TGR5 activator

Abstract

To provide a novel TGR5 activator comprising a naturally derived component as an active ingredient, and to provide food and beverage or the like for TGR5 activation, enhancement of basal metabolic rate, inhibition of lipid accumulation, as well as anti-obesity, anti-fatigue, or anti-diabetic effects.SOLUTION: This invention has been completed based on the finding that: zingiberenes have a TGR5 activating effect.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a TGR5 activator, a method for activating TGR5, and the like. [Background technology]

[0002] TGR5 is widely expressed in the body and is known to receive and activate bile acids. It has been reported that one of the effects of TGR5 activation is improving glucose metabolism, which contributes to anti-obesity and anti-diabetes, and various investigations are being conducted on the components that activate TGR5.

[0003] For example, Patent Document 1 discloses a drug for exerting TGR5 agonism, which contains as an active ingredient an ionone selected from the group consisting of α-ionone, α-methylionone, γ-methylionone, α-allylionone, β-ionone, dihydro β-ionone, and 5,6-epoxy-β-ionone, and Patent Document 2 discloses a TGR5 activator, which contains as an active ingredient at least one selected from gingerol, banaba leaf extract, red perilla extract, red ginger extract, and chaste tree extract. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent No. 5818259 [Patent Document 2] JP 2016-8196 A Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention provides a new TGR5 activator that contains a component derived from a natural product as an active ingredient, and also provides foods, beverages, etc. for activating TGR5, increasing basal metabolic rate, inhibiting lipid accumulation, anti-obesity, anti-fatigue, or anti-diabetes. [Means for solving the problem]

[0006] The inventors discovered that zingiberenes have a TGR5 activating effect, and completed the present invention.

[0007] That is, the present invention relates to the following aspects [1] to [4]. [1] A TGR5 activator whose active ingredient is zingiberene. [2] The TGR5 activator according to [1], which is used for increasing basal metabolic rate, suppressing lipid accumulation, anti-obesity, anti-fatigue or anti-diabetes. [3] A food, drink, medicine, quasi-drug, or feed containing the TGR5 activator described in [1]. [4] A food or drink, medicine, quasi-drug, or feed for activating TGR5, increasing basal metabolic rate, suppressing lipid accumulation, preventing obesity, preventing fatigue, or preventing diabetes, comprising the TGR5 activator described in [1]. Effect of the Invention

[0008] The present invention makes it possible to provide a safe TGR5 activator that contains active ingredients derived from natural products, and by containing zingiberenes as active ingredients, it is possible to provide foods and beverages, pharmaceuticals, quasi-drugs, or feed for activating TGR5, increasing basal metabolic rate, inhibiting lipid accumulation, anti-obesity, anti-fatigue, or anti-diabetes. [Brief description of the drawings]

[0009] [Figure 1] The TGR5 activity of the negative control (NC) and ginger oil at each final concentration is shown in Fig. 1, where SEAP (secretory alkaline phosphatase) activity was measured using a TGR5 (GP-BAR1) Reporter Assay Kit. [Diagram 2] The TGR5 activity of the negative control (NC) and compositions containing zingiberenes at each final concentration is shown in FIG. 13, where SEAP (secretory alkaline phosphatase) activity was measured using a TGR5 (GP-BAR1) Reporter Assay Kit. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] The TGR5 activator of the present invention is a TGR5 activator containing zingiberenes as an active ingredient, and is preferably a TGR5 activator for increasing basal metabolic rate, suppressing lipid accumulation, anti-obesity, anti-fatigue or anti-diabetes.

[0011] The zingiberenes described in the present invention refer to zingiberene represented by the following structural formula and a group of compounds similar to said chemical structure, and are sesquiterpenoids such as sesquiphellandrene, bisabolene, cadinene, etc. The origin of zingiberenes is not particularly limited, and examples thereof include chemical synthesis and extraction from plants, but since ginger-derived ginger oil derived from ginger rhizomes contains zingiberenes at high concentrations, ginger-derived ginger oil is preferred.

[0012] [ka]

[0013] In the present invention, the zingiberens serving as active ingredients are not particularly limited as long as they can exert the TGR5 activation effect, and a substance containing zingiberens may be used, or purified zingiberens may be used, or a ginger extract containing zingiberens may be used. When zingiberens are purified, they can be purified by a general purification method such as reverse phase chromatography. As the ginger extract, for example, fresh ginger rhizomes are dried, extracted by an extraction method such as supercritical extraction, steam distillation, or solvent extraction, and then subjected to solid-liquid separation by filtration using nonwoven fabric, mesh, or the like, centrifugation, or the like, followed by concentrating the ginger extract. Ginger oil is preferred, and for example, commercially available ginger oil containing essential oil components can be used, but an extract containing less 6-shogaol than 6-gingerol is preferred, with a shogaol / gingerol (S / G) ratio of preferably 0.5 or less, more preferably 0.4 or less, and relative to 100 parts by weight of zingiberenes, 6-gingerol is preferably 10 parts by weight or less, more preferably 5 parts by weight or less, and 6-shogaol is preferably 5 parts by weight or less, more preferably 2 parts by weight or less.

