KR20150027648A - Composition for Treating or Preventing Diabetes Comprising Extract from Root of Echinacea Purpurea or Dodeca-2(E),4(E)-Dienoic Acid Isobutylamide as Active Ingredient - Google Patents

Abstract

The present invention relates to a composition for treating, preventing, or alleviating diabetes comprising an extract of root of Echinacea purpurea and dodeca-2(E),4(E)-dienoic acid isobutylamides separated from the same as active ingredients. The extract of root of Echinacea purpurea and dodeca-2(E),4(E)-dienoic acid isobutylamides which are active ingredients of the presnet invention show insulin-like activity and improves insulin resistance, thereby being developed to an antidiabetic active material.

Description

TECHNICAL FIELD [0001] The present invention relates to a composition for treating or preventing diabetes comprising Echinacea root extract or dodeca-2 (E), 4 (E) -dienoic acid isobutylamide as an active ingredient (Composition for Treating or Preventing Diabetes Comprising Extract from Root of Echinacea Purpurea or Dodeca-2 (E), 4 (E) -Dienoic Acid Isobutylamide as Active Ingredient}

The present invention relates to a composition for treating or preventing diabetes comprising Echinacea root extract or dodeca-2 (E), 4 (E) -dienoic acid isobutylamide as an active ingredient.

Due to recent socioeconomic developments, overgrowth, lack of exercise and stress have increased and diabetic population has been rapidly increasing. Diabetes mellitus is the most important chronic disease in the modern age. It causes persistent metabolic syndrome including hyperglycemia due to lack of insulin action, and causes various complications (hypoglycemia, atherosclerosis, diabetic nephropathy, diabetic retinopathy, gangrene, etc.). Diabetes mellitus is classified as type 1 and type 2 diabetes. Currently, more than 85% of diabetic patients are adults with type 2 diabetes, and the number of patients is steadily increasing due to changes in aging and lifestyle.

Currently used diabetes medicines are sulfonylureas drugs that promote insulin secretion in the pancreas, biguanides drugs that slow the absorption of sugars in the small intestine, and thiazolidinediones that increase insulin sensitivity in peripheral tissues (thiazolidinediones) drugs. However, most of these diabetes medications are accompanied by various side effects such as weight gain, hypoglycemia, liver and kidney toxicity.

Accordingly, the development of drugs or functional foods for prevention and treatment of diabetes using natural substances that can effectively manage blood sugar without adverse side effects has recently been attracting attention.

Echinacea is a perennial herbaceous plant found in the northeastern United States of America. Three species of Echinacea species, E. angustifolia , E. pallida and E. purpurea have been used as medicines in the United States and Europe (Qu et al. , 2005; Zhai et al., 2007). Echinacea has a wide variety of therapeutic properties including antiviral, antimicrobial, antifungal, antioxidant, anti-cancer, anti-inflammatory, immunostimulatory and wound healing activities (Barrett, 2003). E. purpurea has been reported to activate PPARγ in 3T3-L1 cells and to increase insulin-stimulated glucose uptake (Christensen et al., 2008; Christensen et al., 2009a). In addition, flowers of E. purpurea have been reported to contain isobutyl amides which can improve insulin resistance (Christensen et al., 2009b).

Adipocyte differentiation of 3T3-L1 adipose precursor cells has been used to study insulin signaling pathways and to screen for compounds with antidiabetic activity. Fat cells store lipids in cells under extreme energy conditions and play an essential role in using lipids that have been stored under nutrient-demanding conditions (Spiegelman and Flier, 2001). Abnormalities in lipid metabolism have been implicated in diseases such as diabetes and obesity (Berg and Scherer, 2005). Adipocyte differentiation is a complex process involving hormone sensitivity and gene expression. When stimulated with IBMX, dexamethasone, and insulin mixture, lipid precursor cells initiate the expression of differentiation-related transcription factors and adipocyte differentiation (Ntambi and Young-Cheul, 2000; Cornelius et al., 1994; Tong and Hotamisligil, 2001). PPARγ and C / EBPα are essential transcription factors that regulate adipocyte differentiation (Ntambi and Young-Cheul, 2000; Tong and Hotamisligil, 2001; Gregoire et al., 1998).

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

Korean Patent No. 10-0497152 Korean Patent Publication No. 10-2010-0117081 Korean Patent Publication No. 10-2011-0018468

