WO2007126293A1

WO2007126293A1 - Use of (-)-catechin for enhancing the expression of adiponectin

Info

Publication number: WO2007126293A1
Application number: PCT/KR2007/002145
Authority: WO
Inventors: Si Young Cho; Pil Joon Park; Hyun Jung Shin; Young-Kyung Kim; Dong Wook Shin; Euiseok Shin; Hyoung Ho Lee; Byeong Gon Lee; Joo-Hyun Baik; Tae Ryong Lee
Original assignee: Amorepacific Corporation
Priority date: 2006-05-01
Filing date: 2007-05-02
Publication date: 2007-11-08
Also published as: KR101558231B1; KR20070106822A

Abstract

(-)-Catechin, a component of green tea, significantly promotes the adiponectin expression in a mammal, and accordingly, it is useful for preventing and treating diabetes, atherosclerosis, or obesity.

Description

USE OF (-)-CATECHIN FOR ENHANCING THE EXPRESSION OF

ADIPONECTIN

FIELD OF THE INVENTION

The present invention relates to a use of (-)-catechin for enhancing the expression of adiponectin, a pharmaceutical composition for enhancing the expression of adiponectin comprising (-)-catechin, and a method for enhancing the adiponectin expression.

BACKGROUND OF THE INVENTION

Adiponectin is one of adipokines secreted by adipocytes, and has been reported to be involved in anti-obesity, anti-diabetes and anti-atherosclerosis mechanisms, and in the regulation of free radical generation (Clin. Nutr. (2004),

23(5):963-974). In particular, adiponectin enhances the insulin sensitivity and reduces the plasma glucose level, while it promotes fatty acid oxidation and suppresses lipogenesis through AMP kinase activation in the liver and skeletal muscle. Thus, it plays a role in the prevention and treatment of diabetes and obesity.

The expression of adiponectin increases during adipocyte differentiation, and it is regulated by several transcription factors such as PPAR-γ (peroxisome proliferative activated receptor-γ), C/EBP-α (CCAAT/enhance binding protein-α), SREBP-Ic (sterol regulartory element binding protein- Ic), LRH-I (liver receptor homolog-1) and KLF7 (kruppel-like factor 7). Among these regulatory transcription factors, KLF7 is a zinc finger protein associated with type 2 diabetes, which is known to be expressed in various tissues to inhibit adipocyte differentiation and adiponectin expression, playing an important role in the pathogenesis of diabetes through impairment of insulin secretion in pancreatic cells {Molecular Endocrinology (2005), 8: 844-856). Although there have been rapid progresses in the understanding of the physiological functions of adiponectin and the signaling pathways mediating adiponectin action, relatively little is known about the mechanisms of its transcriptional regulation, and only a few agents are known to regulate the production of adiponectin. For example, Korean Patent Publication No. 2005- 95411 discloses that a pharmaceutical composition for treatment of type 2 diabetes comprising a protein concentrate derived from Korean millets as an active ingredient can elevate the blood aidponectin level, and Korean Patent Publication No. 2005-115874 relates to an enhancer for the expression of adiponectin comprising a cyanidin compound as an active ingredient. Green tea has been demonstrated to have various therapeutic effects for anti-obesity, anti-diabetes, anti-inflammation and anti-oxidation activities. A recent report has revealed that the injection of a green tea extract into lean and obese Zucker rats significantly lowered the blood glucose and insulin levels, increasing the glucose metabolism in adipocytes (Endocrinology (2000), 141 : 980-987; and J. Agric. Food Chem. (2000), 48: 849-852). Further, there has been a clinical report that about 5 cups of green tea (480 mg of catechin) per day given to diabetes patients have caused a decrease in the blood glucose levels by one half (BMC Pharmacology (2004), 4(18): 1-10). However, the major component of green tea responsible for such therapeutic effects for treating diabetes has not yet been identified. The present inventors have therefore endeavored to develop an effective enhancer for the adiponectin gene expression, and have found that (-)-catechin derived from green tea markedly promotes the adiponectin expression in adipocytes.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to provide an effective enhancer of adiponectin expression in a mammal.

It is another object of the present invention to provide a composition comprising said enhancer for enhancing the expression of adiponectin in a mammal. It is a further object of the present invention to provide an efficient method for enhancing the expression of adiponectin in a mammal.

In accordance with one aspect of the present invention, there is provided a use of (-)-catechin of formula (I) for the manufacture of medicament for enhancing the expression of adiponectin in a mammal.

In accordance with another aspect of the present invention, there is provided a pharmaceutical composition for enhancing the expression of adiponectin, comprising said (-)-catechin as an active ingredient.

In accordance with a further aspect of the present invention, there is provided a method for enhancing the expression of adiponectin in a mammal, comprising the step of administering said (-)-catechin to the mammal.

In accordance with a further aspect of the present invention, there is provided a use of said (-)-catechin for the manufacture of a medicament for suppressing the expression of KLF7 (kruppel-like factor 7) in a mammal. In accordance with a further aspect of the present invention, there is provided a pharmaceutical composition for suppressing the expression of KLF7, comprising said (-)-catechin as an active ingredient.

In accordance with a further aspect of the present invention, there is provided a method for suppressing the expression of KLF7 in a mammal, comprising the step of administering said (-)-catechin to the mammal.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects and features of the present invention will become apparent from the following description of the invention, when taken in conjunction with the accompanying drawings, which respectively show:

FIG 1 : Relative effects of 15 components of green tea on adiponectin expression in 3 T3 -Ll adipocytes;

FIG. 2: Relative inducible effects of 0, 5, 10, 50, 100 and 1000 μM (-)- catechins and 10 μM pioglitazone on the expression of adiponectin in 3T3-L1 adipocytes;

FIG. 3: Relative Effects of 0, 5, 10, 50, 100 and 1000 μM (-)-catechins and 100 nM insulin on glucose uptake in 3T3-L1 adipocytes; and

FIG. 4: Effects of (-)-catechin and pioglitazone on the expressions of Kruppel-like factor 7 (KLF7), peroxisome proliferator-activated receptor-γ (PPAR-γ) and CCAAT enhancer binding protein-α (C/EBP-α) in 3T3-L1 adipocytes.

DETAILED DESCRIPTION OF THE INVENTION (-)-Catechin, (2R,3R)-2-(3,4-dihydroxyphenyl)chromane-3,5,7-triol

(IUPAC nomenclature), is commercially available or can be isolated from plants such as Camellia sinensis (green tea) and Agrimonia eupatolia L. by any one of the known methods, e.g, extraction by heating tee leaves in 100% acetonitrile at

100⁰C for 2 hours (Bull. Korean Chem. Soc. (2007), 28: 276-280).

In an embodiment of the present invention, there are provided the findings of a 2-fold increase in the adiponectin expression and a 4-fold increase in the insulin-stimulated glucose uptake in (-)-catechin-treated 3T3-L1, over untreated control cells (FIGs. 2 and 3). Accordingly, (-)-catechin can promote the adiponectic expression and glucose uptake in a mammal when administered thereto, and in a further aspect, the present invention provides a composition for enhancing the glucose uptake in adipocytes of a mammal, which comprises (-)- catechin as an active ingredient. In another embodiment of the present invention, (-)-catechin- induced increase in the adiponectin expression is mediated by the transcription factor involved in diabetes sensitivity, KLF7 (kruppel-like factor 7) (FIG. 4). In particular, (-)-catechin promotes adipogenesis and augments insulin sensitivity by enhancing the adiponectin expression via the suppression of the expression of KLF7 which has been reported to responsible for type 2 diabetes. Thus, in a further aspect, the present invention provides a composition for suppressing the expression of KLF 7 in adipocytes of a mammal, which comprises (-)-catechin as an active ingredient.

