TGR-5 activating composition, GLP secretion promoter

Abstract

To provide compositions for TGR-5 activation that can activate TGR-5, and to provide a GLP secretion promoting agent.SOLUTION: A composition for TGR-5 activation containing Daucus carota subsp. sativus extract and a GLP secretion promoting agent are provided. Preferably, the extract is derived from the roots or seeds of Daucus carota subsp. sativus. According to the present invention, it is possible to provide a composition for TGR-5 activation that can activate TGR-5, and a GLP secretion promoting agent.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a composition for activating TGR-5 and a GLP secretion promoter. More specifically, it relates to a composition for activating TGR-5 and a GLP secretion promoter used in fields such as pharmaceuticals and health foods.

Background Art

[0002] TGR-5 (Transmembrane G protein-coupled Receptor-5) is a receptor protein that uses bile acids as an endogenous ligand and is a protein expressed in almost all tissues such as the small intestine, adipose tissue, and skeletal muscle. Among them, TGR-5 localized in intestinal L cells is a hormone that promotes the secretion of GLP-1 (Glucagon-like peptide-1), a type of incretin that has the function of promoting insulin secretion when stimulated by bile acids and the like. In recent years, the function of TGR-5 in skeletal muscle has also been becoming clear, and its action has attracted attention.

[0003] For example, Non-Patent Document 1 describes that TGR-5 stimulation affects glucose metabolism and lipid metabolism. Non-Patent Document 2 describes that dietary supplementation of obacunone (a TGR-5 agonist) stimulates muscle hypertrophy and suppresses hyperglycemia and obesity via the TGR-5 and PPARγ pathways. Non-Patent Document 3 describes that activation of TGR-5 in skeletal muscle promotes muscle cell differentiation and muscle hypertrophy.

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Non-Patent Document 2

[0005] As described above, activating TGR-5 can be effective against various symptoms such as hyperglycemia and obesity, and can also promote the secretion of GLP-1. Therefore, there is a need for novel compositions that have the function of activating TGR-5.

[0006] The object of this invention is to provide a TGR-5 activating composition and a GLP secretion promoter that can activate TGR-5. [Means for solving the problem]

[0007] As a result of diligent research into the above-mentioned problems, the inventors of this invention discovered that carrot extract has a strong TGR-5 activating effect, and thus completed the present invention.

[0008] In other words, the present invention provides the following [1] to [4]. [1] A TGR-5 activating composition containing carrot extract, The aforementioned carrot extract is an extract from the plant body of a plant belonging to the genus Daucus in the family Apiaceae. The aforementioned plant of the genus Daucus in the family Apiaceae is at least one species selected from the group consisting of Nantes ginseng, yellow ginseng, Okinawa island ginseng, and wild ginseng. The aforementioned plant body is a root or a seed. The carrot extract is either an extract of the plant body using an organic solvent, or a supercritical extract using carbon dioxide. The organic solvent is at least one selected from the group consisting of ethanol and hexane. The extract of the plant body with the organic solvent is An extraction step in which the organic solvent is added to the plant body to obtain an extract containing insoluble matter. A filtration step to obtain a filtrate by filtering the insoluble matter from the extract, and The method is obtained by a step of removing at least a portion of the organic solvent contained in the filtrate, The supercritical extract is An extraction step in which a supercritical fluid of carbon dioxide is added to the plant body to extract the components of the plant body into the supercritical fluid, The process involves dissolving the supercritical fluid from which the plant components have been extracted in an organic solvent to obtain a mixture, and A TGR-5 activating composition obtained by a method comprising the step of removing at least a portion of the organic solvent from the mixture. The present invention provides a TGR-5 activating composition that can activate TGR-5. [2] The plant of the genus Carrot in the family Apiaceae is Carrot Nantes. The plant is supported by seeds, Furthermore, the organic solvent is ethanol, or the plant of the Apiaceae family, genus Daucus is yellow ginseng. The plant body is a root, Furthermore, the organic solvent is hexane, or the plant of the Apiaceae family, genus Daucus is wild carrot. The plant is supported by seeds, Furthermore, the organic solvent is ethanol, or the plant of the Apiaceae family, genus Daucus is Okinawa Island ginseng. The plant body is a root, The TGR-5 activating composition according to [1], wherein the organic solvent is ethanol. [3] A GLP secretion promoter containing carrot extract, The aforementioned carrot extract is an extract from the plant body of a plant belonging to the genus Daucus in the family Apiaceae. The aforementioned plant of the genus Daucus in the family Apiaceae is at least one species selected from the group consisting of Nantes ginseng, yellow ginseng, Okinawa island ginseng, and wild ginseng. The aforementioned plant body is a root or a seed. The carrot extract is an extract of the plant body with an organic solvent or a supercritical extract using carbon dioxide, The organic solvent is at least one selected from the group consisting of ethanol and hexane, The extract of the plant body with the organic solvent is, An extraction step of adding the organic solvent to the plant body to obtain an extract containing insoluble matter, A filtration step of filtering the insoluble matter from the extract to obtain a filtrate, and, It is obtained by a method including a step of removing at least a part of the organic solvent contained in the filtrate, The supercritical extract is, An extraction step of adding a supercritical fluid of carbon dioxide to the plant body and extracting the components of the plant body into the supercritical fluid, A step of dissolving the supercritical fluid in which the components of the plant body are extracted in an organic solvent to obtain a mixture, and, A GLP secretion promoter obtained by a method including a step of removing at least a part of the organic solvent from the mixture. According to the present invention, the secretion of GLP can be promoted by activating TGR-5. [4] The plant of the genus Daucus in the family Apiaceae is Nantes carrot The plant is supported by seeds, And the organic solvent is ethanol, or the plant of the genus Daucus in the family Apiaceae is yellow carrot The plant body is a root, And the organic solvent is hexane, or the plant of the genus Daucus in the family Apiaceae is wild carrot The plant is supported by seeds, And the organic solvent is ethanol, or the plant of the genus Daucus in the family Apiaceae is Okinawa carrot The plant body is a root, And the organic solvent is ethanol, the GLP secretion promoter according to [3].

