KR20120001100A - The synthetic derivative of danshen components comprising composition inhibiting and preventing obesity - Google Patents

Abstract

PURPOSE: A composition containing Salvia miltiorrhiza Bunge derivatives is provided to reduce expressionof visfatin and resistin and to suppress inflammation. CONSTITUTION: A composition for preventing obesity contains 2-(3'-methoxy-4'-hydroxyphenyl)-6-(3-hydroxypropyl)-5-methoxybenzo[b]furan (5-methoxybenzofuran) which suppresses PPARgamma and C/EBP alpha expression. The compound is prepared by Wittig-Horner reaction, hydrogen reduction, LAH reduction, benzylation, Sonogashira coupling, iodine-induced cyclization, LAH reduction, and debenzylation from 2,5-dimethoxybenzaldehyde.

Description

The synthetic derivative of danshen components comprising composition inhibiting and preventing obesity

The present invention inhibits progenitor cell differentiation and reduces the expression of inflammatory adipokine, bisfatin and resistin [2- (3'-methoxy-4'-hydroxyphenyl) Derivatives of -5- (3-hydroxypropyl) -7-methoxybenzo [ b ] furan-3-carbaldehyde (XH-14)] 2- (3'-methoxy-4'-hydroxyphenyl) -6- (3-hydroxypropyl)- For a composition containing 5-methoxybenzo [ b ] furan (5-methoxybenzofuran). More specifically, XH-14, which is a natural substance of the benozofuran carbaldehyde family, is used as a constituent, and the functional group is changed, or a deformylated synthetic derivative 5-methoxybenzofuran at the C3 position is a progenitor cell (mouse fibroblast 3T3-L1). Bispartin (PBEF), a new adipocaine reported to inhibit the expression of PPARγ and C / EBPα, which are closely related to the differentiation of, and to induce lipogenesis, insulin resistance and inflammatory responses in visceral adipose tissue , by inhibiting the expression of pre-B cell colony-enhancing factor (Nampt, nicotiamide phosphoribosyl-transferase) and resistin, inhibiting the differentiation and accumulation of fat precursor cells and reducing the inflammatory response due to obesity It is about a composition containing 5-methoxybenzofuran derivative.

Recently, with the rapid increase in the obese population, interest in the treatment and prevention of obesity is increasing. Obese people have a high incidence and reduced life expectancy for diseases such as diabetes, hypertension, hyperlipidemia, heart disease, arthritis, respiratory disease, sexual dysfunction, and cancer (Weisberg SP et al., J. Clin. Invest. 112, 1796- 1808, 2003). As metabolic disorders associated with obesity have become a concern, adipose tissue has been studied as an endocrine organ that regulates metabolism as well as a role as a fat reservoir. Adipocytokines (adipokines), such as adiponectin, leptin, resistin, and adipsin, which are produced and secreted from adipose tissue, have been identified and many studies have been conducted. Recently, bispartin and resistin, known as new adipokine, are present in visceral adipose tissue and have been reported as a link between adipose tissue and inflammatory response by increasing lipogenesis and inducing insulin resistance (Lee WJ et al. , Int. J. Obesity 33, 46572, 2009; Bastard JP et al., Eur. Cytokine Netw. 17, 4-12, 2006). In addition, bispartin released from adipose tissue has been reported to increase the expression of adhesion molecules related to atherosclerosis by participating in inflammatory response of vascular tissues (Chudek J & Wiecek A, Pharmacol. Reports 58, 81-88, 2006).

Adipocyte specific transcription factors change in expression level according to differentiation process, and C / EBPa and PPARγ play a central role in the regulation of differentiation of adipose progenitor cells. It is well known that expression is induced at a time point and regulates the expression of specific genes through interaction with each other, and gradually induces activation of fat metabolism and differentiation of fat precursor cells. The fat precursor cell differentiation ability induced by PPARγ is more potent than any other transcription factor, and the expression of PPARγ is regulated by the C / EBP family, in particular C / EBPβ is an important function of inducing early expression of adipocyte differentiation of PPARγ. Do this. Increased expression of this transcription factor increases the expression of PPARγ, which in turn increases the expression of C / EBPa. Therefore, early C / EBPδ and C / EBPβ induce the expression of PPARγ in the process of adipocyte differentiation, and increased expression of PPARγ is responsible for the late stage of adipocyte differentiation by C / EBPa expression. (Cowherd RM et al., Cell DEV. Biol. 10, 3-10, 1999; Tang QQ et al., Biochem. Biophys. Res. Commun. 319, 235239, 2004; Lowell BB, Cell 99, 239-242, 1999).