Echinacea pallida (Nutt.) Nutt., Echinacea purpurea (L.) Moench): a review (see below): a. of their chemistry, pharmacology and clinical properties. J. Pharm. Pharmacol., 57, 929-954 (2005). Barrett, B. Medicinal properties of Echinacea: a critical review. Phytomedicine, 10, 66-86 (2003). Berg, A. H., and Scherer, P. E. Adipose tissue, inflammation, and cardiovascular disease. Circ. Res., 96, 939-949 (2005). Choi, K. M., Lee, Y. S., Sin, D. M., Lee, S., Lee, M. K., Lee, Y. M., Hong, J. T., Yun, Y. P. and Yoo H. S. Sulforaphane inhibits mitotic clonal expansion during adipogenesis through cell cycle arrest. Obesity (Silver Spring), 20, 1365-1371 (2012). Artepillin C, as a PPARgamma ligand, enhances adipocyte differentiation and glucose uptake. J Korean Gastric Cancer Assoc 2006; 10 (1) in 3T3-L1 cells. Biochem. Pharmacol. , 81, 925-933 (2011a). Choi, SS, Cha, BY, Iida, K., Sato, M., Lee, YS, Teruya, T., Yonezawa, T., Nagai, K., and Woo, JT Honokiol enhances adipocyte differentiation by potentiating insulin signaling in 3T3-L1 preadipocytes. J. Nat. Med., 65, 424-430 (2011b). Christensen, K. B., Minet, A., Grevsen, K., Kristiansen, K., and Christensen, L. P. Plants for diabetes: Identification of plant extracts and metabolites as partial PPARγ agonists with potential anti-diabetic effects. Planta Med., 74, SL 79 (2008). Wein, S., Wolffram, S., Kristiansen, K., and Schatz, E., Rimbach, G., and Christensen, KB, Minet, A., Svenstrup, H., Grevsen, K., Zhang, Christensen, LP Identification of plant extracts with potential antidiabetic properties: effect on human peroxisome proliferator-activated receptor (PPAR), adipocyte differentiation and insulin-stimulated glucose uptake. Phytother. Res., 23,1316-1325 (2009a). Christensen, K. B., Petersen, R. K., Petersen, S., Kristiansen, K., and Christensen, L. P. Activation of PPARgamma by metabolites from the flowers of purple coneflower (Echinacea purpurea). J. Nat. Prod., 72, 933-937 (2009b). Clifford, L. J., Nair, M. G., Rana, J., and Dewitt, D. L. Bioactivity of alkamides isolated from Echinacea purpurea (L.) Moench. Phytomedicine, 9, 249-253 (2002). Cornelius, P., Macdougald, O. A., and Lane, M. D. Regulation of adipocyte development. Annu. Rev. Nutr., 14, 99-129 (1994). Gotti, R., Fiori, J., Hudaib, M., and Cavrini, V. Separation of alkamides from Echinacea purpurea extracts by cyclodextrin-modified micellar electrokinetic chromatography. Electrophoresis, 23, 3084-3092 (2002). Gregoire, F. M., Smas, C. M., and Sul, H. S. Understanding adipocyte differentiation. Physiol. Rev., 78, 783-809 (1998). Lehmann, J. M., Moore, L. B., Smith-Oliver, T. A., Wilkison, W. O., Willson, T. M., and Kliewer, S. A. An antidiabetic thiazolidinedione is a high affinity ligand for peroxisome proliferator-activated receptor gamma (PPAR gamma). J Biol Chem, 270, 12953-12956 (1995). Lehrke, M., and Lazar, M. A. The many faces of PPARgamma. Cell, 123, 993-999 (2005). Ntambi, J. M., and Young-Cheul, K. Adipocyte differentiation and gene expression. J. Nutr., 130, 3122S-3126S (2000). Otto, T. C., and Lane, M. D. Adipose development: from stem cell to adipocyte. Crit. Rev. Biochem. Mol. Biol., 40, 229-242 (2005). Perry, N. B., Burgess, E. J., and Glennie, V. L. Echinacea standardization: analytical methods for phenolic compounds and typical levels in medicinal species. J. Agric. Food Chem., 49, 1702-1706 (2001). Qu, L., Chen, Y., Wang, X., Scalzo, R., and Davis, J. M. Patterns of Variation in Alkamides and Cichoric Acids in Roots and Aboveground Parts of Echinacea purpurea (L.) Moench. HortScience, 40, 1239-1242 (2005). Ramji, D. P., and Foka, P. CCAAT / enhancer-binding proteins: structure, function and regulation. Biochem. J., 365, 561-575 (2002). Saltiel, A. R., and Kahn, C. R. Insulin signaling and regulation of glucose and lipid metabolism. Nature, 414, 799-806 (2001). Sasagawa, M., Cech, N. B., Gray, D. E., Elmer, G. W., and Wenner, C. A. Echinacea alkylamides inhibit interleukin-2 production by Jurkat T cells. Int. Immunopharmacol., 6, 1214-1221 (2006). Li, S., and Chen, M. Ginsenoside Rb1 promotes adipogenesis in 3T3-L1 cells by enhancing PPARgamma2 and C / EBPalpha gene expression. Life Sci, 80, 618-625 (2007). 2-E-ene-2-ene-2-ene-2-ene inhibited in T cells by Echinacea-derived undeca-2E-ene- 8,10-dienoic acid isobutylamide. Int. Immunopharmacol., 9, 1260-1264 (2009). Spiegelman, B. M. PPAR-gamma: adipogenic regulator and thiazolidinedione receptor. Diabetes, 47, 507-514 (1998). Spiegelman, B. M., and Flier, J. S. Obesity and the regulation of energy balance. Cell, 104, 531-543 (2001). Tong, Q., and Hotamisligil, G. S. Molecular mechanisms of adipocyte differentiation. Rev. Endocr. Metab. Disord., 2, 349-355 (2001). Tousch, D., Lajoix, AD, Hosy, E., Azay-Milhau, J., Ferrare, K., Jahannault, C., Cros, G. and Petit, P. Chicoric acid, insulin release and glucose uptake. Biochem. Biophys. Res. Commun. , 377, 131-135 (2008). Zhai, Z., Liu, Y., Wu, L., Senchina, D. S., Wurtele, E. S., Murphy, P. A., Kohut, M. L., and Cunnick, J. E. Enhancement of innate and adaptive immune functions by multiple Echinacea species. J. Med. Food, 10, 423-434 (2007). Zuo, Y., Qiang, L., and Farmer, SR Activation of CCAAT / enhancer-binding protein (C / EBP) alpha expression by C / EBP beta during adipogenesis requires a peroxisome proliferator-activated receptor-gamma-associated repression of HDAC1 at the C / ebp alpha gene promoter. J. Biol. Chem. , 281, 7960-7967 (2006).

