KR20110050397A - A pharmaceutical composition comprising lindera erythrocarpa extract, fractions thereof, or compounds isolated from thereform - Google Patents

Abstract

PURPOSE: A composition containing Lindera erythrocarpa extract, fraction, or compounds isolated from the extract and fraction is provided to prevent or treat cancer, arteriosclerosis, rheumatoid arthritis, multiple sclerosis, Alzheimer's disease, or obesity. CONSTITUTION: A pharmaceutical composition for preventing or treating cancer, arteriosclerosis, rheumatoid arthritis, multiple sclerosis, Alzheimer's disease, or obesity contains Lindera erythrocarpa extract, fraction or compounds isolated form the extract or fraction as an active ingredient. The Lindera erythrocarpa extract is isolated using water, C1-C4 alcohol, or mixture solvent thereof.

Description

A pharmaceutical composition containing an extract of a tree, a fraction thereof, or a compound isolated therefrom as an active ingredient.

The present invention relates to a pharmaceutical composition for the prevention or treatment of cancer, atherosclerosis, rheumatoid arthritis, multiple sclerosis, Alzheimer's or obesity, containing as an active ingredient, an extract of a tree, a fraction thereof, or a compound separated therefrom.

Research on physiologically active substances from natural products is being actively conducted, and plant resources are widely used as treatment and prevention or health supplements for diseases.

Lindera erythrocarpa ) is a plant of the family Lauraceae, which is distributed in 1,500 species in 45 genera and is known to grow 6 genera and 12 species in Korea. A birch tree grows in light yellow flowers from April to May and bears about 8 mm of red fruit in September. It is a deciduous tree with a height of 5 m that grows in the southern regions of Korea, Japan and China. The dried fruit has an unusual aroma and bitter taste, and is used in Japan as a pain medicine for gastrointestinal drugs and neuralgia. In addition, it has been reported that the extracts of the leaves and fruits of the birch tree have antifungal activity, and the cyclopentadione compounds isolated from the birch tree have anticancer and anti-inflammatory effects (Wang et al., 2008, Phytotherpy Research 22: 213-216). Was reported.

PPAR-γ (Peroxisome proliferation activated receptor-γ) is one of the Nuclear homone receptor family. PPAR family (PPAR family) is composed of α, β / δ, and γ, PPAR-α is expressed very stably in liver tissue and has an anti-inflammatory function. Other major functions include fatty acid intake and oxidation, macrophage lipid homeostasis, and vascular function. PPAR-γ, on the other hand, is known to be an essential factor in the uptake and storage of fatty acids, regulation of inflammation, gene regulation involved in sugar homeostasis, and lipid metabolism. PPAR-γ regulates cholesterol choresterol homeostasis and regulates adipocyte differentiation and insulin. It has been reported to have important functions such as activity regulation, cancer cell promotion, cardiovascular disease, arteriosclerosis factor and inflammatory factor regulation (Guido Eibl 2008; Michalik and Wahli 2008; Drew et al. 2008; Choi and kim 2006). PPAR-γ can be used in almost all tissues, such as skeletal muscle, liver, breast, prostate, colon, type 2 alveolar pneumocytes, etc. For example, mononuclear leukocytes (Monocyte), non-lymphocytes (B-lymphocyte), endothelial cells (endothelial cells), etc. are characterized by less expression.

PPAR-γ mRNA isoforms (γ1, γ2, γ3) are distinguished by mRNA selective splicing. PPAR is a ligand-dependent transcription factor and forms a heterodimer with RXR, a retinoid acid receptor, to transcribe mRNA. In addition, factors that interact with PPAR-γ in adipocyte differentiation include C / EBP and ADD-1 / SREBP-1, which are fat cell specific genes such as aP2, PEPCK, leptin, ACS and lipoprotein lipase. (Wang et al., 2000; Wright et al., 2000). According to a recent report by Brark et al. (1999), the PPAR-γ gene is an essential gene for organ formation during development through the production of PPAR-γ knockout mice. Mice lacking this gene were observed to die in the gastation stage, 10 days of gestation. In particular, PPAR-γ plays an important role in the regulation of adipocyte differentiation, glucose homeostasis and metabolism. Adipocyte formation is accelerated by PPAR-γ activity when differentiating from adipose cells to adiocytes. Atherosclerosis is a long-term chronic disease that is characterized by the accumulation of lipids and fibrous connective tissues in the arteries, accompanied by an extreme inflammatory response, and in particular, PPAR-γ has been reported to be closely involved. Duval et al., 2002; Fruchart JC 2001).

As functions for PPAR-γ were revealed, PPAR-γ agonists (Troglitazone, Rosiglitazone, JTT501, GW-0295, and GW-7846) were synthesized, and these compounds are generally anti-diabetic, anticancer (anti-carcinogenic), anti-inflammatory and anti-atherosclerotic activity (David and Wry, 2003). The function of agonists in the liver not only reduces fibrosis and hepatitis, but also inhibits liver damage caused by compounds such as dimethylnitrosamine and carbon tetrachloride (CCl ₄ ), and hepatic extracellular. matrix) inhibition of accumulation and enhanced hepatic stellate cell activity (Houseknecht et al., 2002).

Recently, it has been reported that PPAR-γ is characteristically expressed in some cancers and cancer cell lines. An early report of PPAR-γ in human liposarcoma cells in Tontonoz et al. (1997) revealed that colon cancer (colonic cancer, DuBois et al. 1998), breast cancer (breast cencer, Mueller et al. 1998), prostate Research has been conducted in relation to cancer (prostate cancer, Kubota 1998), human malignances (Ikezoe et al. 2001). Therefore, attempts have been made to use PPAR agonists as anticancer agents. In fact, treatment with TZD, one of the qualities in liposarcoma and prostate cancer, has been shown to have antitumor effects (Demtri et al., 1999). Mueller et al., 2000). Fehlberg et al. (2002) reported the effect of inhibiting cancer cell proliferation by treating BADGE, one of agonists, with lymphoma and colon cancer. In addition, GW9662, one of the PPAR antagonists, was used to treat three different types of breast cancer cells (Breast cancer; ER +, ER-, and p53-null), which showed higher growth inhibitory effects than PPAR catalysts. Reported (Seargent et al., 2004).

Recently, PPAR-γ is highly expressed in fibroblast synovial cells of patients with rheumatoid arthritis, and it has been reported that related inflammatory factors are suppressed by treating PPAR ligand (Kawahito et al., 2000; Yamasaki et al. 2002). Multiple Sclerosis is a disease that invades the monocytes, T cells, and demyelination of the central nervous system and refers to diseases with inflammation (Drew et al., 2008), especially PPAR-. γ inhibits the major anti-inflammatory factors STAT-3, NF-kB, Ap-1, etc., and has been reported to inhibit the activity of microglia, macrophage, and monocytes (Minghetti et al., 2002). . PPAR-γ agonists were also treated to reduce the expression of bcl-2, one of the anti-apoptosis molecules of activated T cells in multiple sclerosis patients.