       Currently, various obesity treatments are used for the treatment of obesity, but it causes various side effects such as constipation, greasy stools, abdominal bloating, frequent fart, abdominal pain, dry mouth and insomnia. Therefore, for the treatment of obesity, there are no side effects, and development of drugs or functional raw materials with various mechanisms is required, and the development of therapeutic agents for obesity is required by analyzing active ingredients from natural products containing various components. Using natural products, there is a need to develop new substances that are safe for the human body and have no side effects, and inhibit the differentiation of fat precursor cells and the accumulation of fat in differentiated fat cells.

       In this study, the effect of 5-methoxybenzofuran, a derivative of Salvia Militiorrhiza (XH-14), on the differentiation of adipose progenitor cells was investigated. The effects on the differentiation of adipose progenitor cells were measured by the degree of fat accumulation and the expression of PPARγ and C / EBPa proteins, which are known to increase expression during differentiation. Through this study, 5-methoxybenzofuran, a derivative of salvia, inhibits fat differentiation and reduces the expression of bispartin and resistin, which are secreted from adipocytes and cause inflammatory reactions.

The present invention has been studied to find a derivative of natural products that are more effective than conventionally known obesity inhibitors, and has no side effects, 2- (3'-methoxy-4'-hydroxyphenyl) which is a derivative of sweet ginseng (XH-14) -6- (3-hydroxypropyl) -5-methoxybenzo [ b ] furan (5-methoxybenzofuran) inhibits the differentiation of progenitor cells and the accumulation of intracellular fat, and inhibits the inflammatory adipocaine bispatin and resistin. The present invention has been completed by confirming that it exhibits excellent anti-obesity inhibitory ability without toxicity. Accordingly, an object of the present invention is to provide a composition for preventing and treating obesity, which contains 5-methoxybenzofuran, which is a derivative of Salvia, as an active ingredient.

2- (3 ', which is a derivative of Dansam component [2- (3'-methoxy-4'-hydroxyphenyl) -5- (3-hydroxypropyl) -7-methoxybenzo [ b ] furan-3-carbaldehyde (XH-14)] -methoxy-4'-hydroxyphenyl) -6- (3-hydroxypropyl) -5-methoxybenzo [ b ] furan (5-methoxybenzofuran) inhibits PPARγ and C / EBPα expression and fat accumulation associated with the differentiation of adipose progenitor cells and is inflammatory Reduced expression of bispatin and resistin. As a result, it was confirmed that 5-methoxybenzofuran, which is a derivative of the salvia extract, XH-14, has the role of preventing and improving obesity by inhibiting the differentiation of fat precursor cells and reducing the inflammatory response due to obesity.

2- (3'-methoxy-4'-hydroxyphenyl) -6- (3-hydroxypropyl) -5-methoxybenzo Derivative of Salvia Radix XH-14, a benozofuran carbaldehyde family widely distributed in natural medicinal materials and foods according to the present invention [ b ] furan (5-methoxybenzofuran) inhibited the expression and fat accumulation of PPARγ and C / EBPa related to the differentiation of adipose progenitor cells, and decreased the expression of inflammatory bispartin and resistin. Therefore, 5-methoxybenzofuran is a potent derivative, which has the effect of suppressing the differentiation of fat precursor cells and relieving the inflammatory response due to obesity.

