JP5883599B2 - Method for evaluating or selecting catechin bitterness inhibitor - Google Patents

Description

  The present invention relates to a method for evaluating or selecting a bitterness inhibitor that suppresses bitterness derived from catechins.

  Catechin is attracting attention because it is effective in improving lifestyle-related diseases such as cancer, hypertension, arteriosclerosis, cold prevention, and bactericidal effect on caries (Non-patent Documents 1 and 2), and is included in many beverages. Yes. In order to effectively express the physiological effect, a technique for blending a high concentration of catechins in a beverage is desired in order to easily consume a large amount of catechins.

  On the other hand, since catechins have a bitter taste, there is a problem that the amount of use is limited. Therefore, controlling the bitter taste of catechin is important for increasing the utility value of catechin in a wider range of fields.

  Recognition of bitterness in humans begins with binding to Taste type 2 receptor (TAS2R), a bitter taste receptor expressed on the surface of taste cell membranes in the taste buds of the tongue. TAS2R is a kind of G protein-coupled receptor (GPCR), and 26 kinds of humans and 40 kinds of rats have been identified. TAS2R is known to couple with gustducin, which is classified as Gαi, by binding to a ligand and transmit a signal by causing an increase in intracellular calcium concentration (Non-patent Document 3). ). For some TAS2Rs, bitter substances that serve as ligands have been identified (Non-Patent Document 4).

  As a method for individually observing the activity of a receptor, a method using a gastrointestinal tract cell (non-patent documents 5 and 6) of a non-human animal such as a mouse or rat in which a specific bitter taste receptor is expressed or the purpose thereof There is a method of functionally expressing human bitter taste receptors for other bitter substances in cultured cells (Patent Document 1). In order to suppress human bitterness, a method using a human bitter taste receptor is more preferable.

As a method for detecting the bitter taste receptor activity, a GTPγS binding assay for measuring the amount of GTPγS binding to the amount of activated G protein (Non-patent Document 7) and a trypsin digestion assay for visualizing G protein using a labeled antibody (Non-patent document) Reference 8), activity of phospholipase C (PLC) located downstream of TAS2R receptor, measurement of diacylglycerol and inositol 1,4,5-triphosphate (IP ₃ ) produced from them, phosphodiesterase (PDE) activity Measurement and the like are known, and the calcium imaging method is mainly used (Non-Patent Documents 9 and 10).

  For the development of a material that suppresses the bitter taste of catechin, it is expected that a method of functionally expressing the human bitter taste receptor in cultured cells and individually observing the activity of the receptor can be used. Human bitter taste receptors for catechins must be identified. However, there are few reports on the responsiveness of human bitter taste receptors to catechins. So far, one of the human bitter taste receptors, TAS2R39, has been reported to respond to some catechin species (Non-Patent Document 11), but bitter taste receptors that are responsive to catechins other than TAS2R39. It is not clear whether the body exists.

Special table 2010-508041 gazette

Okabe S, et al., Jpn Cancer Res, 88, 639-643 (1997) Masahiko Hara et al., Journal of the Japan Food Industry Association, 36, 951-955 (1989) Wong GT, et al., Nature, 381, 796-800 (1996) Chandrashekar J, et al., Cell, 100, 703-711 (2000) Wu SV, et al., Physiol Genomics, 22, 139-149 (2005) Wu SV, et al., Proc Natl Acad Sci USA, 99, 2392-2397 (2002) Wessling-Resnick M, and Johnson GL, J. Biol. Chem, 262, 12444-12447 (1987) Ming D, et al., Proc Natl Acad Sci USA, 95, 8933-8938 (1998) Bufe B, et al., Nat Genet, 32, 397-401 (2002) Alexey N, et al., Chem Senses, 29, 583-593 (2004) Narukawa M, et al., Biochem Biophys Res Commun, 405, 620-625 (2011)

  The present invention relates to a method for evaluating or selecting a catechin bitterness inhibitor using a bitter taste receptor that responds to catechins.

  The inventors have succeeded in newly identifying a bitter taste receptor that responds to catechins. And, the responsiveness of the receptor to catechins correlates well with human bitter sensory evaluation, and it has been found that a substance that suppresses the activity of the receptor can be evaluated or screened as a bitterness inhibitor that suppresses the bitter taste of catechins. It was.

That is, the present invention provides the following.
(1) A method for evaluating or selecting a catechin bitterness inhibitor,
Adding a catechin and a test substance to one or more bitter taste receptors selected from the group consisting of TAS2R14 and TAS2R40;
Measuring the response of the receptor to the test substance and catechins;
Adding only the catechins without adding a test substance;
Measuring the response of the receptor to the catechins only; and comparing the response of the receptor to the test substance and catechins with the response to the catechins only. A step of evaluating or selecting the test substance as a bitterness inhibitor by catechins, if the response is less than the response to
Including a method.
(2) The method according to (1), wherein the bitter taste receptor is a bitter taste receptor on a cell that naturally expresses a bitter taste receptor or on a recombinant cell genetically engineered to express a bitter taste receptor. .