[0014] The zingiberene-containing product used in foods, beverages, etc. is preferably a concentrated or dried product, and can be dried and powdered by drum drying, air drying, spray drying, vacuum drying, and / or freeze drying, etc., and a general excipient can be used. Specifically, a powder product obtained by adding an emulsifier to the zingiberene-containing product to emulsify it to increase dispersibility, then blending an excipient such as dextrin and spray drying is preferable. The emulsifier is not particularly limited as long as it is a component having emulsifying ability, and examples thereof include polysaccharides such as acacia gum, ghatti gum, xanthan gum, and starch sodium octenyl succinate, proteins or protein hydrolysates such as casein and soybean protein, phospholipids such as lecithin and enzyme-treated lecithin, polyglycerol fatty acid esters having an HLB value of 10 to 20 or 12 to 18, and sucrose fatty acid esters. The emulsion is preferably an emulsion containing zingiberenes, enzyme-treated lecithin, polyglycerol fatty acid ester, glycerin, reduced starch syrup or other polyol, and water, and two or more kinds of polyols may be used. The emulsification can be carried out by a general emulsification method, and more specifically, the emulsification can be carried out using an emulsification device such as a high-pressure homogenizer, colloid mill, ultrasonic emulsifier, homomixer, homodisper, or the like, or two or more kinds of devices may be combined, and the zingiberenes-containing emulsion can be obtained by the emulsification.

[0015] The TGR5 activator of the present invention can activate TGR5 by ingestion, and activation of TGR5 improves glucose metabolism, increases basal metabolism, and facilitates burning of lipids, thereby providing lipid accumulation suppressing effects, anti-obesity effects that suppress obesity, anti-fatigue effects that suppress fatigue, anti-diabetic effects, etc., and is therefore useful as a TGR5 activator for increasing basal metabolism, suppressing lipid accumulation, anti-obesity, anti-fatigue, or anti-diabetic purposes. The method of ingestion may involve orally ingesting a substance containing zingiberenes, and is not particularly limited as long as the effects of the present invention are observed in the amount of ingestion, and may be taken once a day or in divided doses.

[0016] In the present invention, since the active ingredient zingiberenes are derived from natural products, they can be widely used and can be added to various products, either in liquid form or in dry form. The foods and beverages to which the zingiberenes are added are not particularly limited, and the zingiberenes can be used in various foods and beverages such as beverages, foods, seasonings, functional foods, and supplements, as well as in medicines, quasi-drugs, and feeds, and can be used for TGR5 activation, increasing basal metabolic rate, inhibiting lipid accumulation, anti-obesity, anti-fatigue, or anti-diabetes. The content of the zingiberenes in various products is not particularly limited as long as the effects of the present invention are observed by ingestion, but is preferably 0.00001 to 80% by weight, more preferably 0.0001 to 60% by weight, and even more preferably 0.001 to 50% by weight, and preferably 0.01 to 1,000 ppm of zingiberenes, more preferably 0.02 to 500 ppm, and even more preferably 0.05 to 200 ppm. EXAMPLES

[0017] The present invention will be described in detail below with reference to examples, but the present invention is not limited to the following examples. In the present invention, all % is by weight unless otherwise specified.

[0018] [Preparation 1] Ginger Oil (Ozone Naturals), an extract from ginger rhizome, was applied to an ODS column and eluted with ethanol. A fraction with a relatively high content of zingiberenes was separated and concentrated to obtain a zingiberene-containing composition (zingiberenes: 62.7%) that contains zingiberene, β-sesquiphellandrene, β-bisabolene, γ-cadinene, etc. 6-gingerol and 6-shogaol were not detected.

[0019] [Example 1] The TGR5 activator was measured using the TGR5 (GP-BAR1) Reporter Assay Kit (Cayman Chemical Company). In detail, the expression of the reporter SEAP (secretory alkaline phosphatase) was induced by binding an agonist to TGR5 expressed on cells, and the activity of this SEAP was measured by chemiluminescence to measure the TGR5 activator.

[0020] The HEK293T cells were subcultured in D-MEM medium containing 10% FBS, 100 U / mL penicillin, and 100 U / mL streptomycin. The cells were cultured in a T25 culture flask, with 0.25 to 0.5 × 10 5 The cells were cultured at 37°C in a 5% CO2 incubator.

