JP2014012659A - Myostatin/smad signal inhibitor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a myostatin/Smad signal inhibitor.SOLUTION: A myostatin/Smad signal inhibitor has a fermented tea extract as an active ingredient, the fermented tea extract including polymer polyphenol that at least includes a procyanidin structure and a structure where B rings of catechins are bonded together in a partial structure and having a number average molecular weight of 9,000-18,000.

Description

  The present invention relates to a myostatin / Smad signal inhibitor.

In recent years, with the development of transportation means and the development of information and communication technology, there is a lack of exercise. Decreased muscle mass and strength due to lack of exercise cause a decline in motor function, especially for elderly people, coupled with muscle changes due to aging, and has a serious adverse effect on the quality of life (QOL). it is conceivable that. Muscle atrophy, falls during daily activities caused by a decrease in muscle mass and strength, and accompanying fractures are the main causes of bedridden in the elderly.
In general, as a means for preventing muscle atrophy and a decrease in muscle mass / muscle strength, practice proper exercise or practice rehabilitation. However, a more effective method is desired in practice because of time and physical reasons, difficulty in maintaining motivation, and the like.

  From such a viewpoint, the search of the component which can adjust a motor function by a nutritional approach is performed, for example, the muscular atrophy inhibitor (patent document 1) which uses proanthocyanidin as an active ingredient, and the polyphenol derived from a fruit as an active ingredient A muscle tension enhancer and a body fat adjuster (Patent Document 2) are proposed.

  On the other hand, myostatin belongs to the transforming growth factor-β (TGF-β) superfamily and is thought to play a role in negatively regulating muscle growth. It has been clarified that myostatin inhibits the proliferation and differentiation of myoblasts (Non-patent Document 1), suppresses the proliferation of satellite cells, and puts the satellite cells in a resting state (Non-patent Document 2). . In addition, myostatin-deficient mice have about two-fold increase in skeletal muscle mass due to muscle fiber hyperplasia and hypertrophy compared to normal mice (Non-patent Document 3), and folli, an endogenous inhibitor of myostatin. It has been reported that an increase in muscle mass is observed in mice overexpressing Stalin (Non-patent Document 4).

The activity of myostatin is that myostatin binds to the activin type II receptor (ActRII) and further causes phosphorylation of the type I receptor (ALK4 or ALK5), whereby an intracellular signal cascade works and phosphorylated I Type receptors phosphorylate Smad2 and 3 in the cell, phosphorylated Smad2 and 3 form a heterodimer, and by forming a complex with Smad4, it can be transferred into the nucleus, and the target gene It is known to occur by binding to upstream SBE (Smad binding element) and initiating transcription (Non-patent Document 5).
Therefore, inhibiting any stage of the myostatin signaling pathway, for example, inhibiting myostatin activity by inhibiting the binding of myostatin to ActRII, inactivating the Smad protein, etc. It is considered that the activity of satellite cells, which are stem cells, is increased to promote muscle hypertrophy / regeneration, which can be expected to improve muscle strength, regulate / increase muscle mass, prevent or improve muscle atrophy, and the like.

On the other hand, fermented teas such as oolong tea and black tea have been reported to contain high-molecular polyphenols produced by highly polymerizing catechins during the production process (fermentation, ripening, drying, etc.), of which procyanidin structure And high molecular weight polyphenols having a number-average molecular weight of 9000 to 18000 including at least a structure in which B rings of catechins are bonded to each other, and prevention and treatment of fatty liver (Patent Document 3), abnormal prion It has been reported that it is useful for inhibiting protein formation (Patent Document 4). Recently, it has been reported that the high-molecular polyphenol has an action of promoting the slowing of muscles and is useful for enhancing endurance and recovering from fatigue (Patent Document 5). Furthermore, a method for easily preparing the polymer polyphenol has been reported (Patent Document 6).
However, it has not been known so far that the high-molecular polyphenol has a myostatin / Smad signal inhibitory action.
In Patent Document 5, the promotion of changes in muscle fiber type (transition from IIb to IIa, slow muscle formation) of skeletal muscle when combined with endurance exercise has been studied. It has the action of promoting the slow muscle formation by promoting the expression of PGC-1α based on activation and the like, and what is the rapid muscle fiber type muscle atrophy and muscle strength reduction associated with aging and the like? It is not related.

