KR20110040028A - Pharmaceutical composition and functional health food for preventing or treating diabetes mellitus comprising asarone as effective component - Google Patents

Abstract

PURPOSE: A pharmaceutical composition containing asarone is provided to prevent or relieve diabetes. CONSTITUTION: A pharmaceutical composition for preventing or treating diabetes contains 0.01-50 weight% of asarone as an active ingredient. The pharmaceutical composition further contains a carrier, excipient, and diluent. The pharmaceutical composition is manufactured in the form of powder, granule, tablet, capsule, suspension, emulsion, syrup, aerosol, external use formulation, suppository, and sterilized injection solution. An inhibitor of C/EBP alpha and PPAR gamma contains asarone as an active ingredient. A health food for preventing or relieving diabetes contains asarone as an active ingredient.

Description

Pharmaceutical composition and functional health food for preventing or treating diabetes mellitus comprising asarone as effective component}

The present invention relates to a pharmaceutical composition for preventing or treating diabetes and health functional food containing asaron as an active ingredient, and more particularly to a pharmaceutical composition for preventing or treating diabetes, asa as an active ingredient, asa C / EBPα (CCAAT / enhancer binding protein-α) and PPARγ (peroxisome proliferator activated receptor-γ) expression inhibitor containing Ron as an active ingredient and a health functional food for preventing or improving diabetes containing asaron as an active ingredient will be.

Since obesity due to enhanced differentiation of adipocytes is an established risk factor for type 2 diabetes and cardiovascular disease, molecules that regulate lipoogenesis are important pharmaceutical targets. Therefore, the development of drugs that inhibit lipoogenesis or promote lipolysis is one of the obesity control strategies. Early in the lipogenic differentiation program, adipose progenitor cells undergo mitotic clone expansion (Tang et al . , Proc . Natl . Acad . Sci . USA 2003; 100: 44-49), which is the transcription factor CCAAT / enhancer binding Accompanied by induction of expression of proteins β and δ (C / EBPβ and δ). These factors then induce the expression of the transcription factors C / EBPa and peroxysome growth factor-activated receptor γ (PPARγ) through binding to C / EBP regulatory factors in their promoters. In addition, C / EBPa and PPARγ promote adipocyte specific gene expression, resulting in adipocyte formation. Several natural compounds, including resveratrol and quercetin, exhibit anti-obesity effects by inhibiting the expression of C / EBPa and PPARγ (Rayalam et al., Phytother. Res . 2008; 22: 1367-1371). Hormone-sensitive lipases (HSLs) are key enzymes that contribute to the promotion of lipolysis in adipocytes. It is a rate determining enzyme in the degradation of triacylglycerol (TG) to diacylglycerol (DAG) and free fatty acids (FFA). Lipolytic hormones such as catecholamines and glucagon promote hydrolysis of TG through phosphorylation by cyclic AMP (cAMP) -activated protein kinase A (PKA) and associative activation of HSL. In addition to these hormones, some natural compounds, including phytoestrogens, genistein and procyanidins, have anti-obesity effects by promoting lipolysis through activation of HSL (Szkudelska et al., J. Steroid Biochem . Mol . Biol . 2000; 75: 265-271).

Asarone is a natural plant in India, Acorus A molecule found in certain plants, such as calamus ), drug preparations comprising asaron are used to treat diphtheria, typhoid fever and tuberculosis. Iris roots are used in folk medicine in Dakota and Indonesia for the treatment of diabetes (Acuna et al . , Phytother. Res . 2002; 16: 63-65).

Korean Patent Registration No. 0515746 discloses a diabetic treatment or prevention agent containing Sukchangpo extract, and a pharmaceutical formulation comprising the same. Korean Patent Publication No. 2005-0042769 discloses diabetic and glycemic control foods and feed containing Sukchangpo extract. Is disclosed.

The present invention has been made in accordance with the above requirements, and the present invention was tested for the effect of asaron on adipogenesis and lipolysis in murine 3T3-L1 adipocytes and adipocytes, and combined treatment with asaron. Significantly inhibited the differentiation of 3T3-L1 adipocytes, and post-treatment with asaron completed the present invention by confirming that it increased lipolysis in fully differentiated 3T3-L1 adipocytes.

In order to solve the above problems, the present invention provides a pharmaceutical composition for preventing or treating diabetes containing asarone (asarone) as an active ingredient.

The present invention also provides a C / EBPα (CCAAT / enhancer binding protein-α) and PPARγ (peroxisome proliferator activated receptor-γ) expression inhibitor containing asaron as an active ingredient.

The present invention also provides a dietary supplement for preventing or improving diabetes containing asaron as an active ingredient.

The pharmaceutical composition according to the present invention can be effectively used for treating and preventing various diseases caused by hyperglycemia and hyperglycemia including diabetes.

In addition, asaron of the present invention can be added to various foods or used as food supplements, thereby improving the function of the glycemic control system to obtain the effect of a role as an adjuvant therapy for adult diseases and degenerative diseases.

In order to achieve the object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating diabetes containing asarone (asarone) as an active ingredient.

Asaron inhibits adipogenesis, promotes lipolysis in 3T3-L1 adipocytes, specifically, asaron is a transcription factor C / EBPα (CCAAT / enhancer binding protein-α) and peroxisome proliferator activated receptor- It is possible to prevent or treat diabetes by inhibiting the expression of γ), thereby inhibiting fat formation or promoting lipolysis through increased hormone-sensitive lipase activity.

The present invention also provides a C / EBPα (CCAAT / enhancer binding protein-α) and a peroxisome proliferator activated receptor-γ (PPARγ) expression inhibitor containing asarone as an active ingredient.

The present invention also provides a dietary supplement for preventing or improving diabetes containing asarone (asarone) as an active ingredient.

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

The pharmaceutical composition for preventing or treating diabetes of the present invention may include 0.02 to 50% by weight of the asarone based on the total weight of the composition.

The pharmaceutical composition comprising asaron of the present invention may further include suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

Pharmaceutical dosage forms of the compositions of the present invention may be used in the form of their pharmaceutically acceptable salts, and may be used alone or in combination with other pharmaceutically active compounds, as well as in any suitable collection.

Pharmaceutical compositions comprising asaron according to the present invention, respectively, in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, oral dosage forms, external preparations, suppositories, and sterile injectable solutions according to a conventional method. Can be formulated and used. Carriers, excipients and diluents that may be included in the composition comprising asarone include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium Various compounds or mixtures, including silicates, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil and the like. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate, sucrose ( It is prepared by mixing sucrose or lactose and gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The preferred dosage of the composition of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, the composition of the present invention is preferably administered at 0.0001 to 100 mg / kg, preferably at 0.001 to 100 mg / kg. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.

The composition of the present invention may be administered to mammals such as rats, mice, livestock, humans, and the like in various routes. All modes of administration can be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

In addition, the present invention provides a health functional food comprising the above-mentioned asaron and food acceptable food supplements exhibiting a prevention or improvement effect of diabetes. Examples of foods to which asaron can be added include various foods, beverages, gums, teas, vitamin complexes, and health functional foods.

It may also be added to foods or beverages for the purpose of preventing or improving diabetes. At this time, the amount of the asaron in the food or beverage may be added in 0.01 to 15% by weight of the total food weight, the health beverage composition is added in a ratio of 0.02 to 5 g, preferably 0.3 to 1g based on 100 ml Can be.

The health functional beverage composition of the present invention is not particularly limited to other ingredients except for containing the asarone as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. . Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract, for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. . The proportion of such natural carbohydrates is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

In addition to the above, asaron of the present invention can be used in various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavoring agents, colorants and neutralizing agents (such as cheese, chocolate), pectic acid and salts thereof, alginic acid and Salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like. In addition, the asaron of the present invention may contain a fruit flesh for the production of natural fruit juice and fruit juice beverage and vegetable beverage. These components may be used independently or in combination, and the proportion of such additives is not so critical but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the extract of the present invention.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Materials and methods

Compounds and Reagents

Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from HyClone (Logan, UT). Dexamethasone, 3-isobutyl-1-methylxanthine and insulin were purchased from Sigma-Aldrich (St. Louis, MO). PKA inhibitor H-89 was purchased from Stressgen (Ann Arbor, MI). C / EBPa, C / EBB, PPARγ and adiponectin antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, Calif.) And phospho-HSL, phospho-ERK and ERK antibodies were purchased from Cell Signaling Technology (Danvers, MA). .

Seokchangpo  ( Acorus gramineus  ( Araseae  ( Araceae Isolation of Asaron from Rhizome Extract of))

Seokchangpo ( Acorus The dried root diameter of gramineus (1 kg) was chopped and soaked in 3 L of room temperature methanol (MeOH) for 2 weeks. The resulting MeOH extract was filtered through cotton ball and concentrated in vacuo to give 82 g of a thick syrup-like extract, resuspended in 4 L of distilled water and extracted twice with the same volume of dichloromethane. The extracts were combined and concentrated to dryness to give 32 g of dichloromethane fractions. It was chromatographed on silica gel (230-400 mesh, 300 g) and five fractions (Fr.A-Fr.E) eluted with hexane-ethylacetate (15: 1 to 1: 1). Fr.C (5 g) was purified by repeated silica gel column chromatography, and eluted with hexane-ethyl acetate (10: 1 to 5: 1). The eluate was 1.2 g of yellow oil, which was found to be asaron (1: 1 mixture of α and β isomers) based on NMR spectroscopic analysis (data not shown).

3 T3 - L1  Differentiation of cells

The murine 3T3-L1 adipocyte cell line was obtained from ATCC and cultured in DMEM containing 10% fetal calf serum (HyClone, UT) at 37 ° C. in an atmosphere of 95% air / 5% CO ₂ . . When 3T3-L1 cells were stopped growing in confluence, differentiation was induced by addition of DMEM medium (differentiation medium) supplemented with 0.5 M 3-isobutyl-1-methylxanthine, 2 μM dexamethasone and 1.7 μM insulin. The medium was removed after 2 days of incubation, replaced with DMEM supplemented with 10% FBS and 1.7 μM of insulin for 2 days, after which the medium was changed every two days with DMEM supplemented with 10% FBS. At the time of conducting the experiment on differentiated adipocytes, more than 95% of 3T3-L1 cells were filled with multiple lipid droplets.

oil- Red  O dyeing

Fully differentiated 3T3-L1 adipocytes were washed with phosphate buffered saline (PBS) and fixed for 4 minutes with 4% formaldehyde in PBS. The cells are incubated with an oil-red O dye, which was lysed to 60% isopropanol at room temperature for 15 minutes. Stained lipid droplets were dissolved in isopropanol and quantified by absorbance measurement at 490 nm.

Triglycerides  Level and non- Esterification  Measurement of fatty acids

Fully differentiated 3T3-L1 adipocytes were incubated with 62.5 μM or 125 μM asaron for the indicated time intervals. The cells were washed, harvested in 300 μl of PBS and then sonicated. Triglyceride levels in the sonicated digests were measured using the Cleantech TG-S reaction kit (Asan Pharm, Co., Seoul, Korea) according to the manufacturer's instructions. Non-esterified fatty acids released from mature adipocytes were measured using a cultured adipocyte lipolysis assay kit manufactured by Zen Bio (Research Triangle Park, NC).

cell Viability  black

Adipose progenitor cells 3T3-L1 cells were seeded in 96-well plates at a density of 5000 cells / well. Cells were treated for 48 hours using various concentrations of asarone and then cell viability was measured using the EZ-CyTox Cell Viability Assay Kit (Daeil Lab. Service, Seoul, Korea).

Reverse transcription  Polymerase chain reaction ( PCR )

Total RNA was isolated after asaron treatment in differentiated 3T3-L1 adipocytes using TRI reagents (Molecular Research Center, Inc., Seoul, Korea). 5 μg of RNA was transcribed for 1 hour at 37 ° C. with 0.5 μg of random primers using reverse transcriptase (Promega, Madison, Wis.). The following primer sequences were used for the amplification of adipocyte triglyceride lipase (ATGL) C / EBPβ and C / EBPδ: ATGL forward primer (5'-cagttccacctgctcagaca-3 '; SEQ ID NO: 1) and ATGL reverse primer (5'-ctccgagagagatgtgcaaca -3 '; SEQ ID NO: 2); C / EBPβ forward primer (5'-ctttcgggacttgatgcaatc-3 '; SEQ ID NO: 3) and C / EBPβ reverse primer (5'-aacaaccccgcaggaacat-3'; SEQ ID NO: 4); C / EBPδ forward primer (5′-ctcccgcacacaacatactg-3 ′; SEQ ID NO: 5) and C / EBPδ reverse primer (5′-ggttaagcccgcaaacatta-3 ′; SEQ ID NO: 6). PCR was performed with 30 amplification cycles using a PTC-100 thermal cycler (MJ Research Inc., MA). Each amplification cycle consisted of 30 seconds denaturation at 94 ° C., 30 seconds annealing at 55 ° C., and 30 seconds elongation at 72 ° C. Amplification of β-actin was used as a control. PCR products were taken separately by 1% agarose gel electrophoresis.

Weston Blot  analysis

Differentiated 3T3-L1 adipocytes treated with various concentrations of asarone were washed twice with PBS and 100 μl of 1 × sodium dodecyl sulfate (SDS) denaturing buffer containing phosphatase inhibitors (Sigma-Aldrich, Mo). Cell lysates were prepared using and then sonicated for 15-20 seconds. The sonicated sample was heated at 95 ° C. for 5 minutes and separated by electrophoresis on a 10% SDS-polyacrylamide gel. The protein was then transferred for 2 hours on a 0.45 μm pore size nitrocellulose membrane (Whatman, Dassel, Germany). Subsequently, the membrane was subjected to PPARγ or C / EBPa antibody (Santa Cruz, CA) or phospho-ERK and ERK antibodies or phospho-HSL antibodies (Cell Signaling Technology, MA) or β-actin antibody (Bethyl Laboratories, Inc., TX) Was incubated overnight at 4 ° C with a 1: 1000 dilution. The membranes were then incubated at 25 ° C. for 2 hours with 1: 5000 dilution of anti-rabbit IgG or horseradish peroxidase (Stressgen, MI) conjugated with anti-mouse IgG secondary antibody. Proteins were visualized using enhanced chemiluminescent substrates (Thermo, IL) and analyzed with LAS3000 luminescence imaging analyzer (Fuji Film, Tokyo, Japan).

Example  1: asaron 3 T3 - L1  Inhibits Adipogenesis Differentiation of Adipocytes

Differentiation of the murine adipocyte cell line 3T3-L1 into adipocytes was initiated by incubation of cells for 48 hours using a differentiation cocktail in the presence or absence of asaron. The degree of lipogenesis was measured by microscopic observation of intracellular lipid droplet formation. Asaron treatment significantly inhibited lipoogenesis of 3T3-L1 cells (FIG. 1A). Asaron-induced lipid droplet formation inhibition levels in adipocytes were assessed by oil-red O staining. The amount of oil-red O dye incorporated into the cells was quantified by measuring the absorbance of the isopropanol extract of the cells. Treatment with asaron inhibited the formation of lipid droplets in a dose dependent manner (FIG. 1B). To confirm that asaron mediated inhibition of lipogenesis was not due to the cytotoxic effects of asaron, cell viability was measured with the EZ-CyTox cell viability assay kit. After 48 hours of 125 μM of asarone, no statistically significant cytotoxicity was found (FIG. 1C). The results indicated that the combination of asaron with differentiation cocktails inhibited the differentiation of 3T3-L1 adipocytes, and that the inhibitory effect of asaron on lipid droplet formation was not due to cytotoxicity to 3T3-L1 cells. Suggest.

Example  2: asaron is C / EBP α and PPAR inhibits expression of γ

In order to explain the molecular mechanisms that support the anti-lipogenesis effect of asaron, the differentiation of 3T3-L1 adipocytes into adipocytes was initiated by incubating the cells with a differentiation cocktail for 48 hours with or without asaron. did. C / EBPa and PPARγ transcription factors contribute to adipogenesis progression; Accordingly, expression levels of C / EBPa and PPARγ were assayed after an additional 8 days of incubation in the absence of asaron. Protein levels of both transcription factors decreased in proportion to dose (FIG. 2A), indicating that asarone exhibits its inhibitory effect on lipoogenesis through downregulation of C / EBPa and PPARγ expression. C / EBPβ and C / EBPδ are increased during the initial adipogenesis process, activating C / EBPa and PPARγ transcriptionally to induce complete differentiation of adipocytes (9). Therefore, the effect of asaron on the expression of C / EBβ and C / EBP was investigated. Adipose precursor cells, 3T3-L1 cells, were treated for 8 hours with differentiation cocktails in the presence of asarone, and C / EBPβ and C / EBPδ mRNA levels were assessed using RT-PCR. Asaron significantly inhibited C / EBP expression but did not affect the expression of C / EBPβ (FIG. 2B). The data suggest that asaron-mediated inhibition of C / EBPδ in early lipoogenesis may correlate with downregulation of C / EBPa and PPARγ in later stages of fat formation.