In this regard, the inventor of the present invention has a tree ( Lindera) on Jeju Island. erythrocarpa ), extracts, solvent fractions, and compounds obtained from these fractions were treated on 3T3-L1 cells to confirm the activity of adipose cell biosynthesis and to characterize PPAR-γ agonists, thereby preventing cancer, arteriosclerosis, and rheumatism. It has been confirmed that the present invention can be used as a treatment for arthritis, multiple sclerosis, Alzheimer's disease, or obesity.

The present invention, linera ( Lindera To provide a pharmaceutical composition for the prevention or treatment of cancer, arteriosclerosis, rheumatoid arthritis, multiple sclerosis, Alzheimer's or obesity containing erythrocarpa ) extract, fractions thereof, or compounds isolated therefrom as an active ingredient.

In addition, the present invention provides a method for preparing the extract of the tree, fractions thereof, or compounds isolated therefrom.

In order to achieve the above object, the present invention is a cancer, arteriosclerosis, rheumatoid arthritis, multiple sclerosis, multiple sclerosis, Alzheimer's disease or obesity, containing as an active ingredient extracts of Lindera erythrocarpa , fractions thereof, or compounds isolated therefrom Provided is a prophylactic or therapeutic pharmaceutical composition.

The term "pinewood extract" used in the present invention means that the root, stem, peeling or leaf of the birch tree is extracted with a solvent or the like. The wood is preferably used after washing with running water to remove impurities, and dried, and is preferably ground with a grinder or the like to prepare an extract.

The solvent used may be water, C ₁ ~ C ₄ alcohol, or a mixed solvent thereof, it is preferable to use methanol or ethanol. When ethanol is used, the concentration of ethanol is preferably 65 to 70%.

The term "fraction" as used in the present invention means fractionated birch extract. Suspension of the birch tree may be suspended in distilled water and the like, and then fractionated by hexane, methylene chloride, ethyl acetate, butanol, water, or a combination thereof using a separatory funnel.

The term "compound" used in the present invention means a compound obtained from the birch tree according to the present invention, the chemical structure is represented by the following formula (1) -6.

As used herein, the term "cancer" is a cancer that can be prevented or treated by a PPAR-γ antagonist, gastric cancer (Gastric carcinoma (MGC803)), breast carcinoma (breast carcinomal), pancreatic cancer, lymphoma (lymphoma) cell, liposarcoma, prostate cancer, malignances, colon carcinogenesis, lung carcinogenesis and liver carcinogenesis.

In addition, the present invention comprises the steps of crushing after washing and drying the birch; And extracting the crushed lumber tree with a solvent selected from water, C ₁ -C ₄ alcohol or a mixed solvent thereof to obtain a lumber tree extract.

In order to remove foreign matters and the like of the tree, it is preferable to wash in running water and then dry to remove moisture, and to grind with a grinder or the like to facilitate extraction. In order to prepare the extract, the extract can be obtained from a lumber tree pulverized with water, C ₁ -C ₄ alcohol or a mixed solvent thereof. In order to remove foreign substances and the like, it is preferable to filter, and it is preferable to repeat one to three times to extract and filter the filtered extract again.

In addition, the present invention comprises the steps of crushing after washing and drying the birch; Extracting the crushed lumber tree with a solvent selected from water, C ₁ -C ₄ alcohol or a mixed solvent thereof to obtain a lumber tree extract; And fractionating the non-tree extract into one or more solutions selected from the group consisting of hexane, methylene chloride, ethyl acetate, butanol, and water.

Fractions for each solvent can be obtained when fractionation is performed from the extract of the tree to one or more solutions selected from the group consisting of hexane, methylene chloride, ethyl acetate, butanol, and water. The fractionation step is preferably fractionated sequentially for each solvent.

In addition, the present invention comprises the steps of crushing after washing and drying the birch; Extracting the crushed lumber tree with a solvent selected from water, C ₁ -C ₄ alcohol or a mixed solvent thereof to obtain a lumber tree extract; Fractionating the non-tree extract with methylene chloride to obtain a methylene chloride fraction of the non-tree extract; And performing a silica gel chromatography on the fractions to obtain a cyclopentadione-based compound.

The cyclopentadione-based compound includes the compound of Formulas 1 to 6.

The present invention is a tree ( Linera) erythrocarpa ) extract, a fraction thereof, or a compound isolated therefrom, as a pharmaceutical composition containing the active ingredient and a method of preparing the same, by providing a PPAR-γ antagonist cancer, arteriosclerosis, rheumatoid arthritis, multiple sclerosis, Alzheimer's or obesity It is possible to provide a pharmaceutical composition effective for the prevention or treatment of the.

Figure 1, birch extract (A), ethyl acetate (EtOAc) fraction (B), methylene chloride (CH ₂ Cl ₂ ) fraction (C), hexane (hexane) fraction (D), butanol (BuOH) fraction (E ), And cell (H ₂ O) fraction (F) cell viability and toxicity measurements for each.
Figure 2 shows the results of Oil Red O staining after 8 days of differentiation induction for each of the birch extract (A), ethyl acetate (EtOAc) fraction (B), hexane (hexane) fraction (C).
FIG. 3 shows Oil Red O staining results after 8 days of differentiation induction for butanol (BuOH) fraction (D), water (H ₂ O) fraction (E), and methylene chloride (CH ₂ Cl ₂ ) fraction (F). It is shown.
Figure 4 is a birch extract (A), ethyl acetate (EtOAc) fraction (B), methylene chloride (CH ₂ Cl ₂ ) fraction (C), hexane (hexane) fraction (D), butanol (BuOH) fraction (E ), The result of suppressing the adipocyte differentiation for each of the water (H ₂ O) fraction (F).
Figure 5 shows the cell viability results for compound 1.
Figure 6 shows the Oil Red O staining results (A) and adipocyte differentiation inhibition results (B) for Compound 1.
Figure 7 shows the cell viability results for compound 2.
Figure 8 shows the Oil Red O staining results (A) and adipocyte differentiation inhibition results (B) for the compound 2.
9 shows the cell viability results of Compound 3 (A) and the cell survival results of Compound 4 (B).
10 shows Oil Red O staining results (A) and adipocyte differentiation inhibition results (B; results of Compound 3 on the left and Compound 4 on the right) for Compounds 3 and 4.
11 shows the cell viability results for Compound 5 (A) and the cell survival results for Compound 6 (B).
FIG. 12 shows Oil Red O staining results (A) and adipocyte differentiation inhibition results (B; compound 5 on the left and Compound 6 on the right) for Compounds 5 and 6. FIG.
Figure 13 shows the extent to which the expression of PPAR-γ and C / EBP-α according to the treatment of compound 2 is inhibited.
14 is a result of observing whether or not adipogenesis is inhibited by inhibiting triglyceride formation at the early stage of differentiation-induced treatment of Compound 2. FIG.
FIG. 15 is a graph showing the results of observing whether adipogenesis is inhibited by inhibiting triglyceride formation at the early stage of differentiation induction following the treatment of Compound 2. FIG.
FIG. 16A shows the results of inhibiting adipocyte differentiation according to rogiglitazone 5 uM treatment and simultaneous treatment of rosiglitazone 5 uM and compound 2 50 uM, and FIG. Figure 16C shows the result of inhibiting adipocyte differentiation following treatment with Compound 2 50 uM and rosiglitazone concentration.
FIG. 17A shows the result of adipocyte formation inhibition according to the concentration-specific treatment of rosiglitazone 5 uM and compound 2, and FIG. 17B shows the result of adipocyte formation inhibition following the treatment of 50 μM and rosiglitazone concentrations of Compound 2.
FIG. 18 shows the effect of compound 4 on cell viability in each cell. FIG. 18A shows cell viability in RA cells. FIG. 18B shows cell viability in THP-1 cells. FIG. 18C shows morphological changes according to the concentration of RA cells. Respectively.
FIG. 19 is a graph showing apoptosis inducing effect in each cell of Compound 4. FIG. 19A is an analysis of cell induction in RA cells, and FIG. 19B is an analysis of apoptosis in THP-1 cells.
FIG. 20 is a photograph showing the effect of expression pattern of PPAR-r on Compound 4 in RA cells (FIG. 20A) and THP-1 cells (FIG. 20B). FIG.
21 is a graph showing the antioxidant activity of Compound 2 and Compound 4 on ROS in neurons.
22 is a graph showing the weight gain rate of rats fed HFD with LFD, HFD, and LE extract (Values are mean ± SE ## p <0.01 compared with vehicle-treated LFD rat; * p <0.05 compared with vehicle-treated HFD rat).
FIG. 23 is a graph showing the weight of epididymal WAT in rats fed HFD with LFD, HFD, and LE extracts (#p <0.05 compared with vehicle-treated LFD control rat; * p <0.05 compared with vehicle) -treated HFD control rat)
FIG. 24 is a photograph showing the effect of LE extract associated with fat change in obese rat liver tissue induced by high-fat diet. Representative images are hematoxylin and eosin of vehicle treated LFD control rats (A), vehicle treated HFD control rats (B), LE100 treated HFD rats (C), LE250 treated HFD rats (D). Fat change in sections of the liver stained with (eosin). Scale bar is 100 μm and CV represents central vein.

