JP2013091608A - Myostatin/smad signal inhibitor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a myostatin/Smad signal inhibitor.SOLUTION: The myostatin/Smad signal inhibitor has a black tea extract as an active ingredient.

Description

  The present invention relates to a myostatin / Smad signal inhibitor, a satellite differentiation promoter, a muscle mass increasing agent, and a muscle atrophy 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 based on 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. In addition, a muscle slow muscle accelerator (Patent Document 3) containing a high molecular weight polyphenol extracted from fermented tea as an active ingredient has been proposed.
However, the conventional method cannot be said to be sufficient in terms of effect, and in Patent Document 3, the change in muscle fiber type of skeletal muscle when combined with endurance exercise (transition from IIb to IIa, slow muscle formation) ) Although promotion has been examined, no effect on muscle atrophy or muscle mass reduction due to lack of exercise or aging has been studied.

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).
Therefore, by inhibiting the activity of myostatin in the cells, the activity of satellite cells is increased to promote muscle hypertrophy / regeneration, and the prevention and improvement of muscle atrophy by regulating / increasing muscle mass can be expected.

  Myostatin binds to the activin type II receptor (ActRII) and further causes phosphorylation of the type I receptor (ALK4 or ALK5), thereby causing an intracellular signal cascade. Phosphorylated type I receptor phosphorylates intracellular Smad2 and 3 and phosphorylated Smad2 and 3 form a heterodimer, and then form a complex with Smad4 to enter the nucleus It is known to bind to SBE (Smad binding element) upstream of the target gene and initiate transcription (Non-patent Document 5).

JP 2002-338464 A International Publication No. 2005/074962 JP 2010-37323 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

  In view of such circumstances, 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 found that the tea extract has an excellent myostatin / Smad signal inhibitory action and further differentiation of satellite cells. It has been found that there is an action to promote.

That is, the present invention relates to the following (1) to (4).
(1) A myostatin / Smad signal inhibitor comprising black tea extract as an active ingredient.
(2) A satellite cell differentiation promoter containing black tea extract as an active ingredient.
(3) A muscle mass increasing agent comprising black tea extract as an active ingredient.
(4) A muscle atrophy inhibitor comprising black tea extract as an active ingredient.

  The myostatin / Smad signal inhibitor of the present invention inhibits myostatin signal transduction in cells and promotes the differentiation of satellite cells, and thus can exert effects such as increase in muscle mass and suppression of muscle atrophy. It is useful as a pharmaceutical, quasi-drug, food, etc., or as a material or preparation for blending into these.

It is a figure which shows the myostatin / Smad signal dependent transcriptional activity (luciferase activity) of a black tea extract. It is a figure which shows the expression level of the MyHC1 gene which is concerned in the satellite cell differentiation by a black tea extract. It is a figure which shows the expression level of the myostatin gene by a black 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, “satellite cells” are undifferentiated stem cells that exist between muscle cell membranes, and “satellite cell differentiation promotion” refers to the activation of satellite cells, cell division, and differentiation to existing muscles. It means to lead to myotube formation by cell fusion or by the cell itself.

In this specification, “increase in muscle mass” means, for example, an increase in muscle fiber cross-sectional area or muscle fiber diameter due to proliferation of satellite cells and fusion to muscle fibers, and degradation of muscle protein synthesis rate. Exceeding speed means increasing the protein weight in muscle tissue and increasing muscle weight.
In addition, “muscle atrophy” means that muscle cells decrease / shrink due to the degradation rate of muscle protein exceeding the synthesis rate, and muscle mass decreases, cast fixation by long-term resting bed or fracture, Or it is divided roughly into what is based on microgravity exposure (it is called disuse muscular atrophy), and progressive muscular atrophy 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.

  In the present specification, “non-therapeutic” is a concept that does not include a medical act, that is, a treatment act on the human body by treatment.

  In the present specification, “improvement” refers to improvement of a disease, symptom or condition, prevention, suppression or delay of deterioration of the disease, symptom or condition, or reversal, prevention, suppression or delay of progression of the disease or symptom.