  According to the present invention, it is possible to easily and objectively evaluate or select a substance that suppresses the bitter taste of catechins without depending on subjective sensory evaluation, and it is also possible to perform high-throughput screening. Become.

The graph which shows the response of TAS2R14 and TAS2R40 with respect to EGCg. The graph which shows the correlation with catechin responsiveness and sensory evaluation in a TAS2R14 expression cell. The graph which shows the EGCg response suppression activity of the TAS2R14 expression cell by (beta) -cyclodextrin ((beta) -CD).

  As shown in Examples described later, TAS2R14 and TAS2R40 were newly identified as human bitter taste receptors that respond to catechins. The responsiveness of the receptor to catechins correlates well with the bitter sensory evaluation in humans. This result means that a material that suppresses bitterness based on catechins can be easily evaluated or selected by measuring the response-suppressing activity of cultured human cells in which the receptor is expressed.

  The method for selecting a catechin bitterness inhibitor of the present invention comprises adding a test substance and catechins to one or more bitter taste receptors selected from the group consisting of TAS2R14 and TAS2R40; the test substance and catechins Measuring the response of the receptor to the test substance; adding only the catechins without adding the test substance; measuring the response of the receptor to the catechins only; and receiving the test substance and the catechins The response of the body to the response to only the catechins is compared, and if the response to the test substance and catechins is less than the response to only the catechins, the test substance is evaluated as a bitterness inhibitor by catechins or The step of selecting.

  In the present invention, the catechin bitterness inhibitor means a substance capable of reducing the bitterness caused by catechins. That is, it refers to a substance that reduces the bitterness of catechins when ingested with catechins.

In the present invention, the bitter taste receptor used is at least one selected from TAS2R14 and TAS2R40, which are bitter taste receptors (catechin receptors) that respond to catechins. In addition, the substance selected in the present invention is a substance that suppresses bitterness caused by catechins.
In the present invention, the catechins include, for example, catechin (C), gallocatechin (GC), catechin gallate (Cg), non-epimeric catechins such as gallocatechin gallate (GCg), epicatechin (EC), epigallo Examples include epicatechins such as catechin (EGC), epicatechin gallate (ECg), and epigallocatechin gallate (EGCg). Of these, epigallocatechin gallate (EGCg) is preferred.

TAS2R14 and TAS2R40 are bitter taste receptors expressed in human taste cells, and are registered in GenBank as GI: 12965181 and GI: 28882032, respectively. TAS2R14 is a protein consisting of the amino acid sequence represented by SEQ ID NO: 2, encoded by a gene having the gene sequence represented by SEQ ID NO: 1. TAS2R40 is a protein consisting of an amino acid sequence represented by SEQ ID NO: 4 encoded by a gene having a gene sequence represented by SEQ ID NO: 3. In addition, the amino acids having the sequence identity of 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, still more preferably 98% or more with respect to the amino acid sequences of TAS2R14 and TAS2R40. A polypeptide having a sequence and responsiveness to catechins is treated in the same manner as the catechin receptor of the present invention.
In the method of the present invention, any one of the bitter taste receptors may be used alone, or a plurality may be used in combination.

  The sequence identity of amino acid sequences is calculated by the Lippman-Pearson method (Lipman-Pearson method; Science, 227, 1435, (1985)). Specifically, using the homology analysis (Search homology) program of genetic information processing software Genetyx-Win (Ver.5.1.1; software development), the unit size to compare (ktup) is set to 2 and the calculation is performed. Is done.

  In the method of the present invention, the bitter taste receptor can be used in any form as long as the function of the receptor is not lost. For example, the bitter taste receptor is a tissue or cell that naturally expresses a bitter taste receptor, such as a bitter taste receptor or a taste cell isolated from a living body, or a culture thereof; a membrane of taste cells carrying the bitter taste receptor Used in the form of a recombinant cell or culture thereof genetically engineered to express the bitter receptor; a membrane of the recombinant cell; and an artificial lipid bilayer membrane having the bitter receptor Can be done. All of these forms fall within the range of bitter taste receptors used in the present invention.

In a preferred embodiment, cells that naturally express bitter taste receptors such as taste cells, recombinant cells genetically engineered to express bitter taste receptors, or cultures thereof are used in the methods of the invention. .
The recombinant cell can be prepared by transforming a cell using a vector incorporating a gene encoding a bitter taste receptor. Preferably, in order to improve the responsiveness of the bitter taste receptor expressed in cultured cells, the gene-incorporated vector encoding the chimeric G protein is introduced together with the vector into which the gene encoding the bitter taste receptor is incorporated.

  Examples of the chimeric G protein that can be used for the production of the recombinant cell include, for example, amino acids of amino acids 1 to 330 in the amino acid sequence of human Gα16 (SEQ ID NO: 5) and amