JP6650972B2 - TGR5 activator - Google Patents

Description

  The present invention relates to a TGR5 activator effective for improving endurance, suppressing fatigue, improving muscle strength, and improving motor function.

  TGR5 (G protein-coupled bile acid receptor 1) is a type of G protein-coupled receptor mainly expressed in brown and white adipose tissues and skeletal muscle, and is known to have heat production and energy metabolism regulating effects. (Non-Patent Document 1).

  Bile acids are known as in vivo ligands for TGR5. After synthesis in the liver, bile acids are secreted into the small intestine and reabsorbed in the lower small intestine. Bile acid is present in blood at a concentration of about 5 to 15 μM, and an intracellular signal is activated by binding to TGR5 on the cell membrane of adipose tissue or skeletal muscle (Non-patent Documents 2 to 4). ).

  The TGR5 signal is activated by the following pathway. When an agonist binds to TGR5 present on the cell membrane, the amount of cyclic-AMP (cAMP) in the cell increases, and protein kinase A (PKA) is activated. Subsequently, the activated PKA is activated by phosphorylating cAMP response element binding protein (CREB), and finally the transcription of a target gene having a cAMP response element (CRE) sequence is promoted. Representative examples of these target genes include type II iodothyroneine deiodinase (D2) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α).

D2 is a key molecule for regulating thyroid hormone activity and plays a role in converting inactive thyroxine (T4) to active triiodothyroneine (T3). It has been reported that thyroid hormone has an energy consumption promoting action such as heat production, and that when the expression of D2 gene is increased or the activity is promoted, heat production is increased via increase in T3 (Non-Patent Document 3, 4).
In addition, PGC-1α is a transcription coupling factor, and interacts with CREB and nuclear respiratory factor (NRF), as well as in the nuclear receptor, such as peroxisome promoter-activated receptor gamma (PPAR-γ), which is a nuclear receptor. It is known to perform synthesis and function regulation. The main function of mitochondria is ATP production by oxidative phosphorylation. When the expression of PGC-1α increases, the number of mitochondria increases, oxidative phosphorylation increases, and the amount of ATP increases (Non-Patent Document 5). , 6). It has been reported that an increase in the amount of ATP improves the energy efficiency of skeletal muscle, which is the largest ATP consuming organ in a living body, and suppresses fatigue and improves endurance (Non-Patent Documents 7 and 8). Furthermore, PGC-1α is thought to directly affect muscle mass, endurance, and fatigue. In fact, transgenic mice that express PGC-1α4, one of the isoforms of PGC-1α, in a skeletal muscle-specific manner exhibit phenotypes such as increased skeletal muscle mass and improved fatigue resistance. Has been reported (Non-Patent Document 9). From the above, it is considered that increased expression or activation of PGC-1α leads to an increase in ATP amount, an increase in skeletal muscle mass, suppression of fatigue, and improvement in endurance.
As described above, PGC-1α is a target molecule of TGR5, and it has been revealed that activation of TGR5 increases PGC-1α expression (Non-Patent Document 2). Therefore, activation of TGR5 is thought to bring about improvement of endurance, suppression of fatigue, improvement of muscle strength, and improvement of motor function via PGC-1α.

Generally, it is considered that exercise training and well-balanced nutritional supplementation are important for improving athletic performance such as muscle strength. Recently, in order to improve muscle strength more efficiently for exercise enthusiasts and athletes, attempts have been made to use not only training but also supplementary nutrition such as supplements (Patent Document 1). However, there is a concern that training with excessive intake of some proteins and amino acids may cause adverse effects on renal function and the like (Non-Patent Document 10). On the other hand, non-exercise enthusiasts and athletes also find that excessive dieting results in a shortage of nutrients in the body, a decrease in skeletal muscle, and a decline in motor functions such as muscle strength and endurance. Fatigue associated with the decline is regarded as a problem.
Therefore, not only exercise enthusiasts and athletes aiming to improve performance, but also ordinary people aiming to reduce obesity, an efficient exercise function improving method is desired.
From such a viewpoint, a search for a component having a motor function-improving effect has been conducted. For example, tea catechin has an endurance-improving effect (Patent Document 2) and a polymer fruit polyphenol (Patent Document 3) has a muscle force-improving effect. It has been reported.