  As used herein, “prevention” refers to prevention, suppression or delay of the onset of a disease or symptom in an individual, or reduction of the risk of onset of a disease or symptom in an individual.

The black tea extract used in the present invention means an extract obtained from black tea, and examples of black tea include the genus Camellia, such as C.I. sinensis, C.I. Examples include black tea made from tea leaves obtained from assamica or hybrids thereof through a fermentation process. The tea leaf is not particularly limited, and examples thereof include Nuwara Eliya, Uba, Dimbra, Darjeeling, Assam, Sri Lanka, and the like, and these can be used alone or in combination of two or more.
The method for obtaining the black tea extract is not particularly limited, and means such as solid-liquid extraction, liquid-liquid extraction, dipping, decoction, leaching, reflux extraction, ultrasonic extraction, microwave extraction, and stirring can be used.

  As the solvent for extraction, either a polar solvent or a nonpolar solvent can be used. Specific examples of the solvent include water; alcohols such as methanol, ethanol, propanol and butanol; polyhydric alcohols such as propylene glycol and butylene glycol; ketones such as acetone and methyl ethyl ketone; methyl acetate and ethyl acetate Esters; linear and cyclic ethers such as tetrahydrofuran and diethyl ether; polyethers such as polyethylene glycol; hydrocarbons such as squalane, hexane, cyclohexane and petroleum ether; aromatic hydrocarbons such as toluene; dichloromethane and chloroform And halogenated hydrocarbons such as dichloroethane; and supercritical carbon dioxide; pyridines; organic solvents such as oils and fats and other oils; and mixtures thereof. Suitable examples include water, alcohols, and alcohol-water mixtures, with water being more preferred.

  The temperature of the solvent when extracting from the tea leaves is preferably 70 to 100 ° C. (boiling water), more preferably 80 to 100 ° C. (boiling water) from the viewpoint of flavor. As for the quantity of the solvent at the time of extraction from black tea leaf, 5-70 mass times is preferable with respect to black tea leaf, More preferably, it is 5-60 mass times. The extraction time from the tea leaves is preferably 30 seconds to 60 minutes, more preferably 30 seconds to 40 minutes, and even more preferably 30 seconds to 30 minutes.

  For the black tea extract, the extract or fraction may be used as it is, may be used as a diluted solution diluted with an appropriate solvent, or may be a concentrated extract, a dry powder, or a paste. Also, freeze-dry and dilute with a solvent usually used for extraction, such as water, ethanol, propylene glycol, butylene glycol, water / ethanol mixture, water / propylene glycol mixture, water / butylene glycol mixture, etc. It can also be used. It can also be used by encapsulating in vesicles such as liposomes or microcapsules.

  The black tea extract of the present invention may be a crude product as long as it conforms to food and pharmaceutical acceptable standards and exhibits the effects of the present invention. These purities may be increased by appropriately combining separation and purification methods. Examples of the purification means include organic solvent precipitation, centrifugation, ultrafiltration membrane, high performance liquid chromatograph, column chromatograph and the like.

As shown in Examples described later, the black 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. Moreover, the black tea extract of the present invention enhanced the expression of MyHC1 (Myosin heavy chain1) gene related to myogenic differentiation, while suppressing the expression of myostatin gene.
Myosin heavy chain (MyHC) is a major constituent protein of muscle and plays an important role in muscle contraction. Increased MyHC gene expression enhances muscle contraction protein synthesis, resulting in muscle fiber hypertrophy and skeletal muscle hypertrophy. Moreover, since the myostatin gene expression was suppressed, the muscle loss signal was released, and it is considered that protein synthesis and skeletal muscle hypertrophy were caused.
Therefore, the black tea extract of the present invention is effective in preventing, improving, or treating various diseases and symptoms that are considered to be useful to inhibit the signal transduction of myostatin in cells and to promote the differentiation of satellite cells. Conceivable. For example, the black tea extract of the present invention is considered effective for increasing muscle mass and suppressing muscle atrophy.

  The black tea extract of the present invention can be used for inhibiting myostatin / Smad signal, promoting differentiation of satellite cells, or increasing muscle mass or suppressing muscle atrophy. The use can be in humans or non-human animals, or specimens derived therefrom, and can be therapeutic or non-therapeutic.