As a result of efforts to discover antidiabetic active substances from natural substances, the present inventors have found that Echinacea purpurea root extract and dodeca-2 (E), 4 (E) -dienoic acid isobutylamide Promotes the differentiation of adipose precursor cells and increases the expression of PPARγ and C / EBPα, transcription factors closely related to adipocyte differentiation. Therefore, it has been proved that Echinacea root extract and dodeca-2 (E), 4 (E) -dienoic acid isobutylamide can be effectively used for the treatment and prevention of diabetes by improving insulin resistance. Respectively.

It is an object of the present invention to provide a composition for treating, preventing or ameliorating diabetes comprising Echinacea root extract or dodeca-2 (E), 4 (E) -dienoic acid isobutylamide as an active ingredient.

The objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention provides a pharmaceutical composition comprising Echinacea purpurea root extract or dodeca-2 (E), 4 (E) -dienoic acid isobutylamide [ ) -dienoic acid isobutylamide] as an active ingredient for the prevention and / or amelioration of diabetes.

According to another aspect of the present invention, there is provided a pharmaceutical composition for treating or preventing diabetes comprising Echinacea root extract or dodeca-2 (E), 4 (E) -dienoic acid isobutylamide as an active ingredient .

The composition of the present invention contains, as an active ingredient, an extract obtained from the root of Echinacea purpurea . The term " extract " used herein to refer to Echinacea includes not only the extract obtained by treating an extractive solvent in the roots of Echinacea but also an extract obtained by formulating (for example, pulverizing) such that the Echinacea root itself can be administered to an animal It also has the meaning of including the work of echinacea root.

When the extract of Echinacea root used in the composition of the present invention is obtained by treating an Echinacea root with an extraction solvent, various extraction solvents may be used. Preferably, a polar solvent or a non-polar solvent can be used. Suitable polar solvents are (i) water, (ii) alcohols (preferably methanol, ethanol, propanol, butanol, n-propanol, iso-propanol, n-butanol, 1-pentanol, Or ethylene glycol), (iii) acetic acid, (iv) dimethyl-formamide (DMF) and (v) dimethyl sulfoxide (DMSO). Suitable nonpolar solvents are acetone, acetonitrile, ethyl acetate, methyl acetate, fluoroalkane, pentane, hexane, 2,2,4-trimethylpentane, decane, cyclohexane, cyclopentane, diisobutylene, 1- But are not limited to, pentane, 1-chlorobutane, 1-chloropentane, o-xylene, diisopropyl ether, 2- chloropropane, toluene, 1- chloropropane, chlorobenzene, benzene, diethyl ether, diethylsulfide, Methane, 1,2-dichloroethane, aniline, diethylamine, ether, carbon tetrachloride, and THF. More preferably, the extraction solvent used in the present invention is (a) water, (b) anhydrous or hydrated lower alcohol having 1 to 4 carbon atoms (methanol, ethanol, propanol, butanol, etc.) (E) ethyl acetate, (f) chloroform, (g) butyl acetate, (h) 1,3-butylene glycol, (i) hexane and (j) diethyl ether. . Even more preferably, the extract of the present invention is obtained by treating a lower alcohol to an Echinacea root, and most preferably an Echinacea root extract using ethanol as a solvent.

As used herein, the term " extract " means that it is used in the art as a crude extract as described above, but broadly includes fractions obtained by further fractionating the extract. That is, the Echinacea root extract is obtained not only by using the above-mentioned extraction solvent but also by additionally applying a purification process thereto. For example, a fraction obtained by passing the above extract through an ultrafiltration membrane having a constant molecular weight cut-off value, and a separation by various chromatography (manufactured for separation according to size, charge, hydrophobicity or affinity) The fraction obtained by the purification method is also included in the Echinacea root extract of the present invention.

The extract of Echinacea root used in the present invention can be produced in a powder state by an additional process such as vacuum distillation and freeze drying or spray drying.

The composition of the present invention contains dodeca-2 (E), 4 (E) -dienoic acid isobutylamide as an effective ingredient. The above-mentioned dodeca-2 (E), 4 (E) -dienoic acid isobutylamide may be isolated from Echinacea root extract or chemically synthesized. It is known to those skilled in the art that a compound isolated from an extract and a compound chemically synthesized with respect to dodeca-2 (E), 4 (E) -dienoic acid isobutylamide, an anti-diabetic active compound of the present invention, It is obvious.

The Echinacea root extract or dodeca-2 (E), 4 (E) -dienoic acid isobutylamide, which is an active ingredient of the present invention, promotes the differentiation of adipose precursor cells into adipocytes, Lt; RTI ID = 0.0 > PPARy < / RTI > and C / EBPa, Echinacea root extract or dodeca-2 (E), 4 (E) -dienoic acid isobutylamide, which is an active ingredient of the present invention, increases insulin sensitivity of adipocytes or exhibits insulin-like activity, Prevention, and improvement.

The term "diabetes ", as used herein, refers to a chronic disease characterized by a relative or absolute lack of insulin resulting in glucose-intolerance. The diabetes of the present invention includes all kinds of diabetes, including, for example, type 1 diabetes, type 2 diabetes and diabetes mellitus. Type 1 diabetes is an insulin-dependent diabetes mellitus, which is mainly caused by the destruction of beta -cells. Type 2 diabetes is non-insulin dependent diabetes, resulting from insufficient insulin secretion after meal or by insulin resistance.