1 is cytotoxicity of 2- (3'-methoxy-4'-hydroxyphenyl) -6- (3-hydroxypropyl) -5-methoxybenzo [ b ] furan (5-methoxybenzofuran), which is a derivative of Salvia mildew, in adipocyte differentiation. The figure which shows the safety evaluation of the. This shows the results of cell viability through the MTT assay.
Figure 2 is a derivative of only three components in the adipocyte differentiation process 2- (3'-methoxy-4'-hydroxyphenyl) -5- (3-hydroxypropyl) -7-methoxybenzo [ b ] furan (7-methoxybenzofuran) and
This diagram shows the safety evaluation of cytotoxicity of 2- (3'-methoxy-4'-hydroxyphenyl) -5- (3-hydroxypropyl) -6-methoxybenzo [ b ] furan (6-methoxybenzofuran). This shows the results of cell viability through the MTT assay.
3 is 2- (3'-methoxy-4'-hydroxyphenyl) -5- (3-hydroxypropyl) -7-methoxybenzo [ b ] furan (7-methoxybenzofuran), which is a derivative of only three components in adipocyte differentiation.
This study shows the degree of fat accumulation inhibition through Oil Red O staining of 2- (3'-methoxy-4'-hydroxyphenyl) -5- (3-hydroxypropyl) -6-methoxybenzo [ b ] furan (6-methoxybenzofuran).
Figure 4 is an oil red of 2- (3'-methoxy-4'-hydroxyphenyl) -6- (3-hydroxypropyl) -5-methoxybenzo [ b ] furan (5-methoxybenzofuran) which is a derivative of Salvia extracts during adipocyte differentiation. The results of microscopic observation of accumulated fat formation through O staining and the degree of staining are shown in concentration.
5 is 2- (3'-methoxy-4'-hydroxyphenyl) -5- (3-hydroxypropyl) -7-methoxybenzo [ b ] furan (7-methoxybenzofuran) which is a derivative of only three components.
PPARγ and C / EBPa Expressions Involved in the Differentiation of Adipose Progenitor Cells of 2- (3'-methoxy-4'-hydroxyphenyl) -5- (3-hydroxypropyl) -6-methoxybenzo [ b ] furan (6-methoxybenzofuran) The result obtained by road western blot analysis showing a reduction effect. Quantitative results are measured with a densitometer.
Figure 6 is involved in the differentiation of fat precursor cells of 2- (3'-methoxy-4'-hydroxyphenyl) -6- (3-hydroxypropyl) -5-methoxybenzo [ b ] furan (5-methoxybenzofuran) The results obtained by road western blot analysis showing the effect of reducing PPARγ and C / EBPa expression. Quantitative results are measured with a densitometer.
7 is involved in the differentiation of fat precursor cells of 2- (3'-methoxy-4'-hydroxyphenyl) -6- (3-hydroxypropyl) -5-methoxybenzo [ b ] furan (5-methoxybenzofuran) The results obtained by the reverse reverse transcriptase chain reaction showing the effect of reducing PPARγ and C / EBPa mRNA expression. Quantitative results are measured with a densitometer.
8 is an inflammatory bispatin according to the concentration of 2- (3'-methoxy-4'-hydroxyphenyl) -6- (3-hydroxypropyl) -5-methoxybenzo [ b ] furan (5-methoxybenzofuran) It is a figure which shows the effect of reducing expression. Here, the result obtained by western blot analysis is shown. Quantitative results are measured with a densitometer.
Figure 9 shows the expression of inflammatory resistin according to the concentration of 2- (3'-methoxy-4'-hydroxyphenyl) -6- (3-hydroxypropyl) -5-methoxybenzo [ b ] furan (5-methoxybenzofuran) Figure showing the reduction effect. This represents the result obtained by Western blot analysis. Quantitative results are measured with a densitometer.
10 is an inflammatory bispatin according to the concentration of 2- (3'-methoxy-4'-hydroxyphenyl) -6- (3-hydroxypropyl) -5-methoxybenzo [ b ] furan (5-methoxybenzofuran) Is a diagram showing the effect of reducing the mRNA expression of and resistin. This shows the result obtained by reverse transcriptase chain reaction.

In order to achieve the above object of the present invention, the present invention provides a 2- (3'-methoxy-4'-hydroxyphenyl) -6- (3-hydroxypropyl) -5-methoxybenzo [ b] derivative of the only three component XH-14 of the benozofuran carbaldehyde family. ] An obesity prevention or improvement composition containing furan (5-methoxybenzofuran) as an active ingredient is provided for the differentiation of fat precursor cells (3T3-L1 pre-adipocyte).