(-)-Catechin can be used for prevention or treatment of variable diseases related to the expression of adiponectin or KLF7, including diabetes, atherosclerosis and obesity. Thus, in a further aspect, the present invention provides a composition for preventing or treating an adiponectin or KLF7 expression-related diseases such as diabetes, atherosclerosis or obesity in a mammal, comprising (-)-catechin as an active ingredient.

In the inventive composition of the present invention, (-)-catechin may be employed in an amount ranging from 0.01 to 100% by weight, preferably from

0.01 to 30% by weight, more preferably from 1 to 15 % by weight based on the weight of the inventive compositions, respectively.

The compositions of the present invention may be formulated for oral or parenteral administration in a conventional manner together with one or more pharmaceutically acceptable excipients such as carriers, diluents and forming agents. Representative examples of the pharmaceutically acceptable excipients include forming agents (e.g., starch, glucose and mannitol); fillers (e.g., calcium hosphate and silicic acid derivative); binding agents (e.g., cellulose derivatives including carboxymethylcellulose and hydroxypropylcellulose, gelatin, alginate, polyvinyl pyrrolidone); lubricants (e.g., talc, potassium or magnesium stearate, hydrogenated castor oil and solid polyethylene glycol); disintegrants (e.g., povidone, Croscamellose Sodium, crospovidone); and surfactants (e.g., polysorbate, cetyl alcohol and glycerol monostearate).

In a further aspect, the present invention provides a method for enhancing glucose uptake in adipocytes of a mammal, comprising the step of administering (-)-catechin to the mammal.

In a further aspect, the present invention provides a method for suppressing the expression of KLF7 in adipocytes of a mammal, comprising the step of administering (-)-catechin to the mammal.

In a further aspect, the present invention provides a method for preventing or treating diabetes, atherosclerosis or obesity in a mammal, comprising the step of administering (-)-catechin to the mammal.

In the present invention, a proposed daily dose of (-)-catechin for administration to a mammal including human is about from 0.1 mg/kg to 50 mg/kg, more preferably about from 1 mg/kg to 20 mg/kg, which may be administered once or several times daily. It should be understood that the daily dose should be determined in light of various relevant factors including the condition to be treated, the severity of the patient's symptoms, the route of administration, or the physiological form of the anticancer agent; and, therefore, the dosage suggested above should not be construed to limit the scope of the invention in anyway. The following Examples are intended to further illustrate the present invention without limiting its scope.

Reference Example: Isolation of (-)-catechin from green tea

20 g of green tea leaves was soaked in 400 ml of 100% acetonitrile, and the mixture was heated at 100⁰C for 2 hours. The acetonitrile extract solution thus obtained was purified and concentrated under a reduced pressure to obtain an extract containing (-)-catechin.

Example 1: Enhancement of adiponectin expression by (-)-catechin in adipocytes

Step 1) Adipocyte Differentiation

Mouse 3T3-L1 (ATCC No. CL 173) preadipocyte cells were incubated in DMEM (Dulbecco's modified Eagle's Medium, Gibco 1210-0038) containing 10% calf serum in 10% CO₂ incubator, while changing the medium every other day, until the cells were 70% confluent. For the differentiation, the postconfluent 3T3-L1 cells were incubated in DMEM containing 10% FBS, 0.5 mM 3-isobutyl- 1-methylxanthine (Sigma), 1 μM dexamethasone (Sigma) and 167 nM insulin (Novo-Nordisk) for 2 days, and then in DMEM containing 167 nM insulin and 10% FBS in the following 2 days. Thereafter, the treated cells were maintained for 2 days in DMEM containing 10% FBS, to obtain differentiated adipocytes.