  The black tea extract of the present invention can be used as a myostatin / Smad signal inhibitor, satellite cell differentiation promoter, muscle mass increasing agent, muscle atrophy inhibitor (hereinafter, myostatin / Smad signal inhibitor, etc.) Furthermore, it can be used to produce these agents. At this time, the myostatin / Smad signal inhibitor and the like are acceptable in the following target substance to be blended, such as a carrier selected as necessary alone or in addition to the black tea extract of the present invention. May be used. 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, etc. is a drug for humans or animals, quasi-drugs, foods that exerts the effect of inhibiting myostatin / Smad signal, promoting the differentiation of satellite cells, increasing muscle mass, and suppressing muscle atrophy. Or it can mix | blend and use as an active ingredient of feed. Food and drink, functional food and drink with the concept of physiological functions such as inhibition of myostatin / Smad signal, promotion of differentiation of satellite cells, increase of muscle mass, suppression of muscle atrophy, etc. It can be applied to food and drink for the sick, food for specified health use, etc.

When the myostatin / Smad signal inhibitor of the present invention is used as an active ingredient of a pharmaceutical product (including quasi-drugs), these can be administered in any dosage form. Examples of the dosage form include oral, enteral, transmucosal, injection and the like. Examples of the dosage form of the preparation for oral administration include tablets, coated tablets, capsules, granules, powders, powders, sustained-release preparations, suspensions, emulsions, oral liquids, dragees, pills, fine granules Agents, syrups, elixirs and the like. Examples of parenteral administration include intravenous injection, intramuscular injection, inhalation, infusion solution, suppository, inhalant, percutaneous absorption agent, eye drop, nasal drop and the like.
In order to prepare such pharmaceutical preparations of various dosage forms, the myostatin / Smad signal inhibitor of the present invention may be used alone or in combination with other pharmaceutically acceptable carriers. Examples of such carriers include excipients, binders, extenders, disintegrants, surfactants, lubricants, dispersants, buffers, preservatives, flavoring agents, fragrances, coating agents, diluents, and the like. Is mentioned.

  Among these dosage forms, the preferred form is oral administration, and the content of the black tea extract of the present invention in a preparation containing a myostatin / Smad signal inhibitor or the like (in terms of dried extract) is generally 0. It is preferable to set it as 00001-10 mass%, and it is more preferable to set it as 0.0001-1 mass%.

When the myostatin / Smad signal inhibitor of the present invention is used as an active ingredient of food, the form of the food may be solid, semi-solid or liquid, for example, breads, cakes, noodles, confectionery, jellys In addition to various foods such as frozen foods, ice creams, dairy products, and beverages, the same forms (tablets, capsules, syrups, etc.) as the above-mentioned oral administration preparations can be mentioned.
To prepare various forms of food, the myostatin / Smad signal inhibitor 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 combination. The content of the black tea extract of the present invention in the food (in terms of dried extract) is generally preferably 0.01 to 100% by mass, and preferably 0.1 to 100% by mass. More preferably, it is more preferably 1 to 100% by mass.

In addition, when the myostatin / Smad signal inhibitor of the present invention is used as an active ingredient in feed, the feed includes, for example, livestock feed used for cattle, pigs, chickens, sheep, horses, rabbits, rats, Examples include small animal feeds used for mice and the like, fish and shellfish feeds used for tuna, eel, Thailand, Hamachi, pet foods used for dogs, cats, small birds, squirrels, and the like.
When producing feed, in addition to the myostatin / Smad signal inhibitor of the present invention, meat such as cattle, pigs, sheep, proteins, grains, bran, potatoes, sugars, vegetables, vitamins In addition, feed materials generally used for minerals and the like, and gelling agents, shape-preserving agents, pH adjusters, seasonings, preservatives, nutrient reinforcements and the like generally used for feed can be used in combination.
Moreover, although content of the black tea extract of this invention in feed changes with the usage forms, it is 0.0001-20 mass% normally in dry matter conversion, and 0.001-10 mass% is preferable, 0.01-5 mass% is more preferable