More specifically, 2- (3'-methoxy-4'-hydroxyphenyl) -6- (3-hydroxypropyl) -5-methoxybenzo [ b ] furan (5-methoxybenzofuran), which is a Dansam component XH-14, is derived from benozofurna carbaldehyde. It inhibits the expression of PPARγ and C / EBPa and the proliferation-related proteins PPARγ and intracellular fat accumulation, and prevents and treats obesity as an active ingredient without cytotoxicity while reducing the expression of inflammatory bispatin and resistin and its mRNA level. It provides a composition for.

2- (3'-methoxy-4'-hydroxyphenyl) -6- (3-hydroxypropyl) -5-methoxybenzo [ b ] furan (5-methoxybenzofuran), a Salvia Radix XH-14 derivative, has a structure as shown in Chemical Formula 1 And according to the existing technical method (Patent Registration No. 10-0914379) can be used to synthesize derivatives induced by the modification by synthesizing XH-14.

Formula I

Hereinafter, the present invention will be described in more detail.

Fatty cells of the benozofuran carbaldehyde-based Dansam component XH-14 derivative 2- (3'-methoxy-4'-hydroxyphenyl) -6- (3-hydroxypropyl) -5-methoxybenzo [ b ] furan (5-methoxybenzofuran) of the present invention Differentiation-related proteins of PPARγ and C / EBPa and the accumulation of fat in adipocytes, inhibiting the expression of inflammatory bispatin and resistin and their mRNA levels, and reducing the proliferation of adipose progenitor cells. It regulates and prevents fat accumulation and prevents or improves obesity, which reduces the inflammatory response due to obesity.

The composition of the present invention is composed of 0.01 to 99.9% of XH-14 derivative 2- (3'-methoxy-4'-hydroxyphenyl) -6- (3-hydroxypropyl) -5-methoxybenzo [ b ] furan (5-methoxybenzofuran). It is preferable to contain, and it is more preferable to contain 0.1 to 90%.

However, the composition as described above is not necessarily limited thereto, and may vary according to the condition of the patient and the type and extent of the disease.

The composition comprising the XH-14 derivative 2- (3'-methoxy-4'-hydroxyphenyl) -6- (3-hydroxypropyl) -5-methoxybenzo [ b ] furan (5-methoxybenzofuran) of the present invention is pharmaceutical It may further comprise suitable carriers, excipients and diluents commonly used in the preparation of the composition.

2- (3'-methoxy-4'-hydroxyphenyl) -6- (3-hydroxypropyl) -5-methoxybenzo, which is a derivative of the Salvia extract XH-14, which has anti-obesity and obesity-inhibiting activity according to the present invention The composition containing [ b ] furan (5-methoxybenzofuran) is not particularly limited in its formulation, but is preferably a beverage, powder, concentrate, capsule, or medicine.

Although the present invention is described in more detail based on the following examples, the present invention is not limited thereto.

Example 1 Synthesis of Derivatives of Sweet Salmon

Benzo [ b ] furan is a heterocyclic substance, an interesting substance with alkaloid structure that has special physiological activity in nature. Benzo [ b ] furan is a natural substance that contains 2- (3'-methoxy-4'-hydroxy-phenyl) -5- (3-hydroxypropyl) -7-methoxy-benzo, which is XH-14 in Salvia Miltiorrhiza. [ b ] modified from furan-3-carbaldehyde

2- (3'-methoxy-4'-hydroxy-phenyl) -5- (3-hydroxypropyl) -7-methoxy-benzo [ b ] furan (7-methoxybenzofuran) and

2- (3'-methoxy-4'-hydroxy-phenyl) -5- (3-hydroxypropyl) -6-methoxy-benzo [ b ] furan ( 6-methoxybenzofuran ), and 2- (3'-methoxy-4 ' -hydroxy-phenyl) -6- (3-hydroxypropyl) -5-methoxy-benzo [ b ] furan ( 5-methoxybenzofuran ) was synthesized and their efficacy was compared.

Formula II

Among these, 2- (3'-methoxy-4'-hydroxyphenyl) -6- (3-hydroxypropyl) -5-methoxybenzo [ b ] furan ( 5- methoxybenzofuran ) is Wittig-Horner reaction, hydrogen reduction reaction, LAH reduction reaction, benzylation, regioselective iodide reaction, Sonogashira coupling, iodine-induced cyclization, LAH reduction and debenzylation from 2,5-dimethoxybenzaldehyde in all 9 steps Synthesis by efficient method.