On the other hand, anti-inflammatory and analgesic effects on mint, headache and stress relieving effects on lemon balm, digestive and appetite enhancing effects on marjoram, sedative and hypnotic effects on sansonin, hypotensive and whitening effects on persimmon leaf tea, It is known that dill has appetite and digestive action, spearmint has bactericidal action and digestive action, bacopamoniella has stress reducing action, and loquat tea has pharmacological action such as anti-obesity action.
However, it is not known at all that these have TGR5 activating action, endurance improving action, fatigue suppressing action, muscular strength improving action or motor function improving action.

JP 2002-65212 A JP 2005-89384 A WO 2005/74962

Houten S.M. et al., EMBO J, 25, 1419-1425 (2006) Thomas C. et al., Nature, 7, 678-693 (2008) Watanabe M. et al., Nature, 439, 484-489 (2006) Watanabe M. et al., PLoS One, 7, 1-9 (2012) Wu Z. et al., Cell, 98, 115-124 (1999) Vieau D. et al., World J Diabetes, 2 (9), 149-157 (2011) Bahi L. et al., J Appl Physiol, 96, 59-64 (2004) Tonkonogi M. et al., Exerc Sport Sci Rev, 30, 129-137 (2002) Luas J.L. et al., Cell, 151, 1319-1331 (2012) Anderson C.F. et al., JAMA, 223 (1), 68-72 (1973)

  The present invention relates to providing a TGR5 activator, furthermore, an endurance improver, a fatigue suppressant, a muscle improver, and a motor function improver.

  The present inventors have conducted a search for various natural materials. As a result, a specific plant extract has a TGR5 activating effect, and is used as a drug or drug for improving endurance, suppressing fatigue, improving muscle strength and improving athletic function. It has been found that it is useful as a material for quasi-drugs, food and feed.

That is, the present invention relates to the following (1) to (5).
(1) A TGR5 activator comprising, as an active ingredient, one or more plant extracts selected from mint, lemon balm, marjoram, sansonin, persimmon leaf tea, dill, spearmint, bacopamoniera and loquat tea.
(2) An endurance improver comprising, as an active ingredient, an extract of one or more plants selected from mint, lemon balm, marjoram, sansonin, persimmon leaf tea, dill, spearmint, bacopamoniera and loquat tea.
(3) An anti-fatigue agent comprising, as an active ingredient, one or more plant extracts selected from mint, lemon balm, marjoram, sansonin, persimmon leaf tea, dill, spearmint, bacopamoniella and loquat tea.
(4) A muscle strength improver comprising, as an active ingredient, an extract of one or more plants selected from mint, lemon balm, marjoram, sansonin, persimmon leaf tea, dill, spearmint, bacopamoniella and loquat tea.
(5) A motor function improver comprising, as an active ingredient, an extract of one or more plants selected from mint, lemon balm, marjoram, sansonin, persimmon leaf tea, dill, spearmint, bacopamoniella and loquat tea.

  ADVANTAGE OF THE INVENTION According to this invention, the pharmaceutical or quasi-drug useful for endurance improvement, fatigue suppression, muscular strength improvement, and athletic function improvement, or the raw material used for a pharmaceutical, a quasi-drug, food, or feed is provided. Therefore, according to the present invention, it is possible to activate TGR5 to improve endurance, suppress fatigue, improve muscle strength, and improve motor function.

It is a figure which shows the luciferase activity (TGR5 agonist activity) of PBS (control), the extract of cholic acid and peppermint, lemon balm, marjoram, sansonin, persimmon leaf tea, dill, spearmint, bacopamoniera, and loquat tea.