Example  3: Asaron Enhances Lipolysis Through Activation of Hormone Sensitive Lipase

The effect of asaron on fully differentiated adipocytes was investigated. To determine whether asarone affects lipolysis in differentiated adipocytes, intracellular triglyceride levels were measured in differentiated 3T3-L1 adipocytes treated with 125 μM asarone. Triglyceride levels in the asaron treated group were found to decrease with time (FIG. 3A). To establish whether asaron increases lipolysis, free fatty acid release from the cells was measured. Asaron treatment significantly increased the release of free fatty acids (FIG. 3B). HSL and ATGL both play an important role in lipolysis in isolated mature adipocytes. Thus, expression levels of ATGL mRNA were analyzed using RT-PCR analysis; It was observed that asaron treatment did not significantly affect ATGL expression (FIG. 4A). Subsequently, it was examined whether or not asaron affected the activity of HSL that hydrolyzes triglycerides. Lipolysis in adipocytes is primarily promoted through PKA-mediated phosphorylation and activation of HSL. To test whether HSL is activated, the level of phosphorylated HSL was measured by Western blot analysis using anti-phospho-HSL antibody. Differentiated 3T3-L1 adipocytes were treated with 125 μM asaron for 30 minutes and total cell lysates were obtained by sonication with 1 × SDS denaturing buffer. Phosphorylation of HSL was increased dose dependently by asaron treatment (FIG. 4B). To establish the time kinetics of HSL phosphorylation, differentiated 3T3-L1 cells were treated with 125 μM asaron and total cell lysates were prepared at the indicated time intervals. Phosphorylation of HSL induced by asaron increased with a time-dependent trend (FIG. 4C). Since PKA phosphorylates HSL, we tested the possibility that Asaron promoted PKA-dependent phosphorylation of HSL. Differentiated 3T3-L1 adipocytes were pretreated for 1 hour with H-89, a selective potent cAMP-dependent PKA inhibitor, and incubated for 30 minutes after the addition of 125 μM asarone. Asaron-induced HSL phosphorylation was blocked in the presence of PKA inhibitors (FIG. 4D), suggesting that Asaron actually promotes phosphorylation of HSL through PKA activation. It is also known that HSL, a rate determining enzyme in lipolysis, can be activated by extracellular signal regulatory kinase (ERK). To investigate the effect of asaron on ERK activation, differentiated 3T3-L1 adipocytes were treated with the indicated concentrations of asarone and cell lysates were prepared after 15 minutes. ERK phosphorylation levels increased at 62.5 μM asaron (FIG. 4E), suggesting that PKA and ERK activation by asaron both contribute to HSL activation.

Example  4: Asaron Adiponectin  Increases production

Decrease of adiponectin levels is a risk factor for diabetes and various cancers. To investigate this effect, we examined whether asaron treatment of adipocytes affected adiponectin production. Differentiation of adipocytes 3T3-L1 cells into adipocytes was initiated by incubating the cells with differentiation cocktails for 48 hours in the presence or absence of asaron. The differentiation process was carried out by changing the fresh medium every two days. On day 8 of adipocyte formation, the culture medium was subjected to Western blot analysis to determine the level of secreted adiponectin. Pretreatment with asaron significantly reduced secretion of adiponectin (FIG. 5A). Differentiated 3T3-L1 adipocytes were treated with asarone for 48 hours and secreted adiponectin levels were measured. Interestingly, asaron significantly enhanced the secretion of adiponectin (FIG. 5B). Differentiation of Differentiation Cocktail with Asaron inhibits the differentiation of 3T3-L1 adipocytes into adipocytes. Adiponectin secretion, on the other hand, increased when treated with asarone fully differentiated adipocytes.

