₆ based on the peak at m / z 345.15 [MH] ^- . As a result, Compound 6 was determined as betulatetraol (Lee et al., 1992, Arch Pharm Res, 15: 211-214).

Reference example  1. Materials and reagents

The mushroom used in this study was collected and dried in October 2010 at the estimation site in the southern part of Seoul National University.

The 3T3-L1 cell line used for the measurement of differentiation activity of lipocytic cells was purchased from the American Type Culture Collection (Manassas, VA, USA). The 3T3-L1 cell line 10% BCS, 100 IU / mL penicillin, 100 mg / ml streptomycin, Dulbecco's modified Eagle's medium (DMEM) to a to the air (95%) and CO ₂ (5 at 37 ℃ incubator culture medium containing %) Mixed gas was continuously supplied and cultured. When the cells were confluent at 80-90%, trypsin-EDTA was treated with subculture. The penicillin / streptomycin and trypsin required for cell culture were purchased from Sigma (St. Louis, USA), DMEM and Bovine calf serum were purchased from Hyclone (Franklin, USA).

Reference example  2. Statistical processing

Concentration-by-concentration experiments for each test material were performed over three wells, and the results were expressed as mean ± standard deviation of three measurements. Statistical significance was assessed by one-way ANOVA test and the P value was considered to be statistically significant when the P value was less than 5% (p <0.05).

Experimental Example  1: Evaluation of inhibitory activity of adipocyte formation of active substance

1-1. 3 T3 - L1  Culture of cell line

3T3-L1 mouse mouse embryo fibroblast was cultured by the following procedure (Lin J. et al., Green Tea Polyphenol Epigallocatechin Gallate Inhibits Adipogenesis and Induces Apoptosis in 3T3-L1 Adipocytes, Obesity Research, 13 (6), pp. 982-990, 2005).

The 3T3-L1 cell line 10% BCS, 100 IU / mL penicillin, 100 mg / ml streptomycin, Dulbecco's modified Eagle's medium (DMEM) to a to the air (95%) and CO ₂ at 37 ℃ incubator culture medium containing (5%) was continuously supplied and cultured. When the cells were confluent at 80-90%, trypsin-EDTA was treated with subculture.

1-2. 3 T3 - L1  Adipocyte differentiation of cell line

3T3-L1 cells were seeded at a concentration of ⁵ x 10 ⁴ cells / mL in a 24-well culture plate and then cultured for another two days in a 100% confluent state (day 0). The differentiation inducers (0.5 mM IBMX, 1 μM dexamethasone, (10% FBS, 100 IU / mL penicillin, 100 μg / mL streptomycin) containing 10 μg / mL insulin) were treated with test substances diluted to a concentration of 10 and 50 μg / mL. Two days after the induction of differentiation (day 2), the cells were exchanged with DMEM culture medium (10% FBS, 100 IU / mL penicillin, 100 μg / mL streptomycin) containing 1 μg / mL insulin for differentiation (day 4) The culture medium was replaced every two days with DMEM culture medium (10% FBS, 100 IU / mL penicillin, 100 ㎍ / mL streptomycin) and cultured for another 4 days (day 8) to test mature adipocytes The lipid precursor inhibitory activity of the substance was measured. All the test materials were diluted with the above concentrations (10 and 50 ㎍ / mL) and treated with 5 ㎕ of each culture medium until the 4th day after the induction of differentiation (day 4).

1-3. oil Red  Staining method

3T3-L1 cell line was used as a search system to investigate the effects of the three herbs of fruit, leaf, stem skin extract, (Jeon T, et al., Red yeast rice extracts suppressed adipogenesis by downregulating adipogenic transcription factors and gene expression in 3T3-L1, Life Sci, 12:75 (26), pp. 3195-3203, 2004).

The differentiation potential of 3T3-L1 cells was determined by staining with an oil red reagent. The cultured cells were washed twice with PBS buffer, fixed with 10% formalin for 1 hour and washed with distilled water. 200 쨉 l per well was treated with an oil red oozing solution (isopropyl alcohol: water = 3: 2, 0.6% oil red o), and stained at 37 캜 for 15 minutes. The staining solution was removed, washed twice with distilled water, and then treated with Nonidet P-40 (4% Nonidet P-40 in isopropyl alcohol) for quantification. Absorbance was measured using an ELISA spectrophotometer at 544 nm after extracting the stained fat using a rotary shaker for 15 minutes. The fat production rate (%) of the test group as compared to the control group was calculated according to the following formula.

Fat Production Ratio to Control (%)

= [A _{544 nm} (sample) -A _{544 nm} (ctrl)] / [A _{544 nm} (diff. Ctrl) -A _{544 nm} (ctrl)] x 100

- sample: test group

- ctrl: negative control group (differentiation inducer)

- diff. ctrl: positive control group (differentiation inducer administered group)

1-4. Measurement of inhibitory activity of adipocyte extract against adipocyte differentiation

When methanol extracts of mulberry, leaf and stem shells were treated at 10, 50 and 100 ㎍ / mL, respectively, all three extracts were reddish - (Fig. 5).

1-5. Duck Fruit Extract and Fraction  Measurement of inhibitory activity of adipocyte differentiation

The methanol extract, chloroform fraction, and n - butanol fraction of mulberry fruit significantly decreased the red - stained area compared with the positive control (differentiation - inducing group). The extracts and fractions inhibited 25.6%, 22.3% and 68.6%, respectively, of differentiation from 3T3-L1 adipose precursor cells into adipocytes at a concentration of 50 μg / mL (FIG. 6).