  In the present invention, “TGR5 activation” refers to activation of the transcription of a target gene such as D2 or PGC-1α via an increase in intracellular cAMP, starting from the binding of an agonist to TGR5 present on the cell membrane. To be done. This activation enhances oxidative phosphorylation in mitochondria, leading to an increase in ATP production. Activation of TGR5 can be confirmed by evaluating the activity using a reporter assay or analyzing gene expression downstream of the TGR5 signal.

  In the present invention, “improvement of endurance” refers to an action of improving endurance for exercise in a broad sense including exercise requiring endurance and labor requiring repeated muscular movement. , Endurance for both whole body endurance (running, swimming, etc., when the whole body is under load) and local endurance (exercise of arm to keep guard in boxing) It is. Exercise is based on aerobic energy supply, from aerobic energy supply to long-distance running, swimming, and aerobics-based aerobic exercises, as well as anaerobic energy supply. For example, an exercise that sustains an intense movement, such as continuous punching in boxing, is performed.

  In the present invention, “fatigue” refers to a state in which muscle output (tension) and relaxation speed are reduced due to continuous muscle contraction, that is, “fatigue suppression” refers to suppressing a decrease in tension due to muscle fatigue. Say.

  In the present invention, “improving muscle strength” means that the force generated by contraction of muscles is increased. An increase in muscle mass is different from an increase in muscle strength as a whole.

  In the present invention, “improvement in motor function” means that motor function is maintained or improved by suppressing a decrease in muscle function (muscle strength).

In the TGR5 activator, endurance improver, fatigue suppressant, muscular strength improver and motor function improver of the present invention, mint, lemon balm, marjoram, sansonin, persimmon leaf tea, dill, spearmint, bacopamoniera, loquat tea The extract of each plant is used as an active ingredient.
Here, “mint” is a perennial plant belonging to the genus Lamiaceae, and its scientific name is Mentha arvensis L. var. piperascens Malinv. It is. Examples of the site to be used include a stem, a leaf, a branch, a branch and a leaf, a stem, a bark, a root, a rhizome, a root bark, a mixture thereof, and the like. Among these, a leaf is preferable.
"Lemon balm" is a perennial plant belonging to the genus Lamiaceae, and the scientific name is Melissa
officinalis L. It is. Examples of the site to be used include a stem, a leaf, a branch, a branch and a leaf, a stem, a bark, a root, a rhizome, a root bark, a mixture thereof, and the like. Among these, a leaf is preferable.
"Marjoram" is a perennial plant belonging to the genus Perilla, belonging to the genus Lamiaceae, and its scientific name is Origanum majorana L. It is. Examples of the site to be used include a stem, a leaf, a branch, a branch and a leaf, a stem, a bark, a root, a rhizome, a root bark, a mixture thereof, and the like. Among these, a leaf is preferable.
"Sansonin" is a seed of a deciduous tree of the buckthorn family, Satsuma jujube, and its scientific name is Ziziphizu jujuba.
"Persimmon leaf" is a leaf of a deciduous tree belonging to the genus Persimmonaceae, and its scientific name is Diospyros.
kaki.
"Dill" is an annual plant of the genus Inondo of the Apiaceae family, and has a scientific name of Anethum graveolens L. It is.
"Spearmint" is a perennial plant belonging to the mint genus Lamiaceae, and its scientific name is Mentha spicata L. It is.
"Bacopa moniera" is a perennial plant belonging to the genus Bacopa of the family Scrophulariaceae, and its scientific name is Bacopa.
monniera. The site to be used includes, for example, stems, leaves, branches, branches and leaves, roots, rhizomes, root bark, mixtures thereof, and the like. Of these, leaves are preferred.
“Loquat tea” is a leaf of an evergreen tree of the genus Loquat, and its scientific name is Eriobotrya japonica.