[0021] HEK293T cells precultured in three T25 culture flasks were washed twice with PBS(-). The cells were detached from the walls by trypsin-EDTA treatment, and the resulting cell suspension was transferred to a 50 mL conical tube containing 10 mL of serum-containing culture medium. The tube was centrifuged at room temperature for 5 minutes at 1000 rpm (190 × g), the supernatant was discarded by decantation, and the cells were recovered as a pellet. After loosening the cells by tapping, 10 mL of culture medium was added. The tube was centrifuged at room temperature for 5 minutes at 1000 rpm (190 × g), the supernatant was discarded by decantation, and the cells were recovered as a pellet. 30 mL of culture medium was added to the cell pellet, the cells were uniformly suspended with a pipette, and 200 μL was transferred to a 1.7 mL tube, and the cell count and viability were measured. The remaining liquid was used for the test because the viability was over 90%.

[0022] Based on the measured cell number, 2 x 10 5 The cell number was adjusted to 10 cells / mL. The resulting cell suspension was diluted to 200 μL (4 × 10 4 Two spare plates were transferred to a 37℃ 5% CO2 incubator and pre-cultured for 20 to 24 hours.

[0023] Before the completion of the pre-incubation, a test solution, a negative control (NC) solution, and a positive control (PC) solution were prepared. The test solution was the same ginger oil as in Preparation 1, with a maximum concentration of 0.4%, and six 10-fold dilutions were prepared in a medium containing 0.1% DMSO. The negative control solution was a medium containing 0.1% DMSO, and the positive control solution was the TGR5 Receptor Agonist Positive control included in the kit.

[0024] After the pre-incubation, the culture medium in each well was sucked up and replaced with 150 μL of pre-warmed serum-free medium. 50 μL of each of the test solutions, NC or PC solutions was added to the corresponding wells, which were then transferred to a 5% CO2 incubator at 37°C and cultured for 8 hours (n=3). In addition, 50 μL of serum-free medium was added to wells incubated in the same manner as above to serve as blanks. The final concentrations of each test solution were 0.1%, 0.01%, 0.001%, 0.0001%, 0.00001% and 0.000001%.

[0025] 10 μL of the culture supernatant was transferred to a white plate using an 8-well multi-well pipette, covered, and then heated at 65°C for 30 minutes to inactivate endogenous alkaline phosphatase. After the plate was returned to room temperature, a solution containing a luminescent substrate was added to the plate using an 8-well multi-well pipette at a ratio of 50 μL / well.

[0026] After shaking for 15 minutes at room temperature in the dark, the RLU (Relative Luminescence Units) were measured using a luminescence plate reader under the following measurement conditions: shaking for 5 seconds, measuring for 0.5 seconds / well, and sensitivity of 1 / 100. The average and standard deviation of each measurement value were calculated, and the blank values ​​subtracted from each test solution and NC and the standard deviation are shown in Figure 1. A significant difference test was performed, and a significant difference (*: P<0.05, **: P<0.01) was observed at final concentrations of 0.01% and 0.1%. Furthermore, TGR5 activity was confirmed in the PC, confirming that there were no problems with the experimental system.

[0027] [Example 2] The test solutions were the zingiberene-containing compositions prepared in Preparation 1, and were prepared in a 0.1% DMSO-containing medium in five 5-fold dilutions with a maximum concentration of 0.02%, in the same manner as in Example 1. The final concentrations of the test solutions were 0.005%, 0.001%, 0.0002%, 0.00004%, and 0.000008%.

[0028] The average value and standard deviation of each measured value were calculated, and the blank value was subtracted from each test solution and NC, and the standard deviation is shown in Figure 2. A significant difference test was performed, and a significant difference (*: P<0.05) was observed at a final concentration of 0.005% (zingiberenes: 31.4 ppm). Furthermore, TGR5 activity was confirmed in PC, confirming that there was no problem with the experimental system.

[0029] From the above, it was found that TGR5 is activated by products containing zingiberenes.

Claims

1. A TGR5 activator whose active ingredient is zingiberene.

2. The TGR5 activator according to claim 1, which is used for increasing basal metabolic rate, suppressing lipid accumulation, preventing obesity, preventing fatigue, or preventing diabetes.

3. A food or drink, a medicine, a quasi-drug, or a feed comprising the TGR5 activator according to claim 1.

4. A food or drink, medicine, quasi-drug, or feed for activating TGR5, increasing basal metabolic rate, inhibiting lipid accumulation, preventing obesity, preventing fatigue, or preventing diabetes, comprising the TGR5 activator according to claim 1.

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