The composition of the present invention may be provided in the form of a food composition. In this case, the food composition of the present invention includes components that are conventionally added in the manufacture of food, in addition to the above-mentioned effective ingredients. For example, proteins, carbohydrates, fats, nutrients, seasonings and sweeteners. Examples of the above-mentioned carbohydrates are monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, oligosaccharides and the like; And polysaccharides such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. As a sweetening agent, a natural sweetening agent (e.g., tau Martin, stevia extract (e.g., rebaudioside A, glycyrrhizin)) and a synthetic sweetener (saccharin, aspartame, etc.) may be used. For example, when the food composition of the present invention is prepared as a drink, it may further contain citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, juice, mulberry extract, jujube extract, licorice extract, have.

The composition of the present invention may be provided in the form of a pharmaceutical composition. In this case, the pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier in addition to the active ingredient. Such carriers are those conventionally used in the field of the art and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone , Cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like. The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).

The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate, . On the other hand, the oral dose of the pharmaceutical composition of the present invention is preferably 0.001 to 100 mg / kg (body weight) per day.

The pharmaceutical composition of the present invention can be administered orally or parenterally. When administered parenterally, it can be administered by intravenous injection, subcutaneous injection, muscle injection, intraperitoneal injection, transdermal administration, and the like.

The concentration of the active ingredient contained in the composition of the present invention can be determined in consideration of the purpose of treatment, the condition of the patient, the period of time required, and the like, and is not limited to a specific range of concentration.

The pharmaceutical composition of the present invention may be formulated into a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions in an oil or aqueous medium, or in the form of excipients, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

The features and advantages of the present invention are summarized as follows.

(i) The present invention relates to an antidiabetic use of Echinacea purpurea root extract and dodeca-2 (E), 4 (E) -dienoic acid isobutylamide isolated therefrom.

(Ii) Echinacea root extract of the present invention and dodeca-2 (E), 4 (E) -dienoic acid isobutylamide promote the differentiation of adipocyte precursor cells into adipocytes and promote adipocyte differentiation Lt; RTI ID = 0.0 > PPARgamma < / RTI > and C / EBPa.

(Iii) Echinacea root extract of the present invention and dodeca-2 (E) and 4 (E) -dienoic acid isobutylamide exhibit insulin-like activity and can be developed as antidiabetic active substances that improve insulin resistance have.

The present invention relates to a method for the treatment, prevention or amelioration of diabetes comprising Echinacea purpurea root extract and dodeca-2 (E), 4 (E) -dienoic acid isobutylamide isolated therefrom as an active ingredient ≪ / RTI > Echinacea purpurea root extract, dodeca-2 (E), and 4 (E) -dienoic acid isobutylamide, which are effective ingredients of the present invention, exhibit insulin-like activity and exhibit an insulin resistance- Can be developed.