Figure 1 shows that the combination of differentiation cocktail and asaron inhibits fat formation. (A) Adipose Progenitor Cells Differentiation of 3T3-L1 cells into adipocytes was induced by incubation with differentiation cocktails containing 62.5 μM and 125 μM asaron. On day 8 of differentiation, the morphology of the cells was measured by measuring the morphology of the cells. (B) Adipogenesis was carried out in the presence of asaron. On day 8 of differentiation, differentiated cells were stained with oil-red O and the oil-red O incorporation level was quantified by absorbance measurement at 490 nm. Data is shown as mean ± SEM from three replicates. **, P <0.01, compared to untreated control cells. (C) Adipose progenitor cells 3T3-L1 cells were treated with various concentrations of asaron for 48 hours, and then cell viability was measured. Data is shown as mean ± SEM from three replicates.

2 shows that asaron inhibits the expression of C / EBPa and PPARγ. (A) Adipocytes Differentiation of 3T3-L1 cells into adipocytes was induced by incubation with differentiation cocktails containing 62.5 μM and 125 μM asaron. On day 8 of differentiation, the levels of C / EBPa and PPARγ were measured by Wester blot analysis. (B) Adipose Progenitor Cells 3T3-L1 cells were treated for a time interval defined by 125 μM asaron in the presence of differentiation cocktails. RT-PCR analysis was performed to determine the levels of C / EBPβ and C / EBPδ.

3 shows that asaron promotes lipolysis in differentiated adipocytes. (A) Adipose progenitor cells 3T3-L1 cells were differentiated by incubation using a differentiation cocktail. On day 8 of differentiation, differentiated adipocytes were treated for 125 μM asaron for a time interval. Intracellular triglyceride levels were measured. Data is presented as mean ± SEM from three replicates. **, P <0.01, compared to untreated control cells. (B) Differentiation On day 8, differentiated adipocytes were treated with indicated concentrations of asaron. Levels of non-esterified fatty acids were measured 48 hours after asaron treatment. Data is presented as mean ± SEM from three replicates. **, P <0.01, compared to untreated control cells.

4 shows that asaron promotes phosphorylation of HSL through activation of PKA and ERK. (A) Differentiated 3T3-L1 adipocytes were treated with indicated concentrations of asarone for 24 hours. RT-PCR analysis was performed to determine the level of ATGL mRNA. (B) Differentiated 3T3-L1 mature adipocytes were treated with indicated concentrations of asaron for 30 minutes. Cell lysates were subjected to Western blot analysis and the levels of phospho-HSL were measured. (C) Differentiated 3T3-L1 adipocytes were treated for various time intervals with 125 μM asarone and the levels of phospho-HSL were measured using Western blot analysis. (D) Differentiated 3T3-L1 adipocytes were treated with 10 μM H-89 for 1 hour and then incubated with 125 μM asaron for 30 minutes. Total protein was extracted from differentiated adipocytes and the level of phospho-HSL was measured using Western blot analysis. (E) Differentiated 3T3-L1 mature adipocytes were treated with indicated concentrations of asarone for 30 minutes. Cell lysates were subjected to Western blot analysis and the levels of phospho-ERK and ERK were measured.

5 shows that asaron promotes adiponectin production in differentiated 3T3-L1 adipocytes. (A) Adipose Progenitor Cells Differentiation of 3T3-L1 cells into adipocytes was induced by incubation with differentiation cocktails containing asaron. On day 8 of differentiation, the level of adiponectin in the culture medium was measured using Western blot analysis. (B) 3T3-L1 cells were differentiated by incubation with differentiation cocktails. On day 8 of differentiation, cells were treated with 62.5 μM or 125 μM asarone for 48 hours, after which the level of adiponectin in the culture medium was measured using Western blot analysis.

Claims (4)

A pharmaceutical composition for preventing or treating diabetes, comprising asaron (asarone) as an active ingredient. The method of claim 1, wherein the asaron inhibits the expression of the transcription factors C / ATB (enhancer binding protein-α) and peroxisome proliferator activated receptor-γ (PPARγ) to inhibit fat formation or increase hormone-sensitive lipase activity. Pharmaceutical composition, characterized in that to prevent or treat diabetes by promoting lipolysis. C / EBPα (CCAAT / enhancer binding protein-α) and PPARγ (peroxisome proliferator activated receptor-γ) expression inhibitors containing asarone (asarone) as an active ingredient. A dietary supplement for preventing or improving diabetes containing asarone as an active ingredient.

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