JP 2002-338464 A International Publication No. 2005/07496 Pamphlet JP 2007-320958 A JP 2009-29752 A JP 2010-37323 A JP 2012-5413 A

Thomas et al. , J Biol Chem 2000 275: 40235-40243 McCroskery S et al. , J Cell Biol. 2003 162: 1135-1147 McPherron AC et al. , NATURE 1997 387: 83-90. Gioson H et al. , Am J Physiol Endocrinol Metab 2009 297: E157-E164 Joulia-Ekaza D et al. Curr Opin Pharmacol. 2007 7: 310-5

  The present invention relates to providing a myostatin / Smad signal inhibitor.

  As a result of intensive research on substances that inhibit intracellular signal transduction (myostatin / Smad signal) of myostatin, the present inventor has demonstrated excellent myostatin / Smad signal inhibitory action on fermented tea extracts containing specific high-molecular polyphenols. Found that there is.

  That is, the present invention includes a fermented tea extract containing a high molecular polyphenol having a procyanidin structure and a structure in which B rings of catechins are bonded to each other in a partial structure and having a number average molecular weight of 9000 to 18000. It relates to the myostatin / Smad signal inhibitor.

  Since the myostatin / Smad signal inhibitor of the present invention inhibits myostatin signal transduction in cells, it can be used as a pharmaceutical, quasi-drug, food, etc. that can exert effects such as improving muscle strength and suppressing muscle atrophy. It is useful as a material or a preparation for blending into these.

The figure which shows the myostatin / Smad signal dependent transcriptional activity (luciferase activity) of this invention fermented tea extract.

  As used herein, the term “myostatin / Smad signal” refers to the intracellular signaling pathway of Myostatin involving ActRII, Smad protein, and “myostatin / Smad signal inhibition” refers to, for example, inhibition of binding of Myostatin to ActRII, It means that any of the signal transduction is reduced, regulated or inhibited by inactivation of Smad protein or the like. Thereby, the activity of myostatin in cells can be suppressed. The “myostatin / Smad signal inhibitory effect” can be evaluated by discriminating the binding of the Smad complex to SBE and the expression of the luciferase gene, as shown in Examples below.

In the present specification, “improvement of muscle strength” means that muscle contraction force is enhanced.
“Muscular atrophy” means that muscle cells shrink and muscle mass decreases when the rate of muscle protein degradation exceeds the rate of synthesis. It is roughly divided into those caused by gravity exposure (called disuse muscular atrophy) and progressive muscular atrophy caused by diseases such as amyotrophic side sclerosis (ALS). Furthermore, symptoms similar to muscle atrophy may occur with aging, and this is called age-related muscle weakness (sarcopenia). Therefore, “suppression of muscle atrophy” refers to suppression of a decrease in muscle mass due to inactivity, aging, disease, or the like.

The fermented tea extract of the present invention contains a polymer polyphenol having a procyanidin structure and a structure in which B rings of catechins are bonded to each other in a partial structure and having a number average molecular weight of 9000 to 18000.
Such high molecular weight polyphenols are considered to be produced by highly polymerizing catechins in the production process of fermented tea (fermentation, ripening, drying, etc.).

  Here, fermented tea means tea obtained by causing fermentation of tea leaves, and specific examples thereof include semi-fermented tea oolong tea and fermented tea black tea.

  The procyanidin structure is a structure in which the C ring of catechins and the A ring of other catechins are bonded. Polymer polyphenols extracted from fermented tea include a procyanidin structure and a structure in which the B rings of catechins are bonded to each other, as well as a structure in which the B ring of catechins and the A ring of other catechins are bonded. May be. Specific examples of the partial structure of the polymer polyphenol extracted from fermented tea include the following. The partial structures shown below are merely examples, and the polymer polyphenols extracted from fermented tea are considered to have a wide variety of partial structures including partial structures including benzotropolone, benzoquinone, naphthoquinone, and the like. It is done.