FIG. 1 shows the result of measuring lipid cell lipid accumulation effect of Echinacea root ethanol extract (EEEP) according to insulin concentration. (A) Differentiation of 3T3-L1 adipose precursor cells was induced by adipogenic differentiation inducers, namely IBMX (0.5 mM), dexamethasone (DEX; 1 μM) and insulin (1 μg / Or insulin-free. Cells were either treated with 50 μg / mL EEEP every other day for the first 4 days of differentiation, or not treated with EEEP. (B) Differentiation of lipid precursor cells was induced using various concentrations of insulin (0, 0.1, 0.2, 0.3, 0.4, 0.5 μg / mL) together with IBMX (0.5 mM) and DEX (1 μM). At the same time, cells were either treated with 50 μg / mL EEEP every other day for the first 4 days of differentiation, or not treated with EEEP. On day 6, the cells were stained with Oil Red O dye and the accumulated fat content was quantified. Measurements were expressed relative to those of adipocytes stimulated by a mixture of adipocyte differentiation inducers (0.5 mM IBMX + 1 [mu] M DEX and 1 [mu] g / mL insulin). Experimental results were expressed as mean ± SD of triplicate experiments. Statistical significance: *** P <0.001.
Figures 2A-2C show the effect of EEEP on lipid accumulation in adipocytes. Differentiation of lipoprotein cells was induced by stimulation with a mixture containing 0.5 mM IBMX, 1 [mu] M DEX, and 0.2 [mu] g / ml insulin. 2a: 3T3-L1 cells were treated with adipocyte differentiation inducers at different times for the first 4 days of differentiation of EEEP (0, 10, 30, 50 μg / mL). On day 6, the cells were stained with Oil Red O dye and observed under an optical microscope (x100). Figure 2b: The intensity of the Oil Red O staining was quantified. Figure 2c: The cell content of triglyceride was analyzed using a triglyceride quantification kit. Experimental results were expressed as mean ± SD of triplicate experiments. Statistical significance: * P <0.05, ** P <0.01.
Figure 3 shows the results of measuring the effect of EEEP on the expression of PPARy and C / EBPa in adipocytes. Differentiation of lipoprotein cells was induced using a mixture of adipocyte differentiation inducing substances, including 0.5 mM IBMX, 1 [mu] M DEX, and 0.2 [mu] g / mL insulin. The adipocyte precursor cells were treated with adipocyte differentiation inducers twice daily for the first 4 days of differentiation of EEEP (0, 10, 30, 50 μg / mL). On day 6, cells were harvested and lysed to perform protein expression analysis on PPARy and C / EBPa. Protein expression levels in EEEP-treated cell lysates were expressed as relative ratios compared to the control. Experimental results were expressed as mean ± SD of triplicate experiments. Statistical significance: * P <0.05, ** P <0.01.
Figure 4 shows the chemical structure of isobutylamide and caffeic acid derivatives. Compounds derived from E. purpurea can be prepared by reacting an alkylamide such as dodeca-2 (E), 4 (E) -dienoic acid isobutylamide with an alkylamide such as chicoric acid, chlorogenic acid, cinarin and echinacoside Caffeic acid derivatives.
FIG. 5 shows the effect of dodeca-2 (E), 4 (E) -dienoic acid isobutylamide or caffeic acid derivatives on lipid accumulation of adipocytes. Differentiation of lipoprotein cells was induced using a mixture of adipocyte differentiation inducing substances, including 0.5 mM IBMX, 1 [mu] M DEX, and 0.2 [mu] g / mL insulin. The adipocyte precursor cells were treated with dodeca-2 (E), 4 (E) -dienoic acid isobutylamide, cholic acid, chlorogenic acid, cinarin, and echinacoside in an amount of 0, 30, 50 μM In the first 4 days of differentiation. On day 6, cells were stained with Oil Red O dye and staining intensity was quantitated. Experimental results were expressed as mean ± SD of triplicate experiments. Isobutylamide refers to dodeca-2 (E), 4 (E) -dienoic acid isobutylamide. Statistical significance: *** P <0.001.
Figure 6 shows the effect of dodeca-2 (E), 4 (E) -dienoic acid isobutylamide on lipid accumulation in adipocytes. 3T3-L1 adipose precursor cells were induced with a mixture of adipocyte differentiation inducing substances including 0.5 mM IBMX, 1 [mu] M DEX, and 0.2 [mu] g / mL insulin. (A) Cells differentiated with dodeca-2 (E), 4 (E) -dienoic acid isobutylamide (0, 5, 10, 20 μM) or 5 μM troglitazone with adipocyte differentiation inducers Day. On day 6, cells were stained with Oil Red O dye and visualized under an optical microscope (x100). (B) The cell triglyceride content was analyzed using a triglyceride quantification kit. Experimental results were expressed as mean ± SD of triplicate experiments. Isobutylamide refers to dodeca-2 (E), 4 (E) -dienoic acid isobutylamide. Statistical significance: * P <0.05, ** P <0.01, *** P <0.001.
FIG. 7 shows the results of measurement of the effect of dodeca-2 (E), 4 (E) -dienoic acid isobutylamide on the expression of PPARγ and C / EBPα in adipocytes. Differentiation of lipoprotein cells was induced with a mixture of adipocyte differentiation inducers, including 0.5 mM IBMX, 1 [mu] M DEX, and 0.2 [mu] g / ml insulin. The adipocyte progenitor cells differentiated dodeca-2 (E), 4 (E) -dienoic acid isobutylamide (0, 5, 10, or 20 μM) with adipocyte differentiation inducers Respectively. On day 6, cells were harvested and analyzed for protein expression against PPARy and C / EBPa on cell lysates. The protein expression values of the cell lysates treated with dodeca-2 (E), 4 (E) -dienoic acid isobutylamide were expressed as relative percentages as compared with the control. Experimental results were expressed as mean ± SD of triplicate experiments. Isobutylamide represents dodeca-2 (E), 4 (E) -dienoic acid isobutylamide. Statistical significance: * P <0.05.
Figure 8 shows the effect of E. purpurea and its derived dodeca-2 (E), 4 (E) -dienoic acid isobutylamide on the induction of low-density insulin-induced adipocyte differentiation .

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Experimental Methods and Materials

1. Experimental material

3T3-L1 cells were purchased from the American Type Culture Collection (Manassas, VA, USA). DMEM (Dulbecco's Modified Eagle's Medium), BCS (bovine calf serum) and FBS (fetal bovine serum) were purchased from Invitrogen (Carlsbad, CA, USA). Insulin was purchased from Roche Diagnostics (Mannheim, Germany) and dodeca-2 (E), 4 (E) -dienoic acid isobutylamide [dodeca-2 (E) Were purchased from Chromadex (Irvine, CA, USA). Caffeic acid derivatives such as dexamethasone, IBMX (3-isobutyl-1-methylxanthine), Oil Red O dye, troglitazone and cichoric acid, chlorogenic acid, cynarin and echinacoside, Chemical Co. (St. Louis, MO, USA). Antibodies to PPARγ, C / EBPα and β-actin were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). All compounds were graded for analysis. The triglyceride quantitation kit was purchased from BioVision Research Products (Mountain View, CA, USA).

2. Preparation of Echinacea root ethanol extract (EEEP)

E. purpurea extract (Tasman Extracts Ltd., Nelson, New Zealand) is available from Tasman Extracts Ltd. The extraction process was carried out according to the following procedure. Briefly, the roots of E. purpurea were extracted with 70% (v / v) ethanol for 4 days at 40 ° C, and the extracts were filtered, concentrated and lyophilized. The resulting powder was dissolved in DMSO and used for further study.