  Examples of such plant extracts include various solvent extracts obtained by extraction by a known extraction method, diluents thereof, concentrated solutions thereof, and dried powders thereof. Known extraction methods include, for example, immersion, decoction, leaching, solid-liquid extraction, reflux extraction, supercritical extraction, ultrasonic extraction, microwave extraction, and the like. For example, immersion includes immersion and leaching at 0 ° C. to the boiling point of the solvent (preferably 15 to 40 ° C.) for 1 hour to 4 weeks. Solid-liquid extraction includes 0 ° C. to the boiling point of the solvent (preferably 15 to 40 ° C.). C.) at 30 to 1000 rpm for 30 minutes to 2 weeks. Further, in order to prevent the extract from being oxidized, a means for extracting under a so-called non-oxidizing atmosphere while removing dissolved oxygen by boiling degassing or passing an inert gas such as nitrogen gas may be used in combination. In the case of reflux extraction, extraction can be performed using an extraction instrument such as a Soxhlet extractor.

  As a solvent for extraction, any of a polar solvent and a non-polar solvent can be used. Specific examples of the solvent include, for example, 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; Esters; chain and cyclic ethers such as diethyl ether and tetrahydrofuran; polyethers such as polyethylene glycol; hydrocarbons such as squalane, hexane, cyclohexane, petroleum ether; aromatic hydrocarbons such as toluene; dichloromethane, chloroform And dimethyl sulfoxide; supercritical carbon dioxide; pyridines; organic solvents such as oils such as fats and oils, and waxes; and mixtures thereof. Suitable examples include water, alcohols, and a water-alcohol-based mixed solvent, and ethanol is preferable as the alcohols. Of these, water and an aqueous ethanol solution are preferred. The aqueous ethanol solution preferably has an ethanol concentration of 10% (v / v) or more, more preferably 20% (v / v) or more, more preferably 50% (v / v) or more, and more preferably 90% or more. % (V / v) or more, and preferably 95% (v / v) or less, more preferably 90% (v / v) or less, and preferably 10 to 95% (v / v), more preferably Those having 20 to 95% (v / v), more preferably 50 to 95% (v / v).

  The plant extract is extracted, for example, using 1 to 50 parts by mass of an extraction solvent per 1 part by mass of a plant at 4 ° C to 100 ° C for 0.5 hours to 30 days. It can be done by doing. More specifically, when an aqueous ethanol solution is used as the extraction solvent, it is preferably from 5 parts by mass to 30 parts by mass per 1 part by mass of the plant, and preferably at room temperature for 1 hour to 10 days. Further, these operations may be repeated.

  In the present invention, the above extract may be used as it is, or the extract may be diluted, concentrated, or lyophilized, and then prepared and used as a powder or paste. Also, freeze-dried, at the time of use, diluted with a solvent usually used for extraction, such as water, ethanol, propylene glycol, butylene glycol, a mixture of water and ethanol, a mixture of water and propylene glycol, a mixture of water and butylene glycol, and the like. It can also be used. Further, it can also be used by being encapsulated in vesicles such as liposomes, microcapsules and the like.

  The plant extract of the present invention may be a crude product as long as it conforms to, for example, food and pharmaceutical acceptable standards and exerts the effects of the present invention. In addition, if necessary, removal of inactive contaminants, treatment of deodorization, decolorization, etc., using known means such as liquid-liquid distribution, solid-liquid distribution, filtration membrane, activated carbon, adsorption resin, ion exchange resin, etc. And the purity thereof may be increased by appropriately combining known separation and purification methods. Purification means include organic solvent precipitation, centrifugation, ultrafiltration membrane, high performance liquid chromatography, column chromatography and the like.