The fermented tea extract containing the polymer polyphenol includes (1) a step of mixing a water eluate of fermented tea leaves and an adsorbent, (2) a step of removing non-adsorbed components of the adsorbent, and (3) water. It can be obtained by the step of eluting the adsorbent component of the adsorbent using ethanol which may contain sucrose.
In the above step (1), the water eluate of fermented tea leaves includes, for example, polymer polyphenols contained in fermented tea leaves obtained by adding fermented tea leaves to hot or hot water of 50 ° C. or higher, or boiling water. It is desirable that the aqueous solution contains a water-soluble component.
The adsorbent is not particularly limited as long as it adsorbs a high molecular weight polyphenol, but is preferably a hydrophilic vinyl polymer, more preferably a methacrylate copolymer, and a copolymer of glycidyl methacrylate and ethylene glycol dimethacrylate. Further preferred. Specific examples include hydrophilic vinyl polymer resins (“Toyopearl HW-40” manufactured by Tosoh Corporation).
This step may be performed, for example, in a mode in which an adsorbent is added to a container filled with a water eluate of fermented tea leaves and stirred (the stirring time may be, for example, 5 seconds to 1 hour). You may carry out in the aspect which applies the water eluate of fermented tea leaves to the column filled with. In addition, what is necessary is just to set the mixing ratio of the water eluate of fermented tea leaves, and an adsorbent, for example to 1: 0.1-1: 5 (volume ratio).

  When the step (2) is performed in a mode in which the adsorbent is added and stirred in a container filled with the water eluate of fermented tea leaves in the above step (2), for example, after the adsorbent is filtered on a filter paper, The adsorbent may be washed with a sufficient amount of water. In addition, when the step (1) is performed in such a manner that a water eluate of fermented tea leaves is applied to a column packed with an adsorbent, a sufficient amount of water is passed through the column to wash the adsorbent in the column. Just do it.

In the above step (3), when water-containing ethanol is used as ethanol that may contain water, the ethanol concentration is preferably 40% by volume or more and 60% by volume or more from the viewpoint of elution efficiency of the polymer polyphenol. 90 or less and 80 or less are preferable. Moreover, 60-80 volume% is more preferable and 80 volume% is more preferable.
In addition, when this process performed the process of (1) in the aspect which adds and stirs an adsorbent to the container filled with the water eluate of fermented tea leaves, with respect to the adsorbent wash | cleaned by the process of (2) What is necessary is just to add by adding an elution solvent. Moreover, what is necessary is just to perform by carrying out the elution solvent to the column wash | cleaned at the process of (2), when the process of (1) is performed in the aspect which applies the water eluate of fermented tea leaves to the column filled with the adsorbent.

For the fermented tea extract obtained by the above steps, ethanol is removed by concentration under reduced pressure, for example, and then freeze-dried or spray-dried, whereby the fermented tea extract can be obtained as a powder (pulverization operation). Is not required). Further, if concentrated under reduced pressure instead of freeze drying or spray drying, the fermented tea extract can be obtained as a liquid concentrate. The content of the polymer polyphenol is approximately 7.5% by weight or more (upper limit is approximately 30% by weight), and other components are mainly flavonoid compounds (50% by weight or more). Theaflavins are included. In addition, since this fermented tea extract hardly contains caffeine (1% by weight or less), there is a feature that there is almost no side effect of caffeine.
From such a viewpoint, in the fermented tea extract of the present invention, the mass ratio of the high-molecular polyphenol and caffeine is preferably 15: 2 or more.

As shown in Examples below, the fermented tea extract of the present invention showed an excellent myostatin / Smad signal inhibitory action in a reporter gene assay system using SBE, focusing on the myostatin / Smad signal (Example 1). That is, the fermented tea extract of the present invention inhibits the activity of myostatin by suppressing any stage of the myostatin signal pathway. Thereby, the activity of satellite cells is enhanced, muscle hypertrophy / regeneration is promoted, muscle strength is improved, muscle mass is adjusted / increased, and muscle atrophy is prevented or improved.
Therefore, the fermented tea extract of the present invention can be used for inhibition of myostatin / Smad signal. The use can be in humans or non-human animals, or specimens derived therefrom, and can be therapeutic or non-therapeutic. Here, “non-therapeutic” is a concept that does not include a medical act, that is, a treatment act on the human body by treatment.

  In addition, the fermented tea extract of the present invention can be used as a myostatin / Smad signal inhibitor, and can further be used to produce these agents. At this time, the fermented tea extract of the present invention alone or in addition to the fermented tea extract of the present invention is acceptable in the following target object to be blended, for example, the myostatin / Smad signal inhibitor. You may use what is done. In addition, the said formulation can be manufactured by a conventional method according to the target object which should be mix | blended.

  The myostatin / Smad signal inhibitor may itself be a human or veterinary drug, quasi-drug, food or feed that exhibits the inhibitory effect of myostatin / Smad signal, or the drug, pharmacy It may be a material or a preparation used by blending with an external product or the like. As food, the concept of physiological functions such as inhibition of myostatin / Smad signal, improvement of muscle strength, suppression of muscle atrophy, etc., food / beverage products, functional food / beverage products, food / beverage products for the sick, etc. Includes food for specified health use.

The dosage form of the above-mentioned pharmaceutical (including quasi-drugs) may be any of injections, suppositories, inhalants, transdermal absorption agents, various external preparations, tablets, capsules, granules, powders, syrups, etc. The dosage form may be any of oral administration (internal use) and parenteral administration (external use, injection).
To prepare such pharmaceutical preparations of various dosage forms, the fermented tea extract of the present invention alone or other pharmaceutically acceptable excipients, binders, extenders, disintegrants, interfaces Activators, lubricants, dispersants, buffers, preservatives, flavoring agents, fragrances, coating agents, carriers, diluents, and the like can be used in appropriate combinations.

  Among these dosage forms, the preferred form is oral administration, and the content of the fermented tea extract of the present invention in the preparation is generally preferably 0.00001 to 10% by mass, 0.0001 to More preferably, the content is 1% by mass.

The form of the food may be solid, semi-solid or liquid, for example, various foods such as breads, cakes, noodles, confectionery, jelly, frozen foods, ice creams, dairy products, beverages, Examples include the same forms (tablets, capsules, syrups, etc.) as the above-mentioned oral administration preparations.
In order to prepare various forms of food, the fermented tea extract of the present invention alone or other food materials, solvents, softeners, oils, emulsifiers, preservatives, fragrances, stabilizers, colorants, Antioxidants, humectants, thickeners and the like can be used in appropriate combinations. The content of the fermented tea extract of the present invention in the food (in terms of dry matter of the extract) is generally preferably 0.01 to 100% by mass, and preferably 0.1 to 100% by mass. Is more preferable, and more preferably 1 to 100% by mass.

Examples of the above feed include livestock feed used for cattle, pigs, chickens, sheep, horses, etc., feed for small animals used for rabbits, rats, mice, etc., feed for seafood used for tuna, eel, Thailand, Hamachi, etc. Pet foods used for dogs, cats, small birds, squirrels and the like.
In addition, when producing feed, in addition to the fermented tea extract of the present invention, meat such as cattle, pigs and sheep, proteins, grains, bran, potatoes, sugars, vegetables, vitamins, minerals In general, feed raw materials generally used, and gelling agents, shape-preserving agents, pH adjusters, seasonings, preservatives, nutritional reinforcing agents and the like generally used in feeds can be used in combination.
Moreover, although content of the fermented tea extract of this invention in feed changes with the usage forms, it is 0.0001-20 mass% normally, 0.001-10 mass% is preferable, 0.01- 5 mass% is more preferable.

  The dose or intake of the myostatin / Smad signal inhibitor of the present invention may vary according to the subject's condition, body weight, sex, age or other factors, but for oral administration or intake per adult The fermented tea extract is preferably 0.01 mg to 100 mg / kg per day, more preferably 0.1 mg to 10 mg / kg. Moreover, although the said formulation can be administered or ingested according to arbitrary administration schedules, it is preferable to administer or ingest it once to several times a day.

  The subject of administration or ingestion is not particularly limited as long as it is a person who needs it, but the myostatin / Smad signal inhibitor of the present invention can specifically inhibit myostatin / Smad signal and improve muscle strength. , Exercise enthusiasts and athletes, people with locomotive syndrome, people with age-related muscle weakness (sarcopenia), neuromuscular diseases (inflammatory myopathy, myopathy associated with medical diseases, muscular dystrophy, congenital myopathy, mitochondrial encephalomyopathy , Glycogenosis, etc.), administration, or ingestion is effective in persons with exercise deficiency, bed rest, and rehabilitation trainers after surgical / medical diseases.

With respect to the above-described embodiment, the following aspects are disclosed in the present invention.
<1> Myostatin comprising as an active ingredient a fermented tea extract containing a high molecular polyphenol having a procyanidin structure and a structure in which B rings of catechins are bonded to each other in a partial structure and having a number average molecular weight of 9000 to 18000 / Smad signal inhibitor.
<2> Muscle strength comprising a fermented tea extract containing a high-molecular polyphenol having a procyanidin structure and a structure in which B rings of catechins are bonded to each other in a partial structure and having a number average molecular weight of 9000 to 18000. Improver.
<3> a polymer polyphenol having a procyanidin structure and a structure in which B rings of catechins are bonded to each other and having a number average molecular weight of 9000 to 18000 for producing a myostatin / Smad signal inhibitor Use of fermented tea extract containing.
<4> Fermentation containing a high molecular polyphenol having a procyanidin structure and a structure in which B rings of catechins are bonded to each other, and having a number average molecular weight of 9000 to 18000, for producing a muscle strength improver. Use of tea extract.
<5> Contains a high molecular weight polyphenol having a procyanidin structure and a structure in which B rings of catechins are bonded to each other and having a number average molecular weight of 9000 to 18000, for use in inhibiting myostatin / Smad signal Fermented tea extract.
<6> Fermented tea containing a high molecular weight polyphenol having a procyanidin structure and a structure in which B rings of catechins are bonded to each other and having a number average molecular weight of 9000 to 18000 for use in improving muscle strength Extract.
<7> Myostatin that administers or ingests a fermented tea extract containing a high-molecular polyphenol having a procyanidin structure and a structure in which B rings of catechins are bonded to each other in a partial structure and having a number average molecular weight of 9000 to 18000 / Smad signal inhibition method.
<8> Muscular strength for administering or ingesting a fermented tea extract containing a high molecular polyphenol having a procyanidin structure and a structure in which B rings of catechins are bonded to each other in a partial structure and having a number average molecular weight of 9000 to 18000 How to improve.
<9> Myostatin for administering or ingesting a fermented tea extract containing a high-molecular polyphenol having a procyanidin structure and a structure in which B rings of catechins are bonded to each other in a partial structure and having a number average molecular weight of 9000 to 18000 / Non-therapeutic method of inhibiting Smad signal.
<10> A non-administered or ingested fermented tea extract containing a high-molecular polyphenol having a procyanidin structure and a structure in which B rings of catechins are bonded in a partial structure and having a number average molecular weight of 9000 to 18000. Therapeutic muscle strength improvement method.
<11> Food containing as an active ingredient a fermented tea extract containing a high-molecular polyphenol having a procyanidin structure and a structure in which B rings of catechins are bonded in a partial structure and having a number average molecular weight of 9000 to 18000. Method for non-therapeutic inhibition of myostatin / Smad signal using
<12> Food containing as an active ingredient a fermented tea extract containing a high molecular weight polyphenol having a procyanidin structure and a structure in which B rings of catechins are bonded in at least a partial structure and having a number average molecular weight of 9000 to 18000 A non-therapeutic method for improving muscle strength.
<13> In the above items <1> to <12>, the mass ratio of the polymer polyphenol and caffeine in the fermented tea extract is preferably 15: 2 or more.
<14> In the above items <1> to <12>, the fermented tea extract removes non-adsorbed components of the adsorbent after mixing the water eluate of fermented tea leaves and an adsorbent made of a hydrophilic vinyl polymer. Then, it is obtained by eluting the adsorbing component of the adsorbent using ethanol which may contain water.
<15> In the above <14>, the ethanol that may contain water is preferably hydrous ethanol having an ethanol concentration of 40% or more.
<16> In the above item <14>, the hydrophilic vinyl polymer is preferably a methacrylate copolymer, more preferably a copolymer of glycidyl methacrylate and ethylene glycol dimethacrylate.

Production Example 1 [Preparation of fermented tea extract and analysis of polymer polyphenol]
(A) Preparation of fermented tea extract The preparation method and analysis described in JP 2012-5413 A were performed.
30 g of commercial black tea (Daily Club: trade name of Mitsui Norin) is poured into 1 liter of weakly boiled water, kept as it is for about 1 minute, and further maintained for 10 minutes with occasional stirring, so that Sexual components were extracted. After that, no. Two filter papers (Advantech) were spread and suction filtered to obtain a filtrate (water eluate of black tea). To this filtrate, 250 ml of Toyopearl HW-40F (product name, particle size: 30 μm to 60 μm) of Tosoh Corporation previously washed with water as an adsorbent made of a hydrophilic vinyl polymer was added and stirred for 1 minute, and then Buchner No. Two filter papers (Advantech) were spread on each other, and suction filtration was performed. The adsorbent was collected on the filter paper, and the adsorbent on the filter paper was washed 10 times with 150 mL of water at one time. Next, the operation of adding 150 mL of water-containing ethanol having an ethanol concentration of 80% to the adsorbent on the filter paper and eluting the adsorbed components of the adsorbent was performed 12 times. The eluate of the adsorbent component of 12 adsorbents was concentrated under reduced pressure at 50 ° C. to remove ethanol, and then freeze-dried to obtain 2.96 g of an extract containing high-molecular-weight polyphenol derived from black tea. Got as. The content of the components contained in this black tea-derived extract was quantified by the following method.

(B) Analysis of polymer polyphenol (a) Analytical apparatus: medium pressure pump (SP11, Tokyo Rika Instruments) 2 units, medium pressure liquid chromatography column (φ1 × 30 cm) packed with Toyopearl HW-40F, sample injector ( An apparatus capable of linear concentration gradient of the solvent was assembled using Injector VI (Tokyo Rika Instruments) and fraction collector (CHF161RA, Advantech).
(A) Developing solvent: 90 mL hydrous acetone having an acetone concentration of 20% as solvent A, 90 mL hydrous acetone having an acetone concentration of 50% as solvent B, a flow rate of 1.5 g / 4 minutes, The ratio was changed linearly from 100: 0 to 0: 100.
(C) Sample injection: 40 mg of sample was dissolved in 2 mL of water-containing acetone having an acetone concentration of 20%.
(D) Fractionation: The eluate was fractionated by 1.5 g with a fraction collector (total number of fractions: 115). An elution curve was drawn based on the measurement result of the absorbance at 350 nm of the eluate, and fractionated according to this. Each fraction was concentrated under reduced pressure at 50 ° C. to remove acetone, freeze-dried, weighed, and the ratio of the components constituting the fraction in the sample was determined from the weight.

(C) Analytical result The target polymer polyphenol has a fraction no. It was obtained as a fraction of 75 to 110 and its content in the sample was 25.6% by weight. It should be noted that the fact that this fraction is the target high molecular weight polyphenol indicates that the average molecular weight was measured by the method described in JP-A-2007-320958 (number average molecular weight: 1.36 × 10 ⁴ , weight average molecular weight: 1.58 × 10 ⁴ ) and based on the result of the structural analysis (having the partial structure shown below). Flavonoid compounds are available in fraction no. It was obtained as a fraction of 20 to 74 and its content in the sample was 60.6% by weight.

(D) Analysis of other components Samples were precisely weighed and analyzed using an analysis sample prepared by dissolving in a fixed amount of water-containing acetonitrile having an acetonitrile concentration of 10%.
(A) Analyzer: Shimadzu LC-10A system (pump LC-10AD, column open CTO-10A, detector SPD-M10AVP) was used. As the column, Inertsil ODS-3, φ4.6 × 250 mm, particle size of 5 μm (GL Science) was used.
(A) Analysis conditions: The injection amount of the analysis sample was 4.0 μL, and the flow rate was 0.7 mL / min. The column temperature was 40 ° C.
(C) Developing solvent: A water-containing acetonitrile (containing 0.02% TFA) having an acetonitrile concentration of 5% as the solvent A, and a water-containing acetonitrile (containing 0.02% TFA) having a acetonitrile concentration of 40% as the solvent B And the ratio of the B solvent were changed linearly from 100: 0 to 0: 100 in 70 minutes.
(D) Detection: 280 nm and 375 nm were detected wavelengths, and the ratio of the components contained in the sample in the sample was determined from the number of areas of the standard substance and the number of areas of the analysis sample.
(E) Results: Samples include catechins (epigallocatechin: 3.5% by weight, epigallocatechin gallate: 3.8% by weight) and theaflavins (theaflavin 1: 1.1% by weight, theaflavin 2ab: 1 2% by weight and theaflavin 3: 3.8% by weight). The content of caffeine contained in the sample was 0.38% by weight.

(E) Analysis Results of Other Components From the above results, it was found that about 1/4 of the tea-derived extract obtained by the above-described preparation method is composed of high-molecular polyphenol. In addition, since the caffeine content of this extract was 1% by weight or less, the above-described preparation method contained a high content of black tea-derived high-molecular polyphenol, and the decaffeinated extract. It was found that the method can be easily prepared using only water and ethanol.

  In addition, it was confirmed that the same polymer polyphenol was obtained by a method using oolong tea instead of black tea or a method using hydrous ethanol having an ethanol concentration of 40% instead of hydrous ethanol having an ethanol concentration of 80%.

Example 1 [Myostatin / Smad signal inhibition activity test]
HEK293 cells were seeded in 96-well plates and cultured overnight in DMEM (10% FBS). Signal Reporter plasmid included in Signal ^™ Reporter Assay Kits (manufactured by SABiosciences) (a mixture of a reporter plasmid having a Smad Binding Element upstream of the firefly luciferase gene and a Renilla luciferase for internal standard) in a ratio of 40: 1. Was introduced into the cells using a transfection reagent (Lipofectamine 2000; Invitrogen).
Six hours later, as a test substance, DMEM (0.2%) containing the fermented tea extract prepared in Production Example 1 at final concentrations of 0.001 (w / v)% and 0.002 (w / v)%, respectively. The medium was changed to (FBS), and further 2 hours later, DMEM (0.2% FBS) containing Myostatin (0.05 μg / ml) was added and cultured overnight. On the next day, firefly and Renilla luciferase activities were each measured with a luminometer using Dual-Glo (registered trademark) Luciferase Reporter Assay System (Promega). As a control (CNT), 20 (v / v)% ethanol was used instead of the polymer polyphenol. The results are shown in FIG.

Myostatin / Smad signal-dependent transcriptional activity (luciferase activity) was defined as follows.
Myostatin / Smad signal-dependent transcriptional activity (luciferase activity) = (firefly luciferase activity) / (Renilla luciferase activity)
As a result of the myostatin / Smad signal inhibition activity test, the transcription activation rate when the myostatin-unstimulated luciferase activity was 0% and the myosatin-stimulated luciferase activity was 100% was expressed in%.

  As shown in FIG. 1, myostatin / Smad signal-dependent transcriptional activity (luciferase activity) is suppressed in a concentration-dependent manner by the fermented tea extract, and the fermented tea extract has a significant myostatin / Smad signal inhibitory action. It was confirmed that

Claims (4)

  Myostatin / Smad signal containing as an active ingredient a fermented tea extract containing a high molecular weight polyphenol having a procyanidin structure and a structure in which B rings of catechins are bonded to each other in a partial structure and having a number average molecular weight of 9000 to 18000 Inhibitor.   The myostatin / Smad signal inhibitor according to claim 1, wherein the mass ratio of the high-molecular polyphenol and caffeine in the fermented tea extract is 15: 2 or more.   After the fermented tea extract is mixed with the water eluate of fermented tea leaves and an adsorbent made of hydrophilic vinyl polymer, the non-adsorbed components of the adsorbent are removed, and then ethanol that may contain water is used. The myostatin / Smad signal inhibitor according to claim 1, which is obtained by eluting the adsorbed component of the adsorbent.   The myostatin / Smad signal inhibitor according to claim 3, wherein the ethanol which may contain water is hydrous ethanol having an ethanol concentration of 40% or more.

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