3. Echinacea ( Echinacea purpurea Analysis of Root Extract Ingredients

E. purpurea root extract was obtained from Tasman Extracts Ltd. (Nelson, New Zealand). Chloroic acid, chlorogenic acid, cynarin and echinacoside in E. purpurea root extracts were obtained from Waters SunFire columns (150 X 4.6 mm, 5 μm, Waters Corporation, Milford, Mass., USA) and Agilent 1260 Infinity UV (HPLC) separation module (Agilent Technologies, Palo Alto, Calif., USA) equipped with a UV detector / Vis detector (wavelength UV 330 nm, Agilent Technologies). Dodeca-2 (E), 4 (E) -dienoic acid isobutylamide is directly attached to the Ion Trap Mass Spectrometer (LCQ Fleet, Thermo Scientific) Using a Surveyor HPLC instrument (Thermo Scientific, Waltham, Mass., USA). Chromatographic separation was performed using a Luna C18 column (150 X 2.0 mm, 5 μm, Phenomenex, Inc., Torrance, CA, USA). The SIM transition (m / z) of dodeca-2 (E), 4 (E) -dienoic acid isobutylamide was 252.25. The contents of chicoric acid, chlorogenic acid, cinarin, echinacoside and dodeca-2 (E) and 4 (E) -dienoic acid isobutylamide in E. purpurea root extracts were calculated from the linear regression equation of the calibration curve Respectively.

4. Induction of cell culture and adipocyte differentiation

3T3-L1 adipose precursor cells were cultured and differentiated into adipocytes using a modified method (Choi et al., 2012). Briefly, 3T3-L1 adipose precursor cells from Swiss mouse embryos were cultured in DMEM containing 10% BCS in a 5% CO ₂ incubator at 37 ° C. To induce differentiation, the bottom of the culture vessel was completely covered, and the lipid precursor cells on day 2 were cultured in a differentiation medium containing 10% FBS, 0.5 mM IBMX, 1 M dexamethasone, and 0.2 g / mL insulin for 2 days . The medium was then replaced with DMEM containing 10% FBS and 0.2 μg / mL insulin and cells were further incubated for 2 days. Then, the cells were further cultured in DMEM supplemented with 10% FBS for 2 days.

5. Oil Red O staining

After inducing differentiation of adipocytes, the cells were washed with PBS (phosphate-buffered saline), fixed with 10% formalin for 1 hour at room temperature, stained with Oil Red O dye for 1 hour at room temperature, . For quantitative analysis, Oil Red O staining was dissolved in isopropanol and optical density at 490 nm was measured using a Molecular Devices ELISA reader.

6. Triglyceride analysis

The differentiated cells were washed with PBS, scraped and then dissolved in 5% Triton-X100 solution. The triglyceride content was measured using a triglyceride quantitation kit. For analysis, triglyceride was converted to glycerol and fatty acid, and the glycerol released was oxidized and quantified with a Molecular Devices ELISA reader at 570 nm using a triglyceride standard curve.

7. Protein Expression Analysis

3T3-L1 cells were harvested and lysed with 62.5 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 50 mM dithiothreitol and protease inhibitor cocktail tablet (Roche Diagnostics; Mannheim, Germany) &Lt; / RTI &gt; buffer. Total protein concentrations in the lysates were measured using the BCA protein assay reagent (Pierce; Rockford, IL, USA). Proteins in the lysate were separated by electrophoresis on 12.5-15% SDS polyacrylamide gels and transferred to a polyvinylidene difluoride membrane (GE Healthcare Life Sciences; Piscataway, NJ, USA). Membranes were blocked in 5% BSA (bovine serum albumin, Roche Diagnostics; Mannheim, Germany) and incubated with primary antibodies against PPARγ, C / EBPα, and β-actin for 12 hours at 4 ° C. Membranes were incubated with secondary antibody conjugated with horseradish peroxidase for 12 hours at 4 ° C and visualized with ECL (enhanced chemiluminescence, Amersham Pharmacia Biotech; Buckinghamshire, UK) and analyzed on X-ray film (Eastman Kodak; Rochester, NY, USA) Exposed. The intensity of the band was quantified using the Scion-Image Program for Windows.

8. Statistical analysis

All values are expressed as mean ± standard deviation. Statistical significance was measured by one-way ANOVA using the Newman-Keuls Multiple Comparison test. P - values less than 0.05 were considered statistically significant.

Experiment result

1. Effect of Echinacea root ethanol extract (EEEP) on adipocyte differentiation

Echinacea purpurea root ethanol extract (EEEP) regulates adipocyte differentiation of 3T3-L1 adipose precursor cells. When fat precursor cells were stimulated with a mixture of adipogenic differentiation inducers (0.5 mM IBMX + 1 μM dexamethasone and 1 μg / mL insulin), the fat content of adipocytes treated with 50 μg / mL EEEP was about 69.8% (Fig. 1, A). EEEP also increased the fat content in the cells treated with the insulin-free inducer mixture compared to the control. Thus, these experimental results confirm that EEEP promotes adipocyte differentiation by having similar activity to insulin. When fat precursor cells were treated with 0 - 0.5 μg / mL of insulin, 0.5 mM IBMX and 1 μM dexamethasone, the fat content was significantly increased at all insulin concentrations compared to the control (FIG. 1B). The fat content of adipocytes treated with insulin at 0, 0.1, 0.2, 0.3, 0.4 and 0.5 μg / mL was 7.2%, 19.2%, 30.7%, 27.4%, 26.1% and 25.2% Respectively. Thus, the optimal concentration of insulin required for synergistic induction of adipocyte differentiation with EEEP is 0.2 μg / mL. Preadipocytes were treated with EEEP (0, 10, 30, 50 μg / mL) for the first 4 days of differentiation while inducing differentiation with 0.5 μM IBMX and 1 μM dexamethasone with 0.2 μg / mL insulin. As a result, EEEP significantly increased the accumulation of fat globes in adipocytes in a concentration-dependent manner (Figs. 2a and 2b). In particular, EEEP at 50 μg / mL increased lipid content of adipocytes by about 140% compared to control cells. The triglyceride content of cells as an adipocyte differentiation marker gradually increased with increasing EEEP concentration (Fig. 2C). The triglyceride content of adipocytes treated with 10, 30, and 50 μg / mL of EEEP was 109.4 ± 8.2%, 154.2 ± 8.2%, and 187.7 ± 20.5%, respectively, as compared with the control group.