Hereinafter, preferred embodiments of the present invention will be presented to assist in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the examples.

Example  1: Manufacture of extracts of leaves and peels of birch trees

Lindera which is a terrestrial plant sample used in this embodiment erythrocarpa ) was used by Jeju Hi-Tech Industrial Development Institute Extract Bank, and the solvent used for sample extraction was from Merk Co.

First, the leaves of the non-trees were washed in running water, and then dried at 40 ° C. for hot air for 3 days, and then pulverized with a grinder. 200 g of the dry powder sample was immersed in 70% ethanol and extracted by stirring for 24 hours at room temperature for 3 days, the leachate was filtered through a filter. After repeating the leaching and filtration three times, the filtrate was concentrated under reduced pressure to give 60 g of 70% ethanol extract.

On the other hand, the peeling of the birch tree was also pulverized after drying in the same manner as above, and extracted by the same conditions and methods using 560 g of a dry powder sample to obtain 68.2 g of a 70% ethanol extract.

Example  2: Solvent from the extracts of the birch leaves and the peels Fraction  Produce

Of the extract obtained in Example 1 (70% ethanol extract), 42 g of the birch (leaf) extract and 68.2 g of the birch (peel) extract were each suspended in distilled water, and then n-hexane ( n-hexane), methylene chloride (CH ₂ Cl ₂ ), ethyl acetate (EtOAc), n-butanol (n-BuOH) fractions were separated, filtered, and concentrated under reduced pressure to give each solvent fractions as shown in Table 1 below. Obtained.

Leaf tree extract Birch Tree (Exfoliation) Extract Tree Leaf Extract 42.0 g Birch Tree (Exfoliation) Extract 68.2 g n-hexane fraction 8.56 g n-hexane fraction 5.0 g Methylene chloride (CH ₂ Cl ₂ ) fraction 1.47 g Methylene chloride (CH ₂ Cl ₂ ) fraction 7.0 g Ethyl Acetate (EtOAc) Fraction 4.14 g Ethyl Acetate (EtOAc) Fraction 12.3 g n-butanol (n-BuOH) fraction 10.2 g n-butanol (n-BuOH) fraction 18.4 g Water fraction 14.58 g Water fraction 29.6 g

Example  3: Methylene Chloride of the Tree Extract From fractions  Isolation and Structure Identification of Compounds

The compound was separated from the methylene chloride fraction of the non-wood extract obtained in Example 2 in the following manner.

As a filler used in the separation process, a normal-phase silica gel 60 (0.063-0.200 mm, Merck) was used. The instrument used in the analysis was HPLC (Alliance2695, Waters), and XTerra C ₁₈ 3.5 μm 4.6 × 100 mm was used, and the detector used for analysis was detected using Waters' 2998 model PDA. It was. HPLC grade was used as the solvent and reagents used in the experiment, and NMR (Nuclear Magnetic Resonance) was used as the instrument used for structural analysis. JNM-LA500 of Burker (German) was used. Acetone- d ₆ , methanol- d ₄ , chloroform- d ₁ and DMSO- d ₆ were used.

2.5 g of the methylene chloride fraction of the non-wood-leaf extract obtained in Example 2 was sequentially re-divided into 10 parts under normal silica gel (hexane / ethyl acetate) (v / v) and solvent gradient conditions.

Chromatography was performed using this phase under normal silica gel (hexane / ethyl acetate = 5/1), where compound 5 (12.2 mg) was obtained, and compound 1 (4.7 mg) was obtained as pure crystals obtained by recrystallization. In addition, chromatography was performed under normal conditions of silica gel (chloroform / methanol = 10/1) to give compound 4 (8.7 mg).

7.0 g of the methylene chloride fraction of the extract of the bark of the bark obtained from Example 2 was further fractionated into 8 parts sequentially under normal silica gel (hexane / ethyl acetate) (v / v) and solvent gradient conditions.

Chromatography was carried out using pure silica gel (hexane / ethyl acetate = 3/2) to obtain compound 6 (59.6 mg), and recrystallization to obtain pure crystalline compound 2 (34.4 mg). In addition, Compound 3 (6.4 mg) was obtained from a fraction obtained by chromatography on an ordinary silica gel (hexane / ethyl acetate = 1/1) condition.

It was confirmed that the compound 1-6 had the following chemical structure, respectively.

[Formula 1]

Appearance Pale yellow Molecular formula C ₁₅ H ₁₂ O ₄ Molecular Weight 266.07 Melting point (캜) 166.5-168.5 Solubility Availability Alcohols, DMSO Insoluble Hexane, H ₂ O

¹³ C-NMR (125 MHz, CD ₃ OD): δ 135.0 (C-1). 129.2 (C-2), 111.8 (C-3), 130.9 (C-4), 128.9 (C-5), 129.2 (C-6), 143.2 (C-7), 117.8 (C-8), 168.4 (C-9), 103.1 (C-1 '), 198.9 (C-2'), 171.2 (C-3 '), 108.1 (C-4'), 185.0 (C-5 '), 59.0 (-OCH ₃ )

[Formula 2]

Appearance Pale yellow Molecular formula C ₁₆ H ₁₄ O ₄ Molecular Weight 270.09 Melting point (캜) 126-128 Solubility Availability Alcohols, DMSO Insoluble Hexane, H ₂ O

¹³ C-NMR (125 MHz, CD ₃ OD): δ 191.5, 185.2 (C-3 'and C-6'), 169.8 (C-4 '), 168.6 (C-9'), 142.6 (C-8 ), 135.2 (C-1), 130.2 (C-4), 128.8 (C-3, 5), 128.4 (C-2, 6), 121.31 (C-7), 111.6 (C-5 '), 109.1 (C-1 '), 64.6, 58.5 (4'-OMe and α'-OMe)

(3)

Appearance Pale yellow Molecular formula C ₁₆ H ₁₄ O ₅ Molecular Weight 288.08 Melting point (캜) 92-94 Solubility Availability Alcohols, DMSO Insoluble Hexane, H ₂ O

¹³ C-NMR (125 MHz, CD ₃ OD): δ 135.8 (C-1), 128.5 (C-2), 129.1 (C-3), 130.2 (C-4), 129.1 (C-5), 128.5 (C-6), 121.4 (C-7), 141.4 (C-8), 165.6 (C-9), 64.5 (-OCH ₃ ), 109.6 (C-1 '), 187.4 (C-2'), 149.1 (C-3 '), 148.0 (C-4'), 184.9 (C-5 '), 60.1 (-OCH ₃ )

[Formula 4]

Appearance Pale yellow Molecular formula C ₁₇ H ₁₈ O ₅ Molecular Weight 300.31 Melting point (캜) 84-85 Solubility Availability Alcohols, DMSO Insoluble Hexane, H ₂ O

¹³ C-NMR (125 MHz, CD ₃ OD): δ 135.6 (C-1), 128.4 (C-2), 128.9 (C-3), 130.2 (C-4), 128.9 (C-5), 128.4 (C-6), 121.2 (C-7), 141.4 (C-8), 165.6 (C-9), 64.3 (-OCH ₃ ), 109.5 (C-1 '), 187.4 (C-2'), 149.1 (C-3 '), 148.0 (C-4'), 184.9 (C-5 '), 60.1 (-OCH ₃ × 2)

[Chemical Formula 5]

Appearance Pale yellow Molecular formula C ₁₉ H ₂₀ O ₆ Molecular Weight 344.12 Melting point (캜) 101-102 Solubility Availability Alcohols, DMSO Insoluble Hexane, H ₂ O

¹³ C-NMR (125 MHz, CD ₃ OD): δ 193.5 (1), 148.8 (C-4 '), 145.7 (C-2 "), 134.5 (C-1"), 130.6 (C-3 ") , 128.9 (C-3 ", 5"), 128.5 (C-2 ", 6"), 115.3 (C-1 '), 148.4 (C-2'), 148.0 (C-3 '), 140.3 (C -4 '), 138.6 (C-5'), 146.5 (C-6 '), 62.0 (-OCH ₃ × 2), 61.5 (-OCH ₃ ), 61.3 (-OCH ₃ )

[Formula 6]

Appearance Pale yellow Molecular formula C ₂₀ H ₂₆ O ₆ Molecular Weight 362.17 Melting point (캜) - Solubility Availability Alcohols, DMSO Insoluble Hexane, H ₂ O

¹³ C-NMR (125 MHz, CD ₃ OD): δ 78.9 (C-1). 37.82 (C-2), 32.8 (C-3), 115.3 (C-1 '), 148.4 (C-2'), 148.0 (C-3 '), 140.3 (C-4'), 138.6 (C- 5 '), 146.5 (C-6'), 142.8 (C-1 "), 129.49 (C-2", 6 "), 129.2 (C-3", 5 "), 126.6 (C-4"), 57.5 (-OCH ₃ ), 61.1 (-OCH ₃ ), 61.5 (-OCH ₃ ), 61.4 (-OCH ₃ × 2)

Example  4: Rheumatism  arthritis Cells and monocytes  In cell lines Mehyllucidone Influence of

1) Cell Culture

The human monocyte cell line THP-1 and the rheumatoid arthritis cell line RA cells contain 10% fetal bovine serum (FBS), 100 unit / ml penicillin, 100 ug / ml streptomycin (streptomycin, GIBCO, USA) Cultured in 37%, 5% CO ₂ incubator using RPMI 1640 and DMEM medium.

2) Cell Viability Assay

3T3-L1 battery cells were dispensed into 96 wells at a concentration of 5 × 103 cells / well / 100 μl, and cultured for 24 hours after incubation for 24 hours. After treatment with Ez-CyTox cell viability assay kit (DAEIL LAB SERVICE Co, Korea) solution for 3 hours at 37 ℃ was measured the absorbance at 480nm using ELISA (Bio-Tek, USA). Average absorbance values for each sample group were obtained, and cell viability was calculated by comparison with the absorbance values of the control group.

3) Flow cytometry

After completion of the reaction, the cells were collected, washed with PBS, and fixed with 70% ethanol. After one hour, washed twice with PBS, and treated with RNase A for 30 minutes at 37 ℃. Then, PI (Propidium iodide, Sigma) was treated with a concentration of 50ug / mL and analyzed using FACS calibur (BD Biocsience).

4) RT-PCR (Reverse transcription polymerase chain reaction)

After completion of the reaction, RNA was isolated using a trizol reagent (TRIzol reagent, Invitrogen), and quantified using a UV spectrometer (UV sepectrophotometer, Ultrospec 2100pro, Amersham). After quantification of RNA, cDNA was synthesized using a cDNA synthesis kit (cDNA synthesis kit, Bio-Rad), and PCR was performed using i-Taq DNA polymerase (i-Taq DNA polymerase, intron). As primers for PCR, human PPAR-γ and actin were used.

Example  5: HT -22 in neurons ROS Antioxidant Activity of Compounds 2 and 4 on

Hippocampal neuronal line HT-22 was aliquoted in 96-well plates. Compound 2 and compound 4 were treated for 30 minutes at different concentrations, followed by 30 minutes at 1 mM H 2 O 2 concentration. Based on the result of measuring absorbance after the addition of 50 uM DCF-DA (2 ′, 7′-dichlorofluorescein), the levels of reactive oxygen species (ROS) are shown in Experimental Example 10 (FIG. 21).

DCF-DA (2`, 7`-dichlorofluorescein) is able to permeate cell membrane in acetylated form and when acetate enters cell, acetate group is removed by intracellular esterase. . Although not fluorescing normally, oxidized by ROS generated in the cell, the structure is changed and fluorescence can be used for qualitative and qualitative analysis of the DCF-DA fluorescence. After inducing ROS (Reactive Oxygen Species) in HT-22 neuronal cell lines, 50 uM of DCF-DA was treated to quantitatively and qualitatively analyze fluorescence at excitation / emition wavelength (485nm / 535nm) of FITC series.