1-6. Measurement of inhibitory activity of adipocyte-derived compounds against adipocyte differentiation

As shown in FIG. 7, treatment of compounds 1 to 6 at a concentration of 100 μM during the differentiation of 3T3-L1 adipocyte progenitor cells showed that compounds 2 and 3 inhibited cell differentiation and adipogenesis as compared with the positive control Respectively. In particular, Compound 2 inhibited cell differentiation up to 88.4%, indicating that it has a very high activity. Furthermore, no cell necrosis or the like was observed, and it was confirmed that all of the above active compounds did not show toxicity to cells at an effective concentration.

Experimental Example  2. Quantification PCR  analysis

In order to examine the 3T3-L1 lipoprotein cell differentiation inhibition mechanism and downstream molecular mechanism of Compound 2, gene expression involved in adipocyte formation was evaluated using real-time PCR assay.

RNA extraction was performed using Qiagen's RNease Plus Kit from 3T3-L1 cell line and 1 μg of RNA was synthesized with cDNA using QuantiTech Reverse Transcription Kit (QIAGEN Korea Ltd., Seoul, Korea). cDNA was mixed with QuantiFast SYBR Green PCR master mix (QIAGEN Korea Ltd., Seoul, Korea) and primers. Real-time PCR was repeated 3 times per sample, and 20 μl of reaction solution per well was used. The primers used for PCR quantification and their information are summarized in Table 1. Real-time measurements of fluorescence signals generated during thermal cycling and specific amplification products were performed on an Applied Biosystems 7300 Real-Time PCR System (Life Technologies Corporation, Carlsbad, Calif., USA). The PCR reaction conditions were 40 cycles of 95 ° C for 20 seconds, 60 ° C for 20 seconds, and 72 ° C for 20 seconds, and the reaction was terminated by adding a polymerization reaction at 72 ° C for 5 minutes. After the reaction, the mean value of comparative Ct quantification was analyzed on an amplification plot and GAPDH was used as an internal gene.

name gene Gene description GenBank Cebpa C / EBPα CCAAT / enhancer-binding protein alpha NM_007678 Fabp4 aP2 adipocyte fatty acid binding protein 4 NM_024406 Fasn FAS fatty acid synthase NM_007988 Gapdh GAPDH glyceraldehyde 3-phosphate dehydrogenase NM_008084 Lpl LPL lipoprotein lipase NM_008509 Lep Leptin leptin NM_008493 Pparg PPARγ peroxisome proliferator-activated receptors gamma NM_011146 Scd1 SCD-1 stearoyl coenzyme A desaturase 1 NM_009127 Srebf1 SREBP1 sterol regulatory element binding protein 1 NM_011480

Compound 2 inhibited the gene expression of PPARγ and C / EBPα, which are major transcription factors regulating adipocyte formation, to a concentration-dependent (25 μM to 100 μM) and suppressed the expression of SREBP1, but the expression of SCD-1 and FAS was 100 mu] M, the compound 2 was confirmed to have an effect on the activity of inhibiting fatty acid synthesis (Fig. 8).

Claims (12)

A compound of the following formula 1:

The compound according to claim 1, wherein the compound is Alnus &Lt; / RTI &gt; Japonica ). &Lt; / RTI &gt;

4. The compound according to claim 3, wherein said compound is obtained from a mulberry tree. A group of the group consisting of obesity, hyperlipidemia, dyslipidemia, arteriosclerosis, hepatic hypertrophy, nonalcoholic fatty liver, type 2 diabetes and metabolic syndrome containing the compound of formula 1 of claim 1 or the compound of formula 2 of claim 3 as an active ingredient A pharmaceutical composition for the prevention or treatment of any one of the diseases selected. A group of the group consisting of obesity, hyperlipidemia, dyslipidemia, arteriosclerosis, hepatic hypertrophy, nonalcoholic fatty liver, type 2 diabetes and metabolic syndrome containing the compound of formula 1 of claim 1 or the compound of formula 2 of claim 3 as an active ingredient A food composition for improving any one of the symptoms selected. A pharmaceutical composition for preventing or treating obesity, hyperlipidemia, dyslipidemia, arteriosclerosis, hepatic hypertrophy, nonalcoholic fatty liver disease, type 2 diabetes and metabolic syndrome, Gt; [Claim 8] The method according to claim 7, wherein the extract is obtained by sequentially extracting water, n-hexane, chloroform, and n-butanol from a nut, a leaf, or a stem skin and extracting the extract with a C ₁ to C ₄ lower alcohol or a mixed solvent thereof Wherein the chloroform fraction or the n-butanol fraction is the obtained chloroform fraction or the n-butanol fraction. The pharmaceutical composition according to claim 8, wherein the chloroform fraction contains the compound of formula 1 of claim 1 or the compound of formula 2 of claim 3.  A food composition for the improvement of any one of the diseases selected from the group consisting of obesity, hyperlipidemia, dyslipidemia, arteriosclerosis, diarrhea, nonalcoholic fatty liver, type 2 diabetes and metabolic syndrome, . [Claim 11] The method according to claim 10, wherein the extract is obtained by extracting an extract from mulberry, leaf or stem skin with water, a C ₁ to C ₄ lower alcohol or a mixed solvent thereof in the order of n-hexane, chloroform and n-butanol A chloroform fraction or an n-butanol fraction. The food composition according to claim 11, wherein the chloroform fraction contains the compound of formula 1 of claim 1 or the compound of formula 2 of claim 3.

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