Effect of the Invention

[0009] According to the present invention, it is possible to provide a composition for activating TGR-5 and a GLP secretion promoter that can activate TGR-5.

Brief Description of the Drawings

[0010] [Figure 1] It is a diagram showing the test results of TGR-5 activation by carrot seed extract (ethanol extraction). [Figure 2] It is a diagram showing the test results of TGR-5 activation by carrot root extract (hexane extraction). [Figure 3] It is a diagram showing the test results regarding the effect of promoting GLP-1 secretion by the carrot extract of the present invention. [Figure 4] It is a diagram showing the test results of TGR-5 activation by carrot seed extract (supercritical carbon dioxide extraction). [Figure 5] It is a diagram showing the test results of TGR-5 activation by island carrot root extract (ethanol extraction). [Figure 6] It is a diagram showing the test results of activation of PGC1α induction through the TGR-5 agonist action by carrot root extract. [Figure 7] It is a diagram showing the effect of promoting GLP-1 secretion of carrot root extract in in vivo tests. [Embodiments for Carrying Out the Invention]

[0011] Hereinafter, the composition for activating TGR-5 and the GLP secretion promoter of the present invention will be described. Note that the composition for activating TGR-5 and the GLP secretion promoter described in the embodiments are merely exemplified for explaining the present invention and are not limited thereto.

[0012] [Composition for Activating TGR-5] The composition for activating TGR-5 of the present invention is characterized by containing a carrot extract. The present invention can provide a novel composition for activating TGR-5.

[0013] TGR-5 is a receptor protein that uses bile acids as its endogenous ligand and is expressed in almost all tissues, including the small intestine, adipose tissue, and skeletal muscle. Activation of TGR-5 has several notable effects, including increased GLP-1 secretion in the small intestine, resulting in improved glucose metabolism. In brown adipose tissue and skeletal muscle, it promotes energy metabolism such as β-oxidation, heat production, energy production, muscle cell differentiation, and muscle hypertrophy. Furthermore, TGR-5 activation promotes the browning of white fat cells, transforming them into beige adipocytes that exhibit similar heat production and energy production to brown adipocytes. A decrease in inflammatory cytokines expressed in macrophages has also been observed. Based on these findings, the TGR-5 activating composition of the present invention can provide effects such as improved glucose metabolism, blood glucose reduction, prevention and improvement of glycation, improved lipid metabolism, suppression of obesity, promotion of muscle synthesis, prevention of frailty, increased intestinal absorption and nutrient assimilation, anti-inflammatory activity, healing and repair of the gastrointestinal mucosa, and improved intestinal permeability. In particular, the TGR-5 activating composition of the present invention can provide effects on type 2 diabetes through the secretion of GLP-1.

[0014] The carrot extract used in this invention is an extract of the plant body of a plant belonging to the genus Daucus in the family Apiaceae. The plant body used in the carrot extract of this invention may include the root, seeds, leaves, stems, flowers, etc. From the viewpoint of TGR-5 activation, the root or seed is preferred, and the seed is more preferred.