Example  6: Rat Birch tree Crude extract  High calorie In the diet  Impact

1) Experimental Animal and Experimental Group

Six-week-old Sprague Dawley (SD) rats with an average body weight of 169.3 ± 1.25g were used. A total of 12 rats were used, divided into 4 groups and run for 8 weeks. The composition of the experimental group is shown in Table 1. Feed and drinking water were taken freely, and feeding and drinking water were measured at the same time once a week.

2) feed

Fat-containing diets were used to induce obesity effects. In other words, Rodent diet with 10% kcal fat (D12450B) and Rodent diet with 60% kcal fat (D12492) containing 10% fat in normal diet and 60% fat in high-calorie feed were supplied from central laboratory animals. It was paid.

3) administration of crude wood extract

Crude tree extract was dissolved in cone oil and administered orally by daily dose. The birch extract was orally administered daily at doses of 100 mg / kg (ML100) and 250 mg / kg (ML250).

4) Growth rate analysis

Weight gain was calculated by measuring the weight of the experimental group and the control group twice a week.

5) Abdominal Adipose Tissue Analysis.

After removing the epididymis of the experimental group and the control group, white adipose tissue (epididymal WAT) was separated and weighed. The percentage of body weight was expressed as a percentage by individual.

6) Histological Analysis of Liver

Rats were sacrificed and their livers fixed in 10% neutral formalin for 48 hours. Fixed liver and arteries were tissue-treated in stepwise ethanol (75% -100%) and embedded in paraffin wax. The embedded tissues were cut into 6 μm, stained with haematoxylin and eogene, and observed at 400 times under an optical microscope.

7) statistical analysis

Means were expressed as mean ± SE and comparison between groups was analyzed by Tukey-Kramer method in multiple comparison analysis (ANOVA). When the p value was less than 0.05, it was judged to be significant.

The effect of the following experiment was confirmed using the non-wood extract, fractions thereof, or compounds separated therefrom obtained in Examples 1 to 6 above.

Experimental Example  1: extract of the birch tree and its Fraction  Effect on fat cell survival rate

In order to examine the effect of the extract of the tree and its fractions on the cell viability of 3T3-L1 cells, which are adipocyte lines, the extract of the tree and the fractions prepared in Examples 1 and 2 at various concentrations of 0 ~ 500 ㎍ / ㎖ After 48 hours of treatment and incubation, the survival rate of the cells was observed by MTT method, and toxicity was measured by LDH assay.

The 3T3-L1 fat cell line promotes differentiation-induced fat differentiation and forms triglycerides, making it a suitable cell for anti-obesity and anti-glucose experiments.

In this experimental example, 3T3-L1 cells, which are adipose cell lines, were distributed from ATCC (American Type Culture Collection) and used. Cells were DMEM (Dulbecco's Minimal Essential Medium, Gibco, USA) containing 100 U / ml penicillin, 100 μg / ml streptomycin, 10% elegant serum (BCS: Bovine calf serum, Gibco, USA) Culture was used to incubate in an incubator maintained at 5% CO ₂ and 37 ℃, subculture was carried out every 3 to 4 days.

Cell viability was measured by the following MTT method. 3T3-L1 battery cells were dispensed into 96 wells at a concentration of 5 × 10 ³ cells / well / 100 μl and incubated for 24 hours, and then treated with concentrations of the extracts and fractions prepared in Examples 1 and 2 for 48 hours. . After treatment with Ez-CyTox cell viability assay kit (DAEIL LAB SERVICE Co, Korea) solution for 3 hours at 37 ℃ was measured for absorbance at 480 nm using ELISA (Bio-Tek, USA). The average absorbance value for each sample group was obtained and cell viability was investigated by comparing with the absorbance values of the control group.

In addition, toxicity was measured by the following LDH assay method. 3T3-L1 cells were seeded in 96 wells at a concentration of 5 × 10 ⁴ cells / well and incubated for 24 hours. Thereafter, each sample was treated for each concentration and cultured for 48 hours to measure lactate dehydrogenase (LDH), which is secreted from damaged cell membranes. LDH was measured for absorbance at 490 ~ 690 nm using the Cytotoxcity Detection kit (Roche, Swiss).

Measurement results of the cell viability and toxicity, which are the experimental results, are shown in FIG. 1. As shown in Fig. 1, the extract of the birch tree (A), ethyl acetate (EtOAc) fraction (B), methylene chloride (CH ₂ Cl ₂ ) fraction (C), hexane (hexane) fraction (D), butanol (BuOH) Cell viability of fraction (E), water (H ₂ O) fraction (F) is shown. In FIG. 1, when the cell viability of the control group was 100%, the cell viability of the birch bark extract (A) decreased to about 40% and 70%, respectively, at a concentration of 250 μg / mL or more, which indicates that the cytotoxicity was decreased. This is due to an increase of more than 2 ~ 18%. The cell viability of each fraction of the extracts was measured. As a result, there was no cytotoxicity in the ethyl acetate (B), methylene chloride (C), butanol (E) and water (F) fractions, and the hexane fraction (D) was peeled. It showed similar tendency as the extract.

Experimental Example  2: birch extract and its Fraction  Effect on the inhibition of adipocyte differentiation

In consideration of the cell viability shown in Experiment 1, an appropriate concentration without toxicity was determined to carry out cell differentiation.

Adipocyte differentiation was carried out in the following manner. 3T3-L1 cells were added to 12 wells at a concentration of 7 × 10 ⁴ cells / well and incubated for 2 days. After 2 days post-confluence, differentiation-inducing substance 10 μg / ml insulin (insulin, Sigma, USA), 1 uM dexamethasone (DEX: Dexamethasone, Sigma, USA), 0.5 mM 3-iso Induction of differentiation for 2 days was promoted by exchange with DMEM / 10% fetal bovine serum (FBS) culture containing butyl-1-methylxanthine (3-Isobutyl-1-methylxanthine, IBMX, Sigma, USA). . After 2 days, the culture medium was removed, exchanged with DMEM / 10% FBS culture containing 10 μg / ml insulin, and cultured for 2 days, and then cultured with DMEM / 10% FBS culture. Total differentiation induction was carried out for 8 days, and the samples were treated at 2 days intervals starting with the differentiation induction. Each sample was treated at various concentrations at the beginning of differentiation induction. These concentrations were determined in consideration of each cell viability, and differentiation-induced differentiation patterns were observed.

When differentiation was induced into adipocytes, the accumulation of triglycerides could be observed by the following Oil Red O staining method. Differentiated 3T3-L1 cells were washed with PBS and fixed with 10% formalin / PBS (formalin / PBS) for 1 hour. Then, washed twice with distilled water, stained with 0.6% Oil Red O (Sigma, USA) dye dissolved in isopropanol for 1 hour, and then washed with distilled water. (lipid droplet) was dissolved in isopropanol containing 4% Nonidet P-40 (Amresco, USA) and the absorbance was measured at 520 nm. Lipid synthesis inhibition rate was determined by calculating the OD value in the positive control (PC: 0%), non-induced treatment 100% by concentration, each compound treatment as follows.