2. Effect of EEEP on expression of adipocyte differentiation marker protein

PPARγ and C / EBPα are essential transcription factors for adipocyte differentiation. The expression of PPARγ and C / EBPα in the adipocytes treated with EEEP was gradually increased compared to the control group (FIG. 3). The expression levels of PPARγ in adipocytes treated with 10, 30, and 50 μg / mL of EEEP were about 101.2%, 130.2%, and 142.5%, respectively, as compared with the control group. Expression of C / EBPα in adipocytes treated with EEEP showed a pattern similar to PPARγ. Expression of PPARγ and C / EBPα was significantly increased in adipocytes treated with 10 μg / mL or 30 μg / mL EEEP, and this increase was similar to the increase in fat and triglyceride content. Thus, these experimental results suggest that EEEP promotes the differentiation of 3T3-L1 adipose precursor cells by increasing the expression of PPARγ and C / EBPα.

3. Effect of dodeca-2 (E), 4 (E) -dienoic acid isobutylamide on 3T3-L1 adipocyte differentiation

It was examined whether any of the components of Echinacea extract showed 3T3-L1 adipogenic differentiation promoting effect. The chemical structures of the tested compounds dodeca-2 (E), 4 (E) -dienoic acid isobutylamide, chicory acid, chlorogenic acid, cinarin and echinacoside are shown in FIG. A mixture of adipocyte differentiation inducers comprising E. purpurea derived compounds and 0.5 mM IBMX, 1 μM dexamethasone and 0.2 μg / mL insulin was added to 3T3-L1 adipose precursor cells and the cells were transferred to mature adipocytes Lt; / RTI &gt; Although dodeca-2 (E), 4 (E) -dienoic acid isobutylamide significantly increased fat accumulation, caffeic acid derivatives such as chicory acid, chlorogenic acid, cinarin and echinacoside, . The relative fat content of adipocytes treated with 30 μM of dodeca-2 (E), 4 (E) -dienoic acid isobutylamide increased by ~ 2.8-fold compared to the control (FIG. We analyzed the EEEP components and measured the content of each compound. In EEEP, dodeca-2 (E), 4 (E) -dienoic acid isobutylamide and chicory acid were 218.5 ± 11.7 and 8,665.2 ± 33.1 μg / g, respectively. However, chlorogenic acid, cinarin, and echinacoside were not detected (Table 1).

EEEP (μg / g) Dodeca-2 (E), 4 (E) -dienoic acid isobutylamide 218.5 ± 11.7 Chicory acid 8,665.2 ± 33.1

To examine the effect of dodeca-2 (E), 4 (E) -dienoic acid isobutylamide on adipocyte differentiation, 3T3-L1 adipocytes were treated with IBMX and dexamethasone with 0.2 μg / mL insulin (E), 4 (E) -dienoic acid isobutylamide (0, 5, 10, or 20 μM) or 5 μM troglitazone (antidiabetic agent) at the same time.

(E), 4 (E) -dienoic acid isobutylamide gradually increased the accumulation of lipid spheres in 3T3-L1 adipocytes and increased the accumulation of dodeca-2 (E), 4 (E) The accumulation of fat in the adipocytes treated with dienoic acid isobutylamide increased to a level similar to that accumulated in the adipocytes treated with troglitazone (Fig. 6A). The intracellular triglyceride content was significantly increased by dodeca-2 (E), 4 (E) -dienoic acid isobutylamide (0, 5, 10 μM) in a concentration-dependent manner (FIG. In particular, 20 μM of dodeca-2 (E), 4 (E) -dienoic acid isobutylamide increased the content of triglyceride to a level similar to that of 5 μM of troglitazone. The relative content of triglycerides in adipocytes treated with 5, 10, 20 μM dodeca-2 (E), 4 (E) -dienoic acid isobutylamide or 5 μM troglitazone was 161.2 ± 3.5% 26.1%, 231.9 ± 5.8%, or 248.9 ± 20.3%. These results suggest that dodeca-2 (E), 4 (E) -dienoic acid isobutylamide contributes to the promotion of EEEP-induced adipocyte differentiation in the presence of 0.2 μg / mL insulin.

4. Increased expression of adipocyte-2 (E), 4 (E) -dienoic acid isobutylamide by adipocyte differentiation marker protein

The effect of dodeca - 2 (E), 4 (E) - dienoic acid isobutylamide, which was confirmed to be an adipocyte differentiation - promoting active compound, on the expression of PPARγ and C / EBPα as differentiation marker proteins was investigated. The expression of PPARγ and C / EBPα was significantly increased in adipocytes treated with dodeca-2 (E), 4 (E) -dienoic acid isobutylamide compared to the control group (FIG. 7). The expression levels of PPARγ in adipocytes treated with dodeca-2 (E), 4 (E) -dienoic acid isobutylamide at a concentration of 5, 10, or 20 μM were about 115.1%, 133.3% Or 154.7%. Expression of C / EBPα in adipocytes treated with dodeca-2 (E), 4 (E) -dienoic acid isobutylamide showed a pattern similar to that of PPARγ. Thus, the above results demonstrate that dodeca-2 (E), 4 (E) -dienoic acid isobutylamide promotes the differentiation of 3T3-L1 adipose precursor cells by increasing the expression of PPARγ and C / EBPα It suggests.