The dose or intake of the myostatin / Smad signal inhibitor, etc. of the present invention may vary according to the subject's condition, weight, sex, age or other factors, but in the case of oral administration or intake, tea extraction per adult It is preferable to set it as 0.01 mg-100 mg / kg per day as a thing (dry matter conversion), Furthermore, it is preferable to set it as 0.1 mg-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 inhibits myostatin / Smad signal, promotes differentiation of satellite cells, increases muscle mass. Because it can control muscle atrophy, it is especially suitable for exercise enthusiasts and athletes, patients with locomotive syndrome, people with age-related muscle weakness (sarcopenia), neuromuscular diseases (inflammatory muscle diseases, medical diseases). It is effective for administration or ingestion in patients with concomitant myopathy, muscular dystrophy, congenital myopathy, mitochondrial encephalomyopathy, glycogenopathy, etc., those with exercise deficiency, bed rest, and rehabilitation trainers after surgical / medical diseases.

Production Example 1 [Preparation of black tea extract]
(1) Black tea extract A
SD tea extract powder # 16691 (Kenyan tea spray-dried product, Saneigen FFI Co., Ltd.) dissolved in 20 (v / v)% ethanol water to a concentration of 1 (w / v)%, A test sample was obtained.

(2) Black tea extract B
After extracting 7 g of commercially available tea leaves (manufactured by Mitsui Norin Co., Ltd.) with 400 mL of distilled water at 90 ° C. for 1 minute, the mixture was filtered with No. 2 filter paper (ADVANTEC) to obtain an extract. The obtained extract was freeze-dried to obtain 1.37 g of black tea extract. This was dissolved in 20 (v / v)% ethanol water so as to have a concentration of 1 (w / v)%, and used as a test sample.

(3) Black tea extract C
SD black tea extract powder # 16693 (a Kenya tea spray-dried product, manufactured by San-Ei Gen FFI Co., Ltd.) was dissolved in 20 (v / v)% ethanol water to a concentration of 1 (w / v)%, A test sample was obtained.

(4) Black tea extract D
Dissolve FD black tea extract powder # 16600 (freeze-dried tea from Kenya, Saneigen FFI Co., Ltd.) in 20 (v / v)% ethanol water to a concentration of 1 (w / v)%, A test sample was obtained.

(5) Black tea extract E
FD Chinese tea extract powder # 16342 (Chinese tea freeze-dried product, manufactured by San-Ei Gen FFI Co., Ltd.) was dissolved in 20 (v / v)% ethanol water to a concentration of 1 (w / v)%. A test sample was obtained.

Example 1 [Myostatin / Smad signal inhibition activity test]
HEK293 cells (human embryonic kidney cells) were seeded in 96-well plates and cultured overnight in DMEM (10% FBS). Signal Reporter plasmid included in Signal ^™ Reporter Assay Kits (SABiosciences) (a mixture of reporter plasmid with SBE upstream of firefly luciferase gene and Renilla luciferase for internal standard in a ratio of 40: 1) was transfected. Reagents (Lipofectamine 2000; Invitrogen) were used to introduce into cells. Six hours later, the medium was changed to DMEM (0.2% FBS) each containing black tea extracts A to E having a final concentration of 0.002 (w / v)% as a test substance, and two hours later, myostatin ( DMEM (0.2% FBS) containing 0.05 μg / mL; PEPROTECH) was added and cultured overnight. On the next day, firefly and Renilla luciferase activities were measured with a luminometer using Dual-Glo (registered trademark) Luciferase Reporter Assay System (Promega). As a control, 20 (v / v)% ethanol was used instead of the black tea extract. 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)
The result of the myostatin / Smad signal inhibition activity measurement test shows the transcription activation rate in% when the myostatin-unstimulated luciferase activity is 0%, and the myostatin-stimulated luciferase activity is 100%, indicating a suppression of 50% or more. Was used as a myostatin / Smad signal inhibitor.
The numerical value was shown by the average value +/- standard error (Average (Ave) +/- standard error (SE)). Regarding the statistically significant difference between the groups, t-test with respect to the control group was performed, and when the p value was less than 0.01, there was a significant difference, and ** was shown in the figure.