  As shown in the Examples below, the plant extracts of peppermint, lemon balm, marjoram, sansonin, persimmon leaf tea, dill, spearmint, bacopa moniera and loquat tea have a strong TGR5 activating effect (FIG. 1). . As described above, when the TGR5 signal is activated, PGC-1α transcription is activated, and β-oxidation and oxidative phosphorylation are enhanced in skeletal muscle. By this action, a large amount of ATP is synthesized in the skeletal muscle, and an endurance improving action, a fatigue suppressing action, a muscle strength improving action, and a motor function improving action can be expected (Non-Patent Documents 2-9).

  Accordingly, the plant extracts of the mint, lemon balm, marjoram, sansonin, persimmon leaf tea, dill, spearmint, bacopa monella and loquat tea of the present invention are TGR5 activator, endurance improver, fatigue suppressant, and muscle improver. , Can be a motor function improver (hereinafter referred to as “TGR5 activator and the like”), and can be used for producing a TGR5 activator and the like. Further, it can be used for animals including humans to activate TGR5, improve endurance, suppress fatigue, improve muscle strength, and further improve motor function. Here, the use for humans may be a therapeutic use or a non-therapeutic use. The term "non-therapeutic" refers to a concept that does not include medical practice, that is, does not include a method of operating, treating, or diagnosing humans. , A concept that does not include a method of performing treatment or diagnosis.

The plant extracts of peppermint, lemon balm, marjoram, sansonin, persimmon leaf tea, dill, spearmint, bacopamoniella and loquat tea can be used alone or in any combination.
The composition containing the TGR5 activator of the present invention and the like is TGR5 activation, endurance improvement, fatigue suppression, muscle strength improvement, and further improvement in motor function, becomes a drug, quasi-drug, food or feed, In addition, the TGR5 activator or the like is a raw material or a raw material for producing a pharmaceutical, a quasi-drug, a food or a feed. That is, the TGR5 activator and the like of the present invention exerts effects of improving endurance, suppressing fatigue, improving muscle strength, and improving motor function, such as pharmaceuticals for humans or animals, quasi-drugs, food or feed, etc. It can be blended as an active ingredient. Here, the concept of food is to improve endurance, suppress fatigue, improve muscle strength, and improve athletic function for those who are under exercise, middle-aged and elderly, bed rest, or athlete. Foods, foods for the sick, foods for specified health use, supplements and the like are included.

  The pharmaceuticals and quasi-drugs may be in the form of, for example, tablets, capsules, granules, powders, orally administered preparations such as syrups or injections, suppositories, inhalants, transdermal absorbents, external preparations and the like. Parenteral formulations are included. In order to prepare such various dosage forms, the plant extract of the present invention may be used alone or in any combination, and other active ingredients may be used as long as the TGR5 activating effect is not lost. And pharmacological components, nutritional components, and the like. Further, it may contain a pharmaceutically acceptable carrier, or an additive that can be used for pharmaceuticals or quasi-drugs. Such carriers and additives include excipients, coating agents, binders, extenders, disintegrants, surfactants, lubricants, diluents, osmotic agents, pH adjusters, dispersants, emulsifiers, preservatives Agents, stabilizers, antioxidants, preservatives, coloring agents, ultraviolet absorbers, humectants, thickeners, activity enhancers, anti-inflammatory agents, bactericides, solvents, softeners, fragrances, flavors, flavors, etc. Is mentioned. Of these administration forms, the preferred form is oral administration, and in the case of preparing a liquid preparation for oral use, it can be produced by a conventional method by adding a flavoring agent, a buffer, a stabilizer and the like.

  Examples of the form of the food include milk, processed milk, milk drink, yogurt, soft drink, tea drink, coffee drink, fruit drink, carbonated drink, juice, jelly, wafer, biscuit, bread, noodle, sausage, etc. In addition to various foods such as foods and nutritional foods, further, a nutritional supplement composition in the same form (tablets, capsules, syrups and the like) as the above-mentioned oral administration preparations may be mentioned.