[0015] The plants of the genus Daucus in the family Apiaceae used in this invention are not particularly limited, but examples include carrots (cultivated species, scientific name: Daucus carota subsp. sativus (Hoffm.) Arcang.), wild carrots (wild species, scientific name: Daucus carota), and other carrots. Examples of carrots include Western varieties and Eastern varieties. Examples of Western varieties include Flakkee carrots, Nantes carrots, Gosun carrots, Koyasu Sansun carrots, mini carrots, Parisian carrots, white carrots (e.g., White Belgian), yellow carrots (e.g., Yellow Belgian), and purple carrots. Examples of Eastern varieties include Kintoki carrots, Kumamoto long carrots, Okinawa Island carrots, Kinbi carrots, and black carrots. Examples of wild carrots include wild carrot and wild D. carota subspecies. Among these, from the viewpoint of TGR-5 activation, Flakkee carrot, Nantes carrot, white carrot, yellow carrot, Okinawa Island carrot, wild carrot, and wild carrot subspecies are preferred, and white carrot, yellow carrot, and Okinawa Island carrot are more preferred.

[0016] The carrot extract of the present invention can be an extract with water or various organic solvents, a distillate obtained by steam distillation, a supercritical extract using carbon dioxide, a dried powder, grated carrots, or juice squeezed from grated carrots. Preferably, from the viewpoint of TGR-5 activation, it can be an extract with an organic solvent, a steam distillate, or a supercritical extract using carbon dioxide. Extraction with water or various organic solvents can be carried out using water, various organic solvents, and mixtures thereof as solvents, according to known extraction methods. Before performing the extraction, the raw materials, either whole or roughly chopped, may be washed with a hydrophobic or hydrophilic solvent alone or a mixture thereof to remove unwanted substances such as pigments. Examples of hydrophobic solvents include chloroform, ether, hexane, cyclohexane, toluene, dichloromethane, petroleum ether, and benzene, while examples of hydrophilic solvents include lower alcohols (e.g., methyl, ethyl, n-propyl, isobutyl, or butyl alcohol), ethyl acetate, acetone, isopropyl alcohol, and acetonitrile. Water may be added to the raw materials as needed to wet them.

[0017] To perform extraction with water, for example, 0.5 to 10 parts by weight of water can be added to 1 part by weight of the raw material, and extraction can be carried out at 40 to 120°C for 5 minutes to 2 hours, preferably 10 to 60 minutes, and more preferably 10 to 20 minutes. During this time, stirring can be used to increase the extraction efficiency. Extraction can also be carried out by heating under reflux. After extraction, filtration or pressing is performed using a filter cloth, filter, or mesh screen. In this case, pressurization may be used to increase work efficiency. Alternatively, the extract can also be obtained by centrifugal separation (1000 to 20000 rpm). To increase the recovery rate, this extraction and filtration process can be repeated 1 to 5 times.

[0018] To perform extraction with an organic solvent, for example, 0.5 to 10 parts by weight of solvent can be added to 1 part by weight of the raw material, and the extract can be obtained in the same manner as with water extraction. The solvents used can be the hydrophobic and hydrophilic solvents mentioned above, either alone or in combination of two or more, and aqueous solvents can also be used. In particular, when washing the raw material with a hydrophobic solvent before extraction to remove unwanted substances such as pigments, as described above, and when washing the crude extract after extraction with a hydrophobic solvent, as described below, water, lower alcohols, and 50-80% (v / v) aqueous lower alcohols are preferred as solvents for extraction. Examples of these lower alcohols are the same as those mentioned above. Solvent removal can be carried out by conventional methods such as filtration, centrifugation, and distillation.

[0019] [GLP secretion promoter] The GLP secretion promoter of the present invention is characterized by containing carrot extract. Preferably, the carrot extract is derived from the root or seed. The GLP secretion promoter of the present invention can promote GLP secretion by activating TGR-5. The carrot extract constituting the GLP secretion promoter may be the same as that of TGR-5 in the present invention described above.

[0020] GLP is a type of incretin, a hormone that promotes insulin secretion, and is secreted in the small intestine and other areas upon activation of TGR-5. GLP-1 and GLP-2 are examples of GLP secreted upon activation of TGR-5. Functions of GLP-1 include obesity suppression, improvement of glucose metabolism, blood glucose reduction, and prevention and improvement of glycation. Functions of GLP-2 include maintaining intestinal homeostasis, increasing intestinal absorption and nutrient assimilation, anti-inflammatory activity, healing and repair of the gastrointestinal mucosa, decreased intestinal permeability, and increased mesenteric blood flow. Based on these findings, the GLP secretion-promoting agent of the present invention can provide effects such as improved glucose metabolism, blood glucose reduction, prevention and improvement of glycation, suppression of obesity, increased intestinal absorption and nutrient assimilation, anti-inflammatory activity, healing and repair of the gastrointestinal mucosa, decreased intestinal permeability, and increased mesenteric blood flow. The GLP secretion-promoting agent of the present invention can particularly achieve effects on type 2 diabetes through the secretion of GLP-1.

[0021] [Uses and dosage forms of TGR-5 activating compositions and GLP secretion promoters] The TGR-5 activating composition and GLP secretion promoter of the present invention can be used in applications such as pharmaceuticals, foods, veterinary drugs, and animal feed.