  As shown in FIG. 1, the myostatin / Smad signal-dependent transcriptional activity (luciferase activity) is suppressed by about 50% or more by various black tea extracts, and the black tea extract has a significant myostatin / Smad signal inhibitory action. It was confirmed that

Example 2 [Genetic expression analysis of primary cultured satellite cells derived from human adult skeletal muscle under differentiation-inducing conditions]
Primary cultured satellite cells derived from human adult skeletal muscle (Lonza) are seeded in a 12-well plate, and 24 hours later, to examine the gene expression of MyHC1 (Myosin heavy chain 1), a final concentration of 0.002 (w / w) v) Replacement with DMEM medium (2% Horse Serum) containing each of black tea extracts A to E was performed, and differentiation induction was started. After differentiation induction, the cells were cultured for 48 hours, washed with PBS, and the cells were collected and stored at -80 ° C. In addition, in order to examine the gene expression of myostatin, the differentiation was induced by exchanging with DMEM medium (2% Horse Serum) each containing tea extract A to E having a final concentration of 0.004 (w / v)% as a test substance. Started. After differentiation induction, the cells were cultured for 24 hours, washed with PBS, and the cells were collected and stored at -80 ° C. As a control, 20 (v / v)% ethanol was used instead of the black tea extract.

Gene Expression Analysis RNA was prepared from a sample for gene expression analysis using RNeasy Micro Kit (QIAGEN) according to the attached protocol. The prepared RNAs were prepared at the same concentration, heat-treated at 65 ° C. for 10 minutes, and used after rapid cooling. For reverse transcription, RNA corresponding to 125 ng was used, and 20 μL of reaction solution (1 × PCR buffer II (Roche), 5 mM MgCl 2 (Roche), 1 mM dNTP mix (Takara), 2.5 mM Oligo d (T) 18 mRNA) primer (New England Biolabs), 1 U / μl RNase inhibitor (Takara)) was prepared. The reaction was performed at 42 ° C., 60 minutes → 52 ° C., 30 minutes → 99 ° C., 5 minutes → 4 ° C., and the obtained cDNA was stored at −20 ° C. until use. In addition, as a standard for quantitative PCR, 500 ng of RNA was used, and reverse transcription was performed in the same reaction system.
Quantitative PCR was performed with ABI PRISM7500 Real-time PCR System (Applied Biosystems) using cDNA obtained by reverse transcription reaction as a template. Quantification was performed based on a standard curve prepared using a standard cDNA diluted in 7 steps as a standard. The reaction solution was 10 μL of Fast SYBR Green PCR Master Mix (Applied Biosystems), 100 μM Forward primer 0.2 μL, 100 μM Reverse primer 0.2 μL, dH2O 4.6 μL, and cDNA 5 μL. The temperature conditions for quantitative PCR were 50 ° C. for 2 minutes and 95 ° C. for 10 seconds, followed by 40 cycles of 95 ° C. for 15 seconds and 60 ° C. for 1 minute.
The genes MyHC1 (Myosin heavy chain 1) and myostatin involved in muscle differentiation were measured by quantitative PCR. The results are shown in FIGS.
Table 1 shows the primer sequences used for quantitative PCR.

Results The test results were expressed as mean ± standard error (Average (Ave) ± standard error (SE)), and the expression level of 36B4 was corrected as an internal standard and expressed as a relative mRNA expression level. Significant difference test was performed using t-test, and those having a p value of less than 0.05 and less than 0.01 were considered to have statistical significance (*: p <0.05, **: p <0.01).
2 and 3, the black tea extract significantly increased the expression of MyHC1 gene (FIG. 2), while significantly suppressing the expression of myostatin gene (FIG. 3).
From this, it was confirmed that the black tea extract has an effect of promoting the differentiation of satellite cells.

Claims (4)

  A myostatin / Smad signal inhibitor comprising black tea extract as an active ingredient.   A satellite cell differentiation promoter containing black tea extract as an active ingredient.   A muscle mass increasing agent comprising black tea extract as an active ingredient.   A muscle atrophy inhibitor comprising black tea extract as an active ingredient.

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