Inhibition Rate = (positve OD value-treatment OD value) / (positive OD value) × 100

Oil Red O staining after 8 days of differentiation is shown in FIGS. 2 to 4. In comparison with the positive control group (PC) treated with differentiation induction, up to 80% or more showed inhibition of the concentration-dependent differentiation in the extract of A-tree (A), and up to 60% in the hexane fraction (C) in each fraction. Inhibition rate was shown to (FIG. 2). At the same concentration, butanol fraction (D) showed an inhibition rate of up to 30%, and the rest did not inhibit or tended to decrease slightly (FIG. 3).

Experimental Example  3: Effect of compound 1-6 on adipocyte viability and inhibition of adipocyte differentiation

Compounds 1 to 6 obtained by fractionation from methylene chloride fractions of the lumber tree extract were tested in the same manner as Experimental Examples 1 and 2 on the effect of adipocyte viability and inhibition of adipocyte differentiation.

1) Compound 1

Experimental results for Compound 1 are shown in FIGS. 5 and 6. As shown in Figure 5, there was no reduction rate for each concentration of 3T3-L1 battery cells, it was also found that there is almost no effect on viability because there is no toxicity. As a result of determining the non-cytotoxic concentration to induce adipocyte differentiation, Compound 1 inhibited cell differentiation in a concentration-dependent manner, and showed a suppression rate of at least 75% compared to the control at a lipid content of 50 uM (Fig. 6). Therefore, Compound 1 is evaluated as a substance exhibiting anti-obesity effect by showing an effect of inhibiting adipocyte differentiation without cytotoxicity.

2) compound 2

Experimental results for the compound 2 are shown in Figures 7 and 8. In order to determine the effect of compound 2 on the cell viability of the 3T3-L1 cell line, the survival rate of the cells was observed by MTT method after treatment and incubation at various concentrations of 0-100 uM for 3 days. As shown in FIG. 7, when the cell survival rate of the control group was 100%, the concentration of 100 uM decreased to almost 40-50% of the control group. Compared with the control group, 1.25 ~ 5 uM concentration showed a slight difference, but no significant change. However, 100 uM showed cytotoxicity.

The concentration without cytotoxicity was determined and 3T3-L1 cell-wide cell differentiation was induced. As shown in FIG. 8, the compound 2 showed a concentration-dependent inhibition of differentiation and adipose cell formation, and the concentrations of 6.25 uM, 12.5 uM, 25 uM, and 50 uM were increased by 13.5%, 45.6%, and 88.1%, respectively. It showed a high inhibitory activity of 92.2%. Thus, Compound 2 also showed good results as a fat inhibitory active substance.

3) Compounds 3 and 4

Experimental results for the compounds 3 and 4 are shown in Figures 9 and 10. MTT assay was performed to examine 3T3-Ll cell survival. As shown in FIG. 9 (A), Compound 3 showed a concentration-dependent tendency, but did not decrease the survival rate of all cells, but increased about 20-30% compared to the control group. However, as shown in FIG. 9 (B), Compound 4 showed a concentration-dependent decrease tendency of about 3 to 65% as the concentration was increased, showing the opposite result according to the influence of the methyl group.

The cell viability of each compound was measured to determine the appropriate concentration and 3T3-L1 cell-cell differentiation was induced. As shown in FIG. 10 (A), in the fat cell differentiation pattern after 8 days, Compound 3 showed almost similar tendency as the control group and showed no inhibition of differentiation, and Compound 4 showed cytotoxicity at a concentration of 50 uM or more. The cells died. At lower concentrations, however, it showed no cytotoxicity, as shown by MTT results, and below 25 uM inhibited triglyceride formation by more than 80%. However, although Compound 3 was not inclined to the control group, it is thought that this substance has an antidiabetic effect of absorbing sugar and promoting triglyceride formation.

4) Compounds 5 and 6

Experimental results for the compounds 5 and 6 are shown in Figures 11 and 12. As shown in FIG. 11, it did not appear to affect the cell viability of 3T3-L1 cell cells. As a result of determining the concentration without cytotoxicity and inducing adipocyte differentiation (FIG. 12), there was no concentration-dependent tendency, but the inhibition rate was over 50% at the concentration of 100 uM, and the inhibition rate was lower than 10% at the concentration below. Indicated.

Experimental Example  4 : PPAR -γ, C / EBP -α inhibitory effect

In the process of differentiation of fat cells into morphologically and biochemically mature fat cells, transcriptional activation factors important for the regulation of fat cell genes must be activated. Two of the best known are the Peroxisome proliferator-activated receptors (PPARs) and the CCAAP / enhancer binding protein (C / EBP), which interact with adipocyte gene regulatory sites to promote adipocyte differentiation. Therefore, compound 2 was treated to observe the inhibitory effect of the major factors promoting each differentiation.

The effect was tested the following method. RIPA cell lysis buffer (RIPA lysis buffer: 0.5M Tris-HCl, pH 7.4, 1.5 M NaCl, 2.5% deoxycholic acid, 10% NP-40, 10 mM EDTA, 1 mM PMSF, 1 mM Na ₃ VO ₄ , 1 mM NaF, 1 μg / μl leupeptin) were homogenized for 30 minutes. The supernatant was obtained by centrifugation of homogenized cells, and protein concentration was quantified using a Bio-Rad Protein Assay Kit by standardizing BSA (Bovine serum albumin). Total protein was separated by 30-50 μg of lysate with 10% polyacrylamide gel electrophoresis (SDS-PAGE), which was transferred to a PVDF membrane (polyvinylidene difluoride membrane, Millipore, USA). The PVDF membrane was treated with blocking buffer for 1 hour at room temperature and PPAR-r (Peroxisome proliferator-activated receptor-gamma, to observe the expression pattern of transcription factor important for adipocyte differentiation). Santacruz, USA) and C / EBP-a (CCAAT-enhancer binding protein-alpha, Santacruz, USA) were diluted overnight in 5% skim milk / TTBS. After washing with TTBS, the secondary antibody was diluted with anti-mouse or anti-rabbit IgG (Jackson Immunoresearch, USA) bound to HRP (Horse Radish Peroxidase) and reacted at room temperature for 1 hour. After washing with TTBS, the reaction was performed for 3 minutes using WEST-ZOL (iNtRON, Korea) and was then exposed to an X-ray film.

The effect of compound 2 for 8 days of induction of differentiation is shown in FIG. 13. As shown in FIG. 13, it was confirmed that expression of PPAR-γ and C / EBP-α was inhibited as the concentration of Compound 2 increased. In particular, it started to decrease from the concentration of 25 uM and the expression of transcription factor (transcription factor) was significantly reduced at the concentration of 50 uM. However, the concentration-dependent inhibition of triglyceride formation and the level of transcription factor expression were somewhat different, but Compound 2 inhibited the differentiation by inhibiting the expression of two transcription factors required for differentiation. .