[0022] As described above, the TGR-5 activating composition of the present invention exerts effects such as suppressing obesity, improving glucose metabolism, lowering blood glucose, preventing and improving glycation, improving lipid metabolism, promoting muscle synthesis, and preventing frailty through the activation of TGR-5. Therefore, formulations containing the TGR-5 activating composition can be used as agents for the following applications. (a) Obesity suppressants: Agents that suppress obesity mainly through energy production in skeletal muscle. (b) Hypoglycemic agents: Agents that increase glucose uptake into cells and lower blood glucose levels. (c) Glucose metabolism improving agent: An agent that maintains and improves the homeostasis of glucose metabolism in the body through glucose uptake, energy production in skeletal muscle, etc. (d) Glycation prevention and improvement agents: These agents prevent the binding of excess sugar to proteins in the body, which causes proteins to denature and deteriorate, and produce AGEs (Advanced Glycation End Products), which are aging substances, thereby improving the environment of the body. (e) Lipid metabolism improving agents: Agents that improve lipid metabolism, such as β-oxidation, in skeletal muscle. (f) Muscle synthesis promoters: Agents that promote the differentiation of muscle cells and muscle hypertrophy. (g) Frailty prevention agent: A drug that prevents physical weakness (frailty) due to aging, etc., by promoting muscle cell differentiation and muscle hypertrophy. (h) A gastrointestinal mucosal healing and repair agent is an agent that promotes the proliferation of gastrointestinal mucosal cells to heal and repair damage to the mucosa. (i) Agents that improve intestinal permeability: These are agents that improve the absorption capacity of the intestinal tract.

[0023] Furthermore, as described above, the GLP secretion promoter of the present invention exerts effects such as improving glucose metabolism, lowering blood glucose, preventing and improving glycation, suppressing obesity, increasing intestinal absorption and nutrient assimilation, anti-inflammatory activity, healing and repairing the gastrointestinal mucosa, reducing intestinal permeability, and increasing mesenteric blood flow through the GLP secretion promoter. Therefore, preparations containing the GLP secretion promoter can be used as the aforementioned obesity suppressant, glucose metabolism improving agent, blood glucose lowering agent, glycation prevention and improvement agent, gastrointestinal mucosa healing and repair agent, and intestinal permeability improving agent.

[0024] In addition to ordinary foods, the TGR-5 activating composition and GLP secretion promoter of the present invention can be added to nutritional supplements, functional foods, health foods, and foods for specified health uses for the purpose of achieving the above-mentioned effects.

[0025] The form of the TGR-5 activating composition and GLP secretion promoter is not particularly limited as long as it is acceptable for use in pharmaceuticals, foods, veterinary drugs, feed, etc. Examples include tablets such as sugar-coated tablets, buccal tablets, coated tablets, and chewable tablets; pills, powders, soft capsules, hard capsules, granules, suspensions, emulsions, syrups, liquid preparations such as elixirs; lozenges; processed foods; health foods (nutritional supplements, functional foods, foods for the sick, foods for specified health uses, foods with functional claims, etc.); supplements; foods for the sick (hospital food, sick person food, nursing care food, etc.); confectionery; oils and fats; dairy products; retort foods; microwaveable foods; frozen foods; seasonings; health supplements; beverages; and nutritional drinks. [Examples]

[0026] The following describes embodiments of the present invention, but the present invention is not limited to these embodiments, and various modifications are possible within the technical concept of the present invention.

[0027] Experiment 1. Confirmation of TGR-5 activation To evaluate TGR-5 activation, a luciferase reporter assay was constructed using the following procedure, and TGR-5 activation was evaluated using luminescence induced by luciferase activity.

[0028] (Preparation of carrot extract) The carrot seeds (Nantes variety from the Netherlands) and roots (yellow carrot variety from Germany) were obtained from H. Holstein. For the carrot seeds, 20g of carrot seeds, which had been crushed in a mixer, were mixed with approximately 10 times the amount of ethanol (Fujifilm Wako Pure Chemical Industries) at room temperature using a magnetic stirrer and extracted for about one week. After that, insoluble matter was removed by aspirating with filter paper, and the filtrate was concentrated under reduced pressure using a rotary evaporator to obtain 5.8g of extract (dried). For the carrot root, 20g of the carrot root pulverized in a mixer was mixed with approximately 10 times the amount of hexane (Fujifilm Wako Pure Chemical Industries), stirred at room temperature with a magnetic stirrer, and extracted for about one week. After that, insoluble matter was removed by aspirating with filter paper, and the filtrate was concentrated under reduced pressure using a rotary evaporator to obtain 3.6g of extract (dried). Each extract was redissolved in dimethyl sulfoxide (hereinafter referred to as "DMSO") (Fujifilm Wako Pure Chemical Industries, Ltd.) and subjected to each test.

[0029] (Reagents, plasmid vectors) The pcDNA3.1 expression vector (pcDNA3.1 HA-TGR5), which contains a cDNA designed to affix a hemagglutinin (HA) epitope tag to the N-terminus of human TGR-5, was obtained from GenScrip. The cAMP-dependent luciferase reporter vector (pGL4.29), which ligates a cAMP response element (CRE) to the promoter region, was obtained from Promega.

[0030] (Construction of a luciferase reporter assay system) The following procedure was used to prepare HEK / Luc stable expression cells and HEK / HA-TGR5-Luc stable expression cells for use in the experiment. Human kidney-derived HEK293 cells (ATCC) cultured in 10% FBS-containing DMEM (Gibco) were transfected with pGL4.29, and cells that acquired hygromycin B resistance (final concentration 0.25 mg / mL) were designated as HEK / Luc stable expression cells. Furthermore, these established HEK / Luc stable expression cells were transfected with pcDNA3.1 HA-TGR5, and cells that acquired G418 and hygromycin B resistance were used in experiments as HEK / HA-TGR5-Luc stable expression cells.

[0031] (cAMP-dependent luciferase reporter assay) 2 x 10 on a clear bottom 96-well white plate (Corning). 4 HEK / HA-TGR5-Luc stable expression cells were seeded to form cells, and after 24 hours, carrot seed extract, carrot root extract, or DMSO (control) were added at the respective concentrations. After a reaction at 37°C for 5 hours, luminescence was induced using the Bright-Glo® Luciferase Assay System (Promega), and the intensity was detected using a luminometer (Tecan) to represent luciferase activity. This assay evaluates the increase in intracellular cAMP concentration by measuring the activity of luciferase, which is a reporter gene. The same procedure was also performed on HEK / Luc stable expression cells that do not express TGR-5 to verify the reaction specificity of the test substance to the TGR-5 receptor. The experimental results are shown in Figures 1 and 2.

[0032] (Experimental results) Luciferase reporter assays revealed TGR-5 agonist activity in carrot seed and root extracts. Figure 1 shows that luciferase activity increased in a concentration-dependent manner upon addition of carrot seed extract. This indicates that stimulation with carrot seed extract induces an increase in cAMP concentration in HEK / HA-TGR5 ​​stably expressing cells.

[0033] Furthermore, since this effect was not observed in HEK / Luc stably expressing cells that do not express TGR-5, it was revealed that this is an agonist effect mediated by the TGR-5 receptor. Figure 2 shows the results of the luciferase reporter assay of carrot root extract. Similar to carrot seeds, carrot root extract also exhibits agonist activity mediated by the TGR5 receptor.

[0034] Experiment 2. Confirmation of GLP-1 secretion In Experiment 1, we found TGR-5 agonist activity in carrot seeds and root extracts. Therefore, we evaluated their function using GLP-1 secretion as an indicator, following the procedure described below.

[0035] (Development of a system for evaluating GLP-1 secretion) STC-1 / HA-TGR5 ​​stable expression cells for use in the experiment were prepared using the following procedure. Mouse small intestine-derived STC-1 cells (obtained from ATCC) cultured in 10% FBS-containing DMEM (Gibco) were transfected with the pcDNA3.1 HA-TGR5 ​​plasmid obtained above using Fugene HD (Promega). After 48 hours, G418-sulfate (Wako) was added to a final concentration of 1 mg / mL and cultured. Cells that acquired G418 resistance were used as STC-1 / HA-TGR5 ​​stable expression cells for the following experiments.

[0036] (Evaluation of GLP-1 secretion) The test substances used were the same as in Experiment 1: carrot seed extract and carrot root extract, and DMSO (control) was used as a negative control. The experiment involved using a 24-well plate with 5 × 10⁶ samples. 5STC-1 / HA-TGR5 ​​stable expression cells were seeded to develop into cells, and after 48 hours, the above-mentioned test substances were added at various concentrations (12.5 μg, 25 μg, 50 μg). After a reaction at 37°C for 24 hours, the culture supernatant was collected and centrifuged at 14,000 rpm for 5 minutes, and the supernatant was used as the sample for GLP-1 measurement. GLP-1 detection was performed using the Human GLP-1 (7-36 amide) Immunoassay kit (PerkinElmer Japan), and the Alpha signal was measured by an Alpha chemical amplification type luminescence proximity homogeneous assay (PerkinElmer Japan, Exit: 680 nm, Emission: 615 nm). The experimental results are shown as the ratio of the Alpha signal of the test substance divided by the Alpha signal of the negative control. The experimental results are shown in Figure 3.

[0037] (Experimental results) As shown in Figure 3, carrot seed and root extracts promoted GLP-1 secretion. The GLP-1 secretion-promoting activity of carrot seed and root extracts was stronger with carrot seed extract at lower concentrations, indicating a greater GLP-1 secretion-promoting effect. This result is consistent with the TGR-5 agonist activity results from Experiment 1.