Example 2 Culture Experiment of Adipose Progenitor Cells

       Mouse fibroblast pre-adipocyte cell line 3T3-L1 was purchased from the American Type Culture Collection (ATCC, Manassas, VA) and cultured. Adipose precursor cells 3T3-L1 are high glucose Dulbecco's modified Eagle's Medium (DMEM, Sigma) supplemented with 10% new born calf serum (NCS, Invitrogen Corporation, Auckland, New Zealand), 100 U / ml penicillin and 100 mg / ml streptomycin. Co., St. Louis, Mo., USA). To differentiate into adipocytes, 3T3-L1 at a density of 100,000 cells / ml was post-confluenced for 10 days with 10% fetal bovine serum (FBS) / high glucose DMEM, followed by 0.5 mM IBMX (3-isobytyl-1-methylxanthine). , 0.5 μM dexamethasone and 10% FBS / high glucose DMEM with 5 micrograms / ml insulin (MDI) were added and incubated for 2 days, and added to 10% FBS / high glucose DMEM with 5 μg / ml insulin (I) only. After two more days of incubation, only 10% FBS / high glucose DMEM was added, followed by two more days of incubation, and cultured for a total of six days to differentiate into adipocytes. Each time the medium was changed after post confluence, the Dansam derivative was treated at concentrations of 1, 5, 10 and 25 micromoles.

Experiment Protocol I

Example 3 Safety and Fat Accumulation Inhibitory Effects of Salvia Miltiform Derivatives

 (3-1) Cytotoxicity test MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl-tetrazolium bromide) assay

To determine the effect of 5-methoxybenzofuran, 7-methoxybenzofuran, and 6-methoxybenzofuran on the cell viability of proteomic derivatives, fat precursor cells 3T3-L1 were dispensed into 96 wells at a cell density of 100,000 cells / well, and 1 3 derivatives were treated at 5, 10, and 25 micromolar concentrations, respectively, and cultured for 4 hours in MTT (1 mg / ml PBS) solution at 6 days of differentiation, and then dissolved with 0.04 N HCl / isopropanol at 570 nm. The MTT assay was performed. The MTT assay measures the absorbance of MTT formazan using the mitochondria's ability to reduce the yellow water-soluble substrate MTT tetrazolium to a blue-violet, water-insoluble MTT formazan by dehydrogenase activity. It is well known to reflect the concentration of vigorous cells.

       The results are shown in FIGS. 1 and 2.

As shown in FIGS. 1 and 2, as a result of treating the progenitor derivatives with different concentrations of 1, 5, 10, and 25 micromolar in the process of differentiating progenitor cells, the derivatives were statistically non-cytotoxic and safe.

 (3-2) Determination of Fat Accumulation through Oil Red O Staining

In the process of differentiation of adipose progenitor cells, the degree of inhibition of fat accumulation was measured by treating each of the derivatives of the Salvia Militiorrhiza. On day 6 of differentiation, cultured cells were fixed with 10% formalin and stained with Oil Red O staining solution. Oil Red O staining solution was diluted with distilled water at 60% of 0.7 g / 200 ml isopropanol stock solution and stained at 200X magnification, and the absorbed oil Red O was dissolved in 100% isopropanol and absorbed at 490 nm. The concentration was compared and analyzed.

       The results are shown in FIGS. 3 and 4.

During the differentiation of non-differentiation cells and adipose progenitor cells, oil red O staining was performed on oil fats on the 6th day treated with 1, 5, 10, and 25 micromolar concentrations of saliva derivatives, respectively. The degree was shown by the result measured by absorbance and the result observed with the microscope. 7-methoxybenzofuran and 6-methoxybenzofuran treated at 1, 5, 10, and 25 micromolar concentrations did not inhibit fat accumulation (Fig. 3), but 5-methoxybenzofuran was found to contain fat cells at 5 micromolar or more compared to differentiated cells. Fat accumulation was shown to be suppressed to a significant level (FIG. 4).