  To prepare various forms of food, the plant extract of the present invention is used as a raw material for other food materials, solvents, softeners, oils, emulsifiers, preservatives, fragrances, stabilizers, coloring agents, antioxidants. , Moisturizing agents, thickening agents, food ingredients for improving endurance, foods for suppressing fatigue, foods for improving muscle strength, foods for improving exercise function, pet foods, etc., appropriately combined with medicinal ingredients other than the above plant extract of the present invention. It is possible.

  When the food is a food for the elderly or a bed rest patient who has difficulty supplying an appropriate amount of nutrients, it can be formulated as a nutritional composition such as an enteral nutritional supplement.

  The feed can be prepared in the same form as the above food.

  The content of the plant extract (in terms of dry matter) of the present invention in the above beverages, for example, dairy beverages, soft drinks, tea-based beverages, and the like, is usually 0.0001% by mass or more, preferably 0.001% by mass in the beverage. It is at least 2.0 mass%, preferably at most 1.0 mass%, more preferably at most 0.2 mass%. Further, it is preferably 0.0001 to 2.0% by mass, more preferably 0.001 to 1.0% by mass, and further preferably 0.001 to 0.2% by mass.

  The content of the plant extract (in terms of dry matter) of the present invention in food or feed other than the beverage is usually 0.0001% by mass or more, preferably 0.001% by mass or more, more preferably 0.01% by mass or more. And it is 50% by mass or less, preferably 20% by mass or less, more preferably 10% by mass or less. Further, it is usually preferably 0.0001 to 50% by mass, more preferably 0.001 to 20% by mass, and further preferably 0.01 to 10% by mass.

  In the case of pharmaceuticals or quasi-drugs other than those described above, for example, in the case of oral solid preparations such as tablets, capsules, granules and powders, oral liquid preparations such as internal liquids and syrups, the total weight of the pharmaceuticals etc. The content of the plant extract (in terms of dry matter) of the present invention is usually 0.01% by mass or more, preferably 0.1% by mass or more, more preferably 1.0% or more, and 95% by mass or less, It is preferably at most 80% by mass, more preferably at most 60% by mass. Further, it is usually preferably 0.01 to 95% by mass, more preferably 0.1 to 80% by mass, and further preferably 1.0 to 60% by mass. In addition, the said plant extract may be in a dissolved state or a dispersed state, and the presence state does not matter.

  The administration or intake amount of the TGR5 activator of the present invention or the like varies depending on the dosage form and application, but is 0.01 mg / kg body weight per day for an adult as the plant extract (in terms of dry matter) of the present invention. The above is preferably 0.1 mg / kg body weight or more, 100 mg / kg body weight or less, preferably 50 mg / kg body weight or less, more preferably 20 mg / kg body weight or less. The weight is preferably 0.01 to 100 mg / kg body weight, more preferably 0.1 to 50 mg / kg body weight, and further preferably 0.1 to 20 mg / kg body weight.

  Subjects for administration or ingestion include human or non-human mammals. These subjects may be those who require or desire TGR5 activation, increased endurance, reduced fatigue, increased muscle strength, increased motor function.