[0038] Based on these results, it was revealed that carrot seed extract and carrot root extract possess TGR-5 agonist activity, and that their function is to promote GLP-1 secretion.

[0039] Experiment 3. Confirmation of TGR-5 agonist activity of carrot seed extract using supercritical carbon dioxide. In Experiment 1, TGR-5 agonist activity was found in ethanol extracts of carrot seeds and roots. In Experiment 3, TGR-5 agonist activity was evaluated in extracts obtained using supercritical carbon dioxide.

[0040] (Preparation of carrot extract) 70g of crushed carrot seeds (wild carrots) obtained from H. Holstein Co. (crushed in a mixer) were subjected to a supercritical fluid extraction apparatus (Mitsubishi Chemical Machinery Co., Ltd.) under conditions of 25 MPa, 40°C, and 90 minutes. The supercritical fluid containing the dissolved carrot components was then added to a recovery tank filled with 500 mL of ethanol (Fujifilm Wako Pure Chemical Industries, Ltd., 99.5%) to dissolve the supercritical carbon dioxide extract in the ethanol. Subsequently, the solvent was removed using a rotary evaporator to obtain 3.36 g of supercritical carbon dioxide extract. The TGR-5 agonist activity of the extract was evaluated by redissolving it in DMSO and using it for testing.

[0041] (cAMP-dependent luciferase reporter assay) A cAMP-dependent luciferase reporter assay was performed, similar to Experiment 1. The results are shown in Figure 4.

[0042] (Experimental results) Luciferase reporter assays revealed that carrot seed extract extracted using supercritical carbon dioxide exhibited TGR-5 agonist activity (see Figure 4). Figure 4 shows that supercritical carbon dioxide-extracted carrot seed extract increased luciferase activity in a concentration-dependent manner. This indicates that stimulation with carrot seed extract induced an increase in cAMP concentration in HEK / HA-TGR5 ​​stably expressing cells.

[0043] Furthermore, since this effect was not observed in HEK / Luc stably expressing cells that do not express TGR-5, it became clear that this is an agonist effect mediated by the TGR-5 receptor. These results indicate that, similar to the ethanol extraction method, the supercritical carbon dioxide extraction method also produces carrot seed extract, which exhibits agonist activity via the TGR-5 receptor.

[0044] Experiment 4. Confirmation of TGR-5 agonist activity of island ginseng root extract. In Experiment 1, TGR-5 agonist activity was found in ethanol extracts of Western-type carrot seeds and roots. In Experiment 3, TGR-5 agonist activity was evaluated using root extracts of Okinawa Island carrot, an Oriental-type carrot.

[0045] (Extraction from island carrot root) 420g of Okinawan ginseng root obtained from Ryukyu Seika Co., Ltd. was sliced, and the moisture was removed by freeze-drying to obtain 40.7g of dried product. The dried Okinawan ginseng root was pulverized using a mixer, suspended in 230mL of ethanol (Fujifilm Wako Pure Chemical Industries, 99.5%), stirred with a magnetic stirrer, and extracted at room temperature for one week. Insoluble matter was then removed by aspirating with filter paper, and the solvent was removed from the filtrate using a rotary evaporator. As a result, 3.36g of Okinawan ginseng root extract was obtained. The extract was redissolved in DMSO and used for TGR-5 agonist activity evaluation.

[0046] (cAMP-dependent luciferase reporter assay) A cAMP-dependent luciferase reporter assay was performed, similar to Experiment 1. The results are shown in Figure 5.

[0047] (Experimental results) Luciferase reporter assays revealed TGR-5 agonist activity in ethanol-extracted ginseng root (see Figure 5). Figure 5 shows that ginseng root extract increases luciferase activity in a concentration-dependent manner. This indicates that stimulation with ginseng root extract induces an increase in cAMP concentration in HEK / HA-TGR5 ​​stably expressing cells.

[0048] Furthermore, since this effect was not observed in HEK / Luc stably expressing cells that do not express TGR-5, it became clear that this is an agonist effect mediated by the TGR-5 receptor. These results indicate that the root extract of Okinawan ginseng, an Oriental variety, possesses agonist activity mediated by the TGR-5 receptor.

[0049] Experiment 5. Confirmation of PGC1α induction activity via TGR-5 agonist action. Peroxisome proliferators-activated receptor-γ co-activator-1α (PGC1α), a master regulator of mitochondrial biosynthesis, has been reported to be induced by elevated intracellular cAMP due to TGR-5 agonist activity, contributing to the beigeing of white adipocytes. Therefore, we evaluated whether various carrot extracts possessing TGR-5 agonist activity exhibit PGC1α-inducing activity.