The effect of compound 2 on the regulation of PPAR-γ and C / EBP-α during adipocyte differentiation was investigated over time. After the early induction of differentiation, cells express PPAR-γ and C / EBP-α to form triglycerides, so PPAR-γ expression increased from day 2 of differentiation in cells not treated with Compound 2, and C / EBP-α It was shown that the expression of the time increased from 4 days of differentiation (Fig. 13). However, cells treated with Compound 2 showed no increased expression of PPAR-γ and C / EBP-α from day 2 of differentiation. As a result, compound 2 is believed to inhibit the expression of PPAR-γ and C / EBP-α from the early stage of differentiation, thereby inhibiting the differentiation induction, and is evaluated as a substance with high potential for anti-obesity effect.

Experimental Example  5: Compound 2 Following Repeated Treatment District ball  Differentiation inhibitory effect

The inhibitory effect of 3T3-L1 cell-mediated cell differentiation-inducing compound 2 according to time zone treatment was observed. Since 3T3-L1 cells are cells that promote differentiation-induced fat differentiation and form triglycerides, they are suitable for anti-obesity and anti-diabetic experiments. Once in post-confluence state, it becomes a growth arrest state and no longer grows. In this case, when an inducer such as insulin, isobutylmethylxanthine, and dexamethasone is treated, two cell cycles pass and the triglycerides are expressed by expressing factors necessary for differentiation. At this time, the degree of expression of the factors in the process of differentiation by processing the substance to be examined is observed. By inhibiting the transcription factors appearing during the initial differentiation, it is possible to search for substances that prevent differentiation into adipocytes.

In order to observe whether compound 2 also inhibited triglyceride formation at the early stage of differentiation induction, it inhibited adipogenesis into adipocytes. The inhibitory effect was analyzed by one, two and three treatments for 8 days of differentiation induction. Analysis of inhibition rate of liposynthesis upon repeated treatment of 2 showed that the absorbed lipid droplets were dissolved in isopropanol containing 4% Nonidet P-40 (Amresco, USA) and absorbance was measured at 520 nm. . The results are shown in FIGS. 14 and 15.

As shown in FIG. 14, differentiation inhibition was shown concentration-dependently during the initial single treatment in which differentiation induction was induced. Cytotoxicity was observed at 100 uM concentration, consistent with MTT results, but significantly inhibited triglyceride formation below 50 uM. In addition, the inhibition rate was higher than 90% at 50 uM even up to 4 days after the induction of differentiation, showing higher inhibition rate than the single treatment group. This was superior (Figure 15). As shown in FIG. 15, the effects of inhibiting fat synthesis when Compound 2 was treated once for 8 days of differentiation induction were 5.6%, 32.4%, 53.9%, and 91% in 12.5 uM, 25 uM, 50 uM and 100 uM treatments, respectively. Showed a high inhibitory effect. In the two treatments, 12.5 uM, 25 uM, and 50 uM treatments showed 17.8%, 58.9%, and 92% higher inhibitory effects. Three different treatments at different intervals during differentiation resulted in high inhibitory effects of 44.6%, 88.1% and 96.2% in 12.5 uM, 25 uM and 50 uM treatments, respectively. Therefore, the repeated treatment effect showed more than 2 times the inhibition of fat synthesis.

In particular, the concentration of less than 25 uM showed a suppression rate of about 30-90% higher than the initial single treatment treatment. This not only inhibits the initial differentiation induction of the compound 2, it is judged that it can exhibit more effective anti-obesity activity when present for a long time.

Experimental Example  6: of compound 2 Rosiglitazone  Promotion effect

Rosiglitazone (Rosiglitazone) is a drug of TZD series (thiazolidinediones), which is currently used as a type 2 diabetes drug, and is applied to various diseases such as lipid synthesis, anti-inflammatory as well as diabetes treatment. As shown in FIG. 16 (A), the stimulation of fat cell synthesis and PPAR-γ agonist Rosiglitzaone were treated with 3T3-L1 cell cells to induce differentiation. At this time, Compound 2 was treated to treat PPAR-γ. The agonist function was confirmed.

As shown in FIG. 16, rosiglitazone was a PPAR-γ agonist, which showed 100% differentiation induction even at a concentration of 5 uM (FIG. 16A). Compound 2 was also treated continuously with differentiation induction, and differentiation induction was suppressed even after 8 days of induction. The concentration of rosiglitazone (5 uM) was kept constant and compound 2 was treated according to the concentration, and differentiation was suppressed in a concentration-dependent manner. And inhibited the lipid content of 85% or more at the 50 uM concentration of Compound 2 (FIG. 16B). In addition, Compound 2 was treated at a constant concentration (50 uM) and rosiglitazone concentration (1 ~ 10 uM) treatment resulted in more than 90% differentiation inhibition in all groups. It was found that Compound 2 strongly inhibited the function of rosiglitazone known as PPAR-γ agonist. Therefore, compound 2 has been proved to be an antagonist of PPAR-γ, and may be used in future obesity-related prevention and / or treatment and anticancer drugs and new drugs involving PPAR-γ.

Compound 2 was treated with 2 uM rosiglitazone with concentration-specific treatment to analyze the effect of inhibiting fat synthesis. Lipid synthesis inhibitory effects were 5.5%, 22.8%, 35.9%, 88.2% in 6.25 uM + 2 uM rosiglitazone, 12.5 uM + 2 uM rosiglitazone, 25 uM + 2 uM rosiglitazone, 50 uM + 2uM rosiglitazone treatment, respectively. Seemed. This result showed a somewhat lower inhibitory effect than Compound 2 treatment. In particular, rosiglitazone and Compound 2 treatment showed a 2 to 3 times lower inhibition rate in the low concentration treatment (6.25 to 25 uM) (Fig. 8B. Fig. 17A). These results suggest that rosiglitazone has a competitive relationship between affinity as a PPAR-gamma ligand and PPAR-gamma of compound 2, and thus, the effect of inhibiting fat synthesis is reduced. However, as shown in the above results, compound 2 shows a strong PPAR-γ agonist.

Experimental Example  7: RA  Wow THP Effect of Cell Viability on Compound 4 in --1 Cells

Cell viability was measured after 48 hours of compound 4 treatment. As shown in the results of FIG. 18, both cells showed a tendency to decrease in a concentration-dependent manner, and RA and THP-1 cells showed about 60% cell inhibition rate at 100 uM. The RA cell morphology showed that the cell morphology was changing in a concentration dependent manner.

Experimental Example  8: FACS  Effect of Compound 4 on Apoptosis through Analysis

Each cell was harvested and tested to examine the effect of compound 4 on apoptosis. In RA cells, the number and morphological changes of cells were reduced in a concentration-dependent manner as shown in cell viability, but did not affect cell death. It is thought that compound 4 inhibits cell growth by changing the growth environment rather than inducing cell death. THP-1 cells, on the other hand, showed cell death in a concentration-dependent manner. It can be seen that Compound 4 induces the death of blood cancer cells.