Review

Adipose tissue is involved in the development of insulin resistance and type 2 diabetes, and adipocyte differentiation can be used to search for insulin signaling pathways and compounds with antidiabetic activity (Choi et al., 2011a; Choi et al ., 2011b). The lipid progenitor cells, similar to non-differentiated fibroblasts, form spherical adipocytes and accumulate lipid droplets during adipocyte differentiation (Ntambi and Young-Cheul, 2000; Tong and Hotamisligil, 2000; 2001; Cornelius et al., 1994; Gregoire et al., 1998). The present inventors investigated whether EEEP can regulate low-density insulin-induced adipocyte differentiation in 3T3-L1 adipose precursor cells, and screened active compounds having such activity. Lipogenic precursor cells can be differentiated by a mixture of IBMX, dexamethasone, and insulin. EEEP increased fat content of adipocytes when treated with a mixture of different inducers of insulin, compared to the control group, but did not show any significant difference when treated with a mixture of differentiation inducers lacking IBMX- or dexamethasone. EEEP increased the fat content of adipocytes in the presence of various concentrations of insulin (Fig. 2). Insulin plays an important role in adipocyte differentiation and binds to insulin receptors that are highly expressed on fat cell surfaces (Saltiel and Kahn, 2001). When lipid precursor cells were treated with EEEP, the triglyceride content was also increased (FIG. 3). Differentiation of adipocytes is regulated by various transcription factors. For example, the PPARs family (PPARα, γ, and δ) and the C / EBPs family (C / EBPα, β, and δ) play an important role in adipocyte differentiation. The inventors have found that the expression of PPARy and C / EBPa is gradually increased in adipocytes treated with EEEP (Fig. 4). Thus, EEEP appears to activate the expression of transcription factors necessary for stimulation of adipocyte differentiation under insulin concentrations to the extent of subinduction. In addition, the inventors have identified active compounds that promote adipocyte differentiation in EEEP. Dodeca-2 (E), 4 (E) -dienoic acid isobutylamide significantly increased fat accumulation compared with the control group. However, caffeic acid inducing agents such as chicory acid, chlorogenic acid, cinarin, and echinacoside did not promote adipocyte differentiation (Fig. 5). The lipids accumulated in the adipocytes treated with dodeca-2 (E), 4 (E) -dienoic acid isobutylamide increased more than twice as much as the control group (FIG. 6). Dodeca-2 (E), 4 (E) -dienoic acid isobutylamide also increased the expression of adipocyte differentiation markers, PPARγ and C / EBPα (FIG. 7). Therefore, these results strongly suggest that dodeca-2 (E), 4 (E) -dienoic acid isobutylamide is an active substance that promotes adipocyte differentiation in EEEP.

conclusion

In the present invention, Echinacea purpurea root extract and dodeca-2 (E), 4 (E) -dienoic acid isobutylamide, which are components contained in this extract, To promote adipocyte differentiation. These results suggest that dodeca - 2 (E), 4 (E) - dienoic acid isobutylamide is the active compound responsible for the promotion of adipocyte differentiation in Echinacea root extract. From the experimental results of the present invention, Echinacea root extract and dodeca-2 (E), 4 (E) -dienoic acid isobutylamide increased the expression of PPARγ and C / EBPα and ultimately had antidiabetic activity and insulin sensitizing activity Respectively.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (8)

Echinacea purpurea root extract or dodeca-2 (E), 4 (E) -dienoic acid isobutylamide as the active ingredient. Or a pharmaceutically acceptable salt thereof.
The method according to claim 1, wherein the Echinacea root extract is selected from the group consisting of water, an anhydrous or a lower alcohol having 1 to 4 carbon atoms, a mixed solvent of the lower alcohol and water, acetone, ethyl acetate, chloroform, butyl acetate, Wherein the solvent is an extract of a solvent selected from the group consisting of benzene, toluene, xylene, n-hexane, n-hexane,
The composition according to claim 1, wherein the Echinacea root extract or dodeca-2 (E), 4 (E) -dienoic acid isobutylamide acts to increase insulin sensitivity of adipocytes.
The method according to claim 1, wherein the Echinacea root extract or dodeca-2 (E), 4 (E) -dienoic acid isobutylamide is characterized in that the expression of PPARγ and C / EBPα is increased in adipocytes / RTI &gt;
A pharmaceutical composition for treating or preventing diabetes comprising Echinacea root extract or dodeca-2 (E), 4 (E) -dienoic acid isobutylamide as an active ingredient.
6. The method of claim 5, wherein the Echinacea root extract is selected from the group consisting of water, an anhydrous or a lower alcohol having 1-4 carbon atoms, a mixed solvent of the lower alcohol and water, acetone, ethyl acetate, chloroform, butyl acetate, Wherein the solvent is an extract of a solvent selected from the group consisting of benzene, toluene, xylene, n-hexane, n-hexane,
The composition according to claim 5, wherein the Echinacea root extract or dodeca-2 (E), 4 (E) -dienoic acid isobutylamide acts to increase insulin sensitivity of adipocytes.
6. The method of claim 5, wherein the Echinacea root extract or dodeca-2 (E), 4 (E) -dienoic acid isobutylamide is characterized by increasing the expression of the transcription factors PPARγ and C / EBPα in adipocytes / RTI &gt;

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