[0050] (Preparation of carrot extract) Nantes carrot seed extract: An extract of carrot seeds (Nantes variety from the Netherlands) obtained in the same manner as in Experiment 1 was used. Wild carrot seed extract: 20g of wild carrot seeds (H. Holstein Co., Ltd.), which had been crushed in a mixer, were mixed with approximately 10 times the amount of ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.), stirred with a magnetic stirrer at room temperature, and extracted for about one week. After that, insoluble matter was removed by aspirating with filter paper, and the filtrate was concentrated under reduced pressure using a rotary evaporator to obtain 2.8g of extract (dried). Island ginseng root extract: An island ginseng root extract obtained in the same manner as in Experiment 4 was used. Each extract was redissolved in DMSO and used in the test.

[0051] (Transfection and addition of test substance) Human liver-derived Hep3B cells (obtained from ATCC) cultured in 10% FBS-containing DMEM (Gibco) were divided into 5 × 10⁶ cells. 4Cells were seeded in 24-well plates to a density of cells / well, and pcDNA3.1 HA-TGR5 ​​of the plasmid described in Experiment 1 was added to a density of 0.4 μg DNA / well using Fugene HD (Promega). 48 hours after transfection, test substances of various concentrations (Nantes ginseng seeds, wild ginseng seeds, and island ginseng roots) were added, and RNA was collected after 4 hours. A control was provided with 0.1% DMSO, and a TGR-5 receptor agonist (abcam:ab142091) was used as a positive control.

[0052] (RNA extraction and real-time PCR) RNA extraction was performed using the RNeasy Plus Mini Kit purchased from Qiagen, and the RNA concentration was quantified using NanoDrop (Thermo Fisher Scientific). Subsequently, single-stranded cDNA was synthesized using PrimeScript RT Reagent Lit (Takara) and subjected to real-time PCR. Real-time PCR was performed using Premix EX Taq (Takara), Taqman probes, and a real-time PCR instrument (Thermo Fisher Scientific). Taqman probes for the target gene PGC1α (Hs00173304_m1) and the housekeeping gene 18S (Hs99999901_s1) were obtained from Thermo Fisher Scientific. Gene expression levels were evaluated using the ΔΔCt method by setting a threshold at an appropriate point in the exponential amplification region of the PCR product, calculating the number of cycles (threshold cycle: Ct value) obtained from the intersection of the threshold and the PCR product amplification curve, and then measuring the expression levels between each test substance. For evaluation of expression levels between each test substance, the PGC1α mRNA expression level in 0.1% DMSO (the control) was divided by the 18S expression level and set to 1, and this value was compared with the expression level when the test substance was added.

[0053] (Experimental results) Real-time PCR using a Taqman probe revealed that ethanol extracts of Nantes ginseng seeds, wild ginseng seeds, and Okinawan ginseng roots exhibited PGC1α-inducing activity compared to the control. In particular, Okinawan ginseng root extract induced PGC1α mRNA expression more strongly than the positive control TGR-5 receptor agonist. The results suggest that various carrot extracts possessing TGR-5 agonist activity have PGC1α-inducing activity, which is the master regulator of mitochondrial biosynthesis, and contribute to the enhancement of heat production and energy production associated with the beigeing of white adipocytes.

[0054] Experiment 6. Confirmation of GLP-1 secretion-promoting effect (in vivo) In Experiment 1, carrot seed extract was identified as a material possessing TGR-5 agonist activity. In Experiment 2, cell studies confirmed that this extract promotes GLP-1 secretion, one of the functions of TGR-5 (see Experiment 2, Figure 3). Furthermore, in Experiment 6, the effect of oral administration of carrot seed extract on increasing blood GLP-1 secretion was confirmed.

[0055] (Preparation of carrot extract) An extract of wild carrot seeds obtained in the same manner as in Experiment 5 was used.

[0056] (in vivo study) C57BL / 6J mice (8 weeks old, male) were fasted and administered the test substance orally (10 mL / kg) after dissolving it in a solvent (water for injection, 5% DMSO, 0.5% carboxymethylcellulose (hereinafter referred to as CMC), 1% Tween-80). Thirty minutes after administration of the test substance, glucose dissolved in water for injection was administered (2000 mg / kg / 5 mL), and blood was collected from the posterior vena cava 15 minutes later. The collected blood was transferred to a tube containing 1.5 mg / mL LEDTA-2Na (Tokyo Chemical Industry Co., Ltd.), 500 KIU / mL aprotinin (Merck), and DPP IV Inhibitor (Merck), and centrifuged at 3000 rpm, 4°C, for 10 minutes to obtain plasma, which was stored at -80°C until GLP-1 measurement. GLP-1 in plasma was detected using the Human GLP-1(7-36amide) Immunoassay kit (PerkinElmer Japan).