With respect to the above-described embodiment, the present invention further discloses the following aspects.
<1> a TGR5 activator, an endurance improver comprising an extract of one or more plants selected from mint, lemon balm, marjoram, sansonin, persimmon leaf tea, dill, spearmint, bacopamoniera and loquat tea as an active ingredient; Fatigue inhibitor, muscle strength improver or motor function improver.
<2> Endurance enhancer by activating TGR5, comprising as an active ingredient one or more plant extracts selected from mint, lemon balm, marjoram, sansonin, persimmon leaf tea, dill, spearmint, bacopa monniera and loquat tea. , A fatigue inhibitor, a muscle strength improver, or a motor function improver.
<3> Mint, lemon balm, marjoram, sansonin, persimmon leaf tea, dill, spearmint, bacopa monniera and loquat for producing TGR5 activator, endurance improver, fatigue suppressor, muscle strength improver or motor function improver Use of one or more plant extracts selected from tea.
<4> selected from mint, lemon balm, marjoram, sansounin, persimmon leaf tea, dill, spearmint, bacopa monniera and loquat tea for use in TGR5 activation, endurance improvement, fatigue suppression, muscle strength improvement or motor function improvement One or more plant extracts.
<5> selected from mint, lemon balm, marjoram, sansonin, persimmon leaf tea, dill, spearmint, bacopa monniera and loquat tea for use in endurance improvement, fatigue suppression, muscle strength improvement or motor function improvement by TGR5 activation One or more plant extracts.
<6> A method for activating TGR5 by administering or ingesting an effective amount of one or more plant extracts selected from mint, lemon balm, marjoram, sansonin, persimmon leaf tea, dill, spearmint, bacopamoniella and loquat tea. , Endurance improvement method, fatigue suppression method, muscle strength improvement method or motor function improvement method.
<7> Activation of TGR5 by administering or ingesting an effective amount of one or more plant extracts selected from mint, lemon balm, marjoram, sansonin, persimmon leaf tea, dill, spearmint, bacopamoniera and loquat tea. Endurance improvement method, fatigue suppression method, muscle strength improvement method or motor function improvement method.
<8> In the above item <3>, the use is a non-therapeutic use.
<9> In the above item <6> or <7>, the method is a non-therapeutic method.
<10> In the above item <6> or <7>, the subject of administration or ingestion is a human who needs or desires activation of TGR5, improvement of endurance, suppression of fatigue, improvement of muscular strength or motor function. <11> In the above item <6> or <7>, the target of administration or ingestion is a person lacking exercise, middle-aged and elderly, a bed rest person, or an athlete.
<12> In the above item <6> or <7>, the dose or the intake is 0.01 to 100 mg / kg body weight, preferably 0.1 to 50 mg / kg body weight as a plant extract per day for an adult. And more preferably 0.1 to 20 mg / kg body weight.

1. Preparation of each plant extract (1) Mint extract:
To 100 g of dried peppermint leaves (obtained from Shinwa Bussan Co., Ltd.), 1000 mL of a 50% aqueous ethanol solution was added, and the mixture was extracted at room temperature and standing for 7 days, and the extract obtained by filtration was used. . The solid content concentration of the extract was 1.65% (w / v).
(2) Lemon balm extract:
Lemon balm extract (obtained from Kenko Tsusho Co., Ltd.) was dissolved in a 20% aqueous ethanol solution to a concentration of 1% (w / v) and used.
(3) Marjoram extract:
To 10 g of dried whole marjoram (obtained from Tokaimoto Tenkaido Co., Ltd.), 100 mL of a 50% aqueous ethanol solution was added, and the mixture was extracted at room temperature and standing for 7 days. Using. The solid content concentration of the extract was 2.22% (w / v).
(4) Sansunin extract:
To 100 g of Sansoun seeds (obtained from Shinwa Bussan Co., Ltd.), 1000 mL of a 50% aqueous ethanol solution was added, and the mixture was extracted at room temperature and under static conditions for 7 days, and the extract obtained by filtration was used. The solid content concentration of the extract was 0.95% (w / v).
(5) Persimmon leaf tea extract:
1000 mL of a 50% aqueous ethanol solution was added to 100 g of persimmon leaves (obtained from Tokaimoto Tenkaido Co., Ltd.), and the mixture was extracted at room temperature for 7 days under static conditions. The solid content concentration of the extract was 1.0% (w / v).
(6) Dill extract:
Dill liquid SP-77544 (obtained from San-Ei Gen FFI Co., Ltd.) obtained by steam distillation of whole dill grass is dissolved in a 50% aqueous ethanol solution to a concentration of 1% (w / v). Was.
(7) Spearmint extract:
To 100 g of whole spearmint (obtained from Tokaimoto Tenkaido Co., Ltd.), 1000 mL of a 50% aqueous ethanol solution was added, and the mixture was extracted at room temperature and standing for 7 days, and then the extract obtained by filtration was used. The solid content concentration of the extract was 2.10% (w / v).
(8) Bacopa monniera extract:
Bacopa monniera extract (obtained from Organodanisco Food Techno Co., Ltd.), which is a water-containing ethanol extract of bacopa monniera leaves, was dissolved in a 50% aqueous ethanol solution to a concentration of 1% (w / v) and used.
(9) Loquat tea extract:
Loquat leaf extract powder (obtained from Matsuura Pharmaceutical Co., Ltd.) was dissolved in a 50% aqueous ethanol solution to a concentration of 1% (w / v) and used.