Example 4 Inhibition of Expression of PPARγ and C / EBPa Associated with Differentiation of Adipose Progenitor Cells

(4-1) Western blot analysis

In order to measure the expression level of PPARγ and C / EBPa that are increased by adipocyte differentiation, the progenitor precursor 3T3-L1 was treated with 200,000 cells / 35 mm dish cell density. Cell extracts were prepared by adding lysis buffer to the cultured cells, and 10-12% SDS-PAGE gel was performed. Electrophoretic protein bands were transferred to the nitrocellulose membrane, treated with 5% skim milk to prevent nonspecific binding, and then mouse primary antibodies PPARγ and C / EBPa (Cell Signaling Technology Inc., Beverly, MA, USA) was diluted 1,000-fold and reacted with stirring and reacted with a secondary antibody of goat anti-rabbit horseradish peroxidase (1: 10,000, Jackson ImmunoReasearch Laboratory, West Grove, PA, USA) for 1 hour. Proteins of PPARγ and C / EBPa in the nitrocellulose membrane were detected by Supersignal West pico chemiluminescence (Pierce Biotechnology, Rockford, IL, USA) and photographed by X-ray film (Konica Co., Tokyo, Japan).

       The results are shown in FIGS. 5 and 6.

The expression of PPARγ and C / EBPa in differentiated adipocytes increased 4 to 6 times compared to undifferentiated cells. However, the expression level of these transcription factors was increased in cells treated with the 5-methoxybenzofuran derivative of Dansam. However, 7-methoxybenzofuran and 6-methoxybenzofuran derivatives were ineffective (FIG. 6).

(4-2) Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis]

       Trizol reagent kit (Invitrogen, Carlsbad, CA, USA) was used for total RNA extraction of cells. Reverse transcription reaction to synthesize cDNA from 5 μg of extracted RNA was performed using 200 units of reverse transcriptase and 0.5 mg / mL oliogo- (dT) 15 primer (Bioneer Co., Korea). The base sequences of primers used for mRNA expression of PPARγ and C / EBPa are as follows.

       PPARγ (forward primer: 5'-ACCACTCGCATTCCTTTGAC

-3 ', reverse primer: 5'-TCAGCGGGAAGGACTTTATG

-3 ', 606 bp), C / EBPa (forward primer: 5'-GGTGCTGGAGTT GACCAGTG-3', reverse primer: 5'-CGGAATCTCCTAGTCCTGGC-3 ', 256 bp), GAPDH (forward primer: 5'-AACTTTGGCATTGTGGAAGGG-3 reverse primer: 5'-GACACATTGGGG GTAGGAACA-3 ', 224 bp) and β-actin (primer: 5'-GACTACCTCATGAAGATC-3', reverse primer: 5'-GATCCACATCTGCTGGAA-3 ', 516 bp) A chain reaction was performed. The polymerase chain reaction was carried out at 30-33 cycles, and the reaction was confirmed on 1% agarose gel containing ethidium bromide.

As a result, PPARγ and C / EBPa mRNAs were increased in adipocytes differentiated for 6 days than in undifferentiated adipocytes, but significantly reduced PPARγ and C / EBPa mRNAs after treatment with more than 10 micromoles of 5-methoxybenzofuran derivative. (FIG. 7).

Example 5 Inhibitory Effect of Inflammatory Bispartin and Resistin Inducing Inflammation and Insulin Resistance

 (5-1) Western blot analysis

       5-methoxybenzofuran was treated in the differentiation process at 1, 5, 10, 25 micromolar concentrations. Cell extracts were prepared by adding lysis buffer to the cultured cells and electrophoresed with 10-12% SDS-PAGE gel at 80 V. The protein on the gel was transferred to the nitrocellulose membrane and then blocked for 5 hours in 5% skim milk to prevent nonspecific binding. Incubate with bispatin primary antibody (Phoenix Pharmaceuticals, Belmont, CA, USA) and resistin primary antibody (Santa Cruze, Santa Cruze, CA, USA) for 3 hours with agitation with nitrocellulose membrane. After incubation at room temperature for 3 hours, goat anti-rabbit horseradish peroxidase and donkey anti-goat horseradish peroxidase (Jackson ImmunoResearch Laboratory, West Grove, Pa., USA) were incubated for 1 hour. Proteins transferred to the nitrocellulose membrane were detected by Supersignal West pico chemiluminescence (Pierce Biotechnology, Rockford, IL, USA) and photographed with X-ray film (Konica Co., Tokyo, Japan).