Experimental Example  9: Effect of Compound 4 on Each Cell PPAR -r expression effect

The expression pattern of PPAR-γ in RA and THP-1 cells was analyzed. As shown in the result of FIG. 19, RA cells showed a tendency to decrease PPAR-γ expression in a concentration-dependent manner. It does not induce cell death but inhibits the expression of PPAR-γ, which can be said to play an antagonist role in RA cells. However, in THP-1 cells, compound 4 did not affect the expression of PPAR-γ. Therefore, it can be said that Compound 4 is not involved in the mechanism of causing cell death without inhibiting the expression of PPAR-γ in THP-1 cells.

Experimental Example  10: DCF - DA  By measuring method ROS Quantitative measurement of

The hippocampal neuronal cell line HT-22 was cultured in 96-well plates and treated with Compound 2 and Compound 4 for 30 minutes at different concentrations, followed by 30 minutes at 1 mM H2O2. As a result of absorbance measurement after adding 50 uM DCF-DA (2`, 7`-dichlorofluorescein), as shown in FIG. 21, the levels of ROS in neurons were remarkably increased when 5 uM of Compound 2 and Compound 4 were treated. Decreased.

Experimental Example  11: Rat Birch tree Crude extract  High calorie In the diet  Impact

1) Experimental progress.

Subjects with mortality during the 8-week experimental period were not included in the experimental results. The number of animals in the experimental group and the control group used in the results is shown in Table 9.

2) weight change

Compared to the results of 60% high-calorie feed of obese control, the results of the administration of crude wood extract did not show a significant difference from the high-calorie control group when administered 100 mg per body weight. It became. Therefore, it was confirmed that the effect of weight gain may be obtained when oral administration of crude wood extract is performed for 8 weeks or more at an appropriate concentration (FIG. 22).

3) white adipose tissue rate

As a result of analyzing the abdominal fat, the ratio of white adipose tissue (WAT) per body weight of the high calorie diet group tended to increase significantly (Table 10). When comparing the WAT ratio per weight of the ML100 group and the ML250 group on the average tends to decrease the ratio (Fig. 23), especially in the case of the ML250 group was significantly lower than the control group (p <0.05).

4) Histological observation

Fat degeneration was observed in all livers of the high calorie diet group and little in the normal diet diet group. Fat degeneration in the MC50 group and MC100 group was observed at a relatively low level of fat degeneration compared to the control group and a very low level of fat degeneration was observed in the MC250 group (FIG. 24).

5) Comprehensive Review

The sustained intake of high calorie diets showed a significant increase in body weight compared to the diet fed normal diets, which is consistent with previous studies that high calorie intake fed obesity (Xu et al. , 2008). In addition, the weight of the epididymal white fat in the abdomen was significantly higher in the high-calorie diet group than in the non-caloric diet group. Support the theory of storage (Schrauwen-Hinderling et al., 2005). The trend of weight gain in obesity model was different depending on the concentration of crude extract, and there was a significant decrease in weight gain at the highest concentration. In addition, the weight per weight of the epididymal white fat was effectively reduced in the weight per weight of the white fat of the 250MC group. In the histological findings of fat distribution and fat degeneration, hepatic crude extracts were estimated to inhibit liver damage due to high calorie intake.

Claims (17)

Lindera erythrocarpa ) A pharmaceutical composition for the prophylaxis or treatment of cancer, arteriosclerosis, rheumatoid arthritis, multiple sclerosis, Alzheimer's or obesity, comprising as an active ingredient extract, fractions thereof, or compounds isolated therefrom.
The method of claim 1, wherein the extract is an Item of expenditure Item of expenditure tree wood water, C ₁ ~ C ₄ alcohols, or a pharmaceutical composition, characterized in that the prepared and extracted with a mixed solvent thereof.
The pharmaceutical composition of claim 2, wherein the alcohol is methanol or ethanol.
The pharmaceutical composition of claim 3, wherein the concentration of ethanol is 65 to 75%.
The pharmaceutical composition according to claim 1, wherein the fraction is a hexane fraction, a methylene chloride fraction, an ethyl acetate fraction, a butanol fraction, a water fraction or a combination thereof.
According to claim 1, wherein the compound is a pharmaceutical composition, characterized in that at least one compound selected from the group consisting of compounds represented by the formula (1-6).
[Formula 1]

(2)

(3)

[Chemical Formula 4]

[Chemical Formula 5]

[Formula 6]

The pharmaceutical composition of claim 6, wherein the compound of Formula 1 is applied at 6.25 to 100 uM.
The pharmaceutical composition of claim 6, wherein the compound of Formula 2 is applied at 6.25 to 50 uM.
The pharmaceutical composition of claim 6, wherein the compound of Formula 3 is applied at 6.25 to 100 uM.
The pharmaceutical composition of claim 6, wherein the compound of Formula 4 is applied at 6.25 to 50 uM.
The pharmaceutical composition of claim 6, wherein the compound of Formula 5 is applied at 50 to 100 uM.
The pharmaceutical composition of claim 6, wherein the compound of Formula 6 is applied at 6.25 to 100 uM.
The pharmaceutical composition according to claim 1, wherein the extract of the tree, the fraction thereof, or the compound isolated therefrom is a PPAR-γ antagonist.
According to claim 1, wherein the cancer (Gastric carcinoma (MGC803)), breast cancer (breast carcinomal), pancreatic cancer (pancreatic cancer), lymphoma (lymphoma cell), liposarcoma (liposarcoma), prostate cancer, tumor (malignances), colon cancer (colon carcinogenesis), lung cancer (lung carcinogenesis) and liver cancer (liver carcinogenesis), the pharmaceutical composition, characterized in that any one selected from the group consisting of.
Washing, drying and grinding the birch; And
Extracting the crushed birch tree with a solvent selected from water, a lower alcohol of C ₁ ~ C ₄ or a mixed solvent thereof to obtain a birch tree extract.
Washing and drying the wood, and then grinding it with a grinder;
Extracting the crushed lumber tree with a solvent selected from water, a lower alcohol of C ₁ -C ₄ , or a mixed solvent thereof to obtain a lumber tree extract; And
A method for producing a fraction of the lumber extract comprising the step of fractionating the nectar extract into one or more solutions selected from the group consisting of hexane, methylene chloride, ethyl acetate, butanol, water.
Washing and drying the wood, and then grinding it with a grinder;
Extracting the crushed lumber tree with a solvent selected from water, a lower alcohol of C ₁ -C ₄ , or a mixed solvent thereof to obtain a lumber tree extract; And
Fractionating the non-tree extract with methylene chloride to obtain a methylene chloride fraction of the non-tree extract; And
Method for preparing a cyclopentadione-based compound separated from the birch tree comprising the step of performing a silica gel chromatography on the fractions to obtain a cyclopentadione-based compound

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