[0057] The settings for each group (n=7) are as follows. (1) Vehicle group (water for injection, 5% DMSO, 0.5% CMC, 1% Tween-80) (2) Control group (glucose alone, 2000 mg / kg) (3) Glucose group (2000 mg / kg) + Carrot seed (wild carrot) extract (500 mg / kg) administration group (4) Glucose (2000 mg / kg) + APD597 (GPR119 agonist: Namiki Shoji) 30 mg / kg administration group

[0058] (Experimental results) The results are shown in Figure 7. The control group, administered glucose alone, showed increased GLP-1 secretion compared to the vehicle group, administered only the solvent. Under these conditions, carrot seed extract showed an even stronger GLP-1 secretion-promoting effect compared to the control group, and its strength was almost equivalent to that of APD597, a GPR119 agonist. These results suggest that carrot seed extract has a GLP-1 secretion-promoting effect. [Industrial applicability]

[0059] The present invention can provide a novel TGR-5 activating composition and a GLP secretion promoter. Furthermore, it can provide pharmaceuticals, quasi-drugs, oral cosmetics, oral beauty products, health foods (nutritional supplements, functional foods, foods for the sick, foods for specified health uses, foods with functional claims, etc.), supplements, animal feed, etc., containing the novel TGR-5 activating composition and GLP secretion promoter.

Claims

1. A TGR-5 activating composition containing carrot extract, The aforementioned carrot extract is an extract from the plant body of a plant belonging to the genus Daucus in the family Apiaceae. The aforementioned plant of the genus Daucus in the family Apiaceae is at least one species selected from the group consisting of Daucus nanthes, Daucus carota, Daucus mongolica, and Daucus carota. The aforementioned plant body is a root or a seed. The carrot extract is either an extract of the plant body using an organic solvent, or a supercritical extract using carbon dioxide. The organic solvent is at least one selected from the group consisting of ethanol and hexane. The extract of the plant body with the organic solvent is An extraction step in which the organic solvent is added to the plant body to obtain an extract containing insoluble matter. A filtration step to obtain a filtrate by filtering the insoluble matter from the extract, and The method is obtained by a step of removing at least a portion of the organic solvent contained in the filtrate, The supercritical extract is An extraction step in which a supercritical fluid of carbon dioxide is added to the plant body to extract the components of the plant body into the supercritical fluid, The process involves dissolving the supercritical fluid from which the plant components have been extracted in an organic solvent to obtain a mixture, and A TGR-5 activating composition obtained by a method comprising the step of removing at least a portion of the organic solvent from the mixture.

2. The plant of the genus Carrot in the family Apiaceae is Carrot nanthes, the plant body is a seed, and the organic solvent is ethanol. The plant of the genus Daucus in the family Apiaceae is yellow ginseng, the plant body is a root, and the organic solvent is hexane. The plant of the genus Daucus in the family Apiaceae is wild carrot, the plant body is a seed, and the organic solvent is ethanol, or The TGR-5 activating composition according to claim 1, wherein the plant of the genus Daucus in the family Apiaceae is Okinawa Island ginseng, the plant body is a root, and the organic solvent is ethanol.

3. A GLP secretion promoter containing carrot extract, The aforementioned carrot extract is an extract from the plant body of a plant belonging to the genus Daucus in the family Apiaceae. The aforementioned plant of the genus Daucus in the family Apiaceae is at least one species selected from the group consisting of Daucus nanthes, Daucus carota, Daucus mongolica, and Daucus carota. The aforementioned plant body is a root or a seed. The carrot extract is either an extract of the plant body using an organic solvent, or a supercritical extract using carbon dioxide. The organic solvent is at least one selected from the group consisting of ethanol and hexane. The extract of the plant body with the organic solvent is An extraction step in which the organic solvent is added to the plant body to obtain an extract containing insoluble matter. A filtration step to obtain a filtrate by filtering the insoluble matter from the extract, and The method is obtained by a step of removing at least a portion of the organic solvent contained in the filtrate, The supercritical extract is An extraction step in which a supercritical fluid of carbon dioxide is added to the plant body to extract the components of the plant body into the supercritical fluid, The process involves dissolving the supercritical fluid from which the plant components have been extracted in an organic solvent to obtain a mixture, and A GLP secretion promoter obtained by a method comprising the step of removing at least a portion of the organic solvent from the mixture.

4. The plant of the genus Carrot in the family Apiaceae is Carrot nanthes, the plant body is a seed, and the organic solvent is ethanol. The plant of the genus Daucus in the family Apiaceae is yellow ginseng, the plant body is a root, and the organic solvent is hexane. The plant of the genus Daucus in the family Apiaceae is wild carrot, the plant body is a seed, and the organic solvent is ethanol, or The GLP secretion promoter according to claim 3, wherein the plant of the genus Daucus in the family Apiaceae is Okinawa Island ginseng, the plant body is a root, and the organic solvent is ethanol.

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