Step 2) Effects on adiponectin expression by the treatment with green tea components

The differentiated adipocytes obtained in Step 1 were incubated in DMEM (GIBCO-BRL) containing 5% (wt/vol) fatty acid-free FBS for 16 hours, and washed with PBS 3 times. 15 compounds known as components of green tea, i.e., (-)-Epigallocatechin 3-gallate (-EGCG), (-)-epigallocatechin (-EGC), (-)- epicatechin 3 gallate (-ECG), (+)-epicatechin (+EC), (-)-epicatechin (-EC), (-)- gallocatechin 3-gallate (-GCG), (-)-gallocatechin (-GC), (-)-catechin 3-gallate (- CG), (-)-catechin (-C), (+)-catechin (+C), quercetin, gallic acid, theobromine, theophylline and theanine (all purchased from Sigma), were each dissolved in DMSO and dispersed in a medium to a concentration of 1/1000 (v/v). The washed cells were treated with 100 μM of each compound for 24 hours, and the adiponectin level in each diluted medium was determined using a mouse adponectin quantikine kit (Quantikine immunoassay kit, R&D Systems). 10 μM pioglitazone (Sigma), a therapeutic agent for type 2 diabetes, known to increase adiponectin expression and 0.1% (v/v) DMSO for 24 hours were used as positive and negative controls, respectively. The relative adiponectin levels of the treated cells were calculated based on that of the untreated negative control. The results are shown in FIG. 1.

As shown in FIG 1, the adiponectin protein expression is significantly enhanced only in the cells treated with (-)-catechin at a level comparable to that of the positive control, whereas such increase was not detectable in any of the cells treated with other components.

Example 2: Enhancement of adiponectin expression by dose of (-)-catechin in adipocytes

The differentiated adipocytes obtained in Step 1 of Example 1 were incubated in DMEM (GIBCO-BRL) containing 5% (wt/vol) fatty acid-free FBS for 16 hours, washed 3 times with PBS, and treated with 5, 10, 50, 100 and 1000 μM (-)-catechin in 0.1% (v/v) DMSO for 24 hours, respectively. The washed cells were also treated with 10 μM pioglitazone and 0.1% (v/v) DMSO for 24 hours as positive and negative controls, respectively.

Each of the media of the treated cells was harvested and diluted 10,000- fold, and the adiponectin level in each diluted medium was determined using a mouse adponectin quantikine kit (Quantikine immunoassay kit, R&D Systems). The relative adiponectin levels of the treated cells were calculated based on that of the untreated negative control. The results are shown in FIG. 2.

As shown in FIG. 2, (-)-catechin increases the expression of adiponectin protein in differentiated adipocytes in a dose-dependent manner, and in particular, the adiponectin expression level of the cells treated with 50 or 100 μM (-)- catechin was 2 fold higher than that of the negative control. Meanwhile, in the cells treated with 1000 μM (-)-catechin, the adiponectin expression decreased, which may be due to the cytotoxicity of (-)-catechin at a concentration more than 500 μM.

Example 3: Elevation of insulin-stimulated glucose uptake by (-)-catechin in adipocytes

The differentiated adipocytes obtained in Step 1 of Example 1 were incubated in DMEM (GIBCO-BRL) containing 5% (wt/vol) fatty acid-free FBS for 16 hours, washed 3 times with PBS, and treated with 5, 10, 50, 100 and 1000 μM (-)-catechin in 0.1% (v/v) DMSO for 24 h, respectively. The washed cells were also treated with 10O nM insulin for 1 h and 0.1% (v/v) DMSO for 24 hours as positive and negative controls, respectively.

Then, after removing the culture media from the treated cells, each of the cells was washed 3 times with PBS and incubated with 10 μM (2 μCi/ml) 2- deoxy-D-[¹⁴C]glucose for 10 min in HEPES buffer-saline (140 mMNaCl, 5 mM KCl, 2.5 mM MgCl₂, 1 mM CaCl₂, 20 mM HEPES, pH 7.4) to initiate glucose uptake. After three washes in ice-cold PBS, the cells were extracted with 10% SDS and subjected to liquid scintillation counting for ¹⁴C radioactivity to determine glucose uptake in each of the treated cells. Relative glucose uptake levels of the treated cells were calculated based on that of the untreated negative control. The results are shown in FIG 3.

As shown in FIG. 3, (-)-catechin increases glucose uptake in differentiated adipocytes in a dose-dependent manner, and particularly, the glucose uptake in 100 μM (-)-catechin-treated cells was 4 fold higher than that in the untreated negative control. Meanwhile, the glucose uptake in cells treated with 1000 μM (-)-catechin was decreased, which may be due to the cytotoxicity of (-)-catechin at a concentration more than 500 μM.

Example 4: Inhibitory effect of (-)-catechin on KLF7 expression in adipocytes

The differentiated adipocytes obtained in Step 1 of Example 1 were incubated in DMEM (GIBCO-BRL) containing 5% (wt/vol) fatty acid-free FBS for 16 hours, washed 3 times with PBS, and treated with 50 μM (-)-catechin in 0.1% (v/v) DMSO for 24 hours. The washed cells were also treated with 10 μM pioglitazone and 0.1% (v/v) DMSO for 24 hours as positive and negative controls, respectively. The treated cells was washed with PBS, and lysed in 200 μl of a protein extraction buffer (8 M urea, 2% CHAPS (3-[(3-chloramidopropyl) dimethylammonio]-l-propane-sulfonate), 50 mM DTT (dithiothreitol), 2 M thiourea, 2 mM PMSF (phenyl methane sulfonyl fluoride), 100 μg/μl leupeptine cocktail) at room temperature for 10 min. Then, each of the cell lysate was centrifuged at 15,000 g and 4 "C for 10 min, followed by harvesting the supernatant, and the protein content of the supernatant was determined by BIO- Rad Protein Dye Reagent™-based assay. 100 μg of the total protein from each supernatant was resolved by 8% SDS-PAGE, and transferred to PDF membrane (BioRad) at 50 V for 12 hours. The transferred blots were blocked with 5% skim milk solution for 1 hour, and incubated for 1 hour with anti-PPAR-γ antibody (Upstate), anti-C/EBP-α antibody (Cell signaling), anti-KLF7 antibody (Abnova Corporation) or anti-β-actin monoclonal antibody (Sigma) as a primary antibody, and with HRP (horse radish peroxidase)-conjugated anti-rabbit IgG (Amersham) as a secondary antibody. The immunoreactive blots on the membrane were revealed by ECL (enhanced chemiluminescence) kit (Amersham

Biosciences), scanned by PowerLook 2100 XL (Umax), and quantified using

ImageMaster 2D Elite (Amersham Biosciences). The results are shown in FIG 4.

As shown in FIG. 4, the treatment of (-)-catechin remarkably reduces the KLF7 protein expression, compared with the untreated negative control, but does not alter the expression levels of the PPAR-γ and C/EBP-α in differentiated adipocytes. Meanwhile, the expression of PPAR-γ was significantly increased in the pioglitazone-treated cells.

These results suggest that unlike pioglitazone, which enhances adiponectin expression by directly activating PPAR-γ, the ability of (-)-catechin to enhance adiponectin expression may be attributable to the suppression of KLF7 expression.

Preparation Example: Preparation of pharmaceutical medication comprising (-)- catechin

120 mg of the extract containing (-)-catechin obtained in Reference Example, 33 mg of microcrystalline cellulose, 33 mg of dibasic calcium phosphate, 12 mg of sodium starch glycollate and 2 mg of magnesium stearate were mixed, and the mixture was compressed using a conventional tablet-making machine to obtain a tablet comprising (-)-catechin as an active ingredient.

While the invention has been described with respect to the above specific embodiments, it should be recognized that various modifications and changes may be made to the invention by those skilled in the art which also fall within the scope of the invention as defined by the appended claims.