       The results are shown in FIGS. 8 and 9.

As shown in FIG. 8, the expression of bispartin protein was hardly expressed in undifferentiated adipocytes, but increased significantly in differentiated adipocytes. In comparison, the expression pattern was significantly inhibited when 5-methoxybenzofuran was treated with 5 micromoles or more. In addition, in FIG. 9, there was almost no expression of resistin protein in undifferentiated adipocytes, but markedly increased in differentiated adipocytes. In comparison, the expression pattern was significantly suppressed when more than 10 micromole of 5-methoxybenzofuran was treated.

(5-2) Reverse Transcriptase Chain Reaction (RT-PCR)

       In order to measure the mRNA level of intracellular bispartin, RNA was extracted by treatment with Trizol reagent kit (Invitrogen) on 3T3-L1 fat precursor cells of 200,000 cells / 35 mm dish cell density. Reverse transcription reaction to synthesize cDNA from the extracted RNA 5 micrograms was performed using 200 units of reverse transcriptase and 0.5 mg / ml oligo- (dT) 15 primer (Bioneer Co.). The base sequences of the primers used to express bispatin and resistin mRNA are as follows.

       Bispartin (forward primer: 5'-CAGTGCCTGTGTCTGTGGTCA-3, reverse primer: 5'-CTAATGAGGTGCCACGTCCTG-3 ', 665 bp) and resistin (forward primer: 5'-ACACACACCCTTCTCCACTA)

-3, reverse primer: 5'-CAAACAAGACTTCAACTCCC

-3 ', 415 bp) and reverse transcriptase polymerase chain reaction of GAPDH (forward primer: 5'-AACTTTGGCATTGTGGAAGGG-3', reverse primer: 5'-GACACATTGGGG GTAGGAACA-3 ', 224 bp) were performed. Polymerase chain reaction was performed by 25-30 PCR cycles, and the reaction was confirmed on 1% agarose gel containing ethidium bromide.

       The results are shown in FIG.

       The mRNA levels of bispartin and resistin were found to increase bispartin mRNA expression in differentiated adipocytes, while inhibiting bispartin mRNA expression in 5-methoxybenzofuran-treated cells. In addition, increased resistin mRNA expression in differentiated adipocytes was shown to inhibit the expression of resistin mRNA at 25 micromolar concentration in 5-methoxybenzofuran treated cells.

Claims (4)

2- (3'-methoxy-4'-hydroxyphenyl) -6- (3-hydroxypropyl) -5-methoxybenzo, a derivative of Salvia soybean, characterized by inhibiting the expression of PPARγ and C / EBPa, which are differentiation regulating proteins of adipocytes [ b ] Furan (5-methoxybenzofuran) containing obesity prevention and suppression composition. 2- (3'-methoxy-4'-hydroxyphenyl) -6- (3-hydroxypropyl) -5-methoxybenzo, a derivative of Salmon, which inhibits the expression of inflammatory adipocaine, bispartin, and resistin in adipocytes [ b ] Furan (5-methoxybenzofuran) containing obesity prevention and suppression composition. The method of claim 1,
2- (3'-methoxy-4'-hydroxyphenyl) -6- (3-hydroxypropyl) -5-methoxybenzo [ b ] furan (5-methoxybenzofuran) is Wittig-Horner reaction, hydrogen reduction, LAH reduction, benzylation, regioselective iodide, Sonogashira coupling, iodine-induced cyclization, LAH reduction and debenzylation from 2,5-dimethoxybenzaldehyde A composition for preventing and inhibiting obesity, containing derivatives of Salvia mildew component. The method of claim 2,
2- (3'-methoxy-4'-hydroxyphenyl) -6- (3-hydroxypropyl) -5-methoxybenzo [ b ] furan is Wittig-Horner reaction, hydrogen reduction, LAH reduction, benzylation, regioselective iodide, Sonogashira coupling, iodine-induced cyclization, LAH reduction and debenzylation from 2,5-dimethoxybenzaldehyde A composition for preventing and inhibiting obesity, containing derivatives of Salvia mildew component.

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