2. Evaluation of TGR5 Activating Effect The TGR5 activating effect of each of the plant extracts of peppermint, lemon balm, marjoram, sansonin, persimmon leaf tea, dill, spearmint, bacopamoniella and loquat tea prepared in 1 above was evaluated by a luciferase assay. In this assay system, human TGR5 activity is determined by the transcriptional activation of CREB, which is a signal downstream of TGR5, and can be quantified by luciferase activity.
The human TGR5 expression vector was prepared according to the following procedure. CDNA was prepared by reverse transcription of Total RNA extracted from HepG2 cells (human liver cancer cell line) using a High Capacity RNA-to-cDNA Kit (Applied biosystems). Using the prepared cDNA as a template, the protein coding region of the human TGR5 (hTGR5) gene (NM_170699) was amplified by PCR. The primers shown in Table 1 below were used for PCR, and Kpn I and Xho I recognition sequences (described in uppercase letters) were added as restriction enzyme sites on the 5 ′ side of the forward primer and reverse primer, respectively. It is.

The amplified DNA fragment was treated with a restriction enzyme ( Kpn I, Xho I), incorporated into a pcDNA3.1 / Zeo (+) (Invitrogen) multicloning site, and treated with pcDNA3.1-h. TGR5 Vector. The firefly luciferase expression vector used was pGL4.29 [luc2P / CRE / Hygro] Vector (Promega). As a Renilla luciferase expression vector used for correcting the gene transfer efficiency, phRL-TK-Vector (Promega) was used.
The sequence of the luciferase assay is as follows. HEK293 cells (human embryonic kidney cell line) were seeded at 5.0 × 10 ⁴ cells / well in a 24-well plate, and DMEM medium (Life Technology) containing 5% charcoal-treated FBS (Life Technologies) was seeded.
(Technologies) for 1 day. Then, pcDNA3.1-h. 40 ng of TGR5 Vector, 200 ng of pGL4.29 [luc2P / CRE / Hygro] Vector and 100 ng of phRL-TK-Vector, 3 μL of superfect (QIAGEN) were mixed, and the mixture was added to HEK293 cells. After 3 hours, the medium was changed to DMEM medium containing 5% charcoal-treated FBS. Four hours after the medium was changed, a positive control (5 μM) or a test substance (0.001%) was added, and the cells were cultured for 12 to 18 hours. As a positive control, cholic acid (Wako Pure Chemical Industries, Ltd.), a main component of bile acids, was added. Then, the intracellular firefly luciferase activity was measured using Dual-Luciferase Reporter Assay System (Promega). In addition, the obtained numerical value was corrected by Renilla luciferase activity. Luciferase luminescence was quantified using a luminometer (BERTHHOLD LB9506). The results obtained are shown in FIG.
As is apparent from FIG. 1, the plant extracts of peppermint, lemon balm, marjoram, sansonin, persimmon leaf tea, dill, spearmint, bacopamoniera and loquat tea were as potent as TGR5 activation as the positive control, cholic acid. Has an action.

Claims (2)

A TGR5 activator comprising an aqueous ethanol extract of lemon balm leaves as an active ingredient. A TGR5 activating food or feed comprising an aqueous ethanol extract of lemon balm leaves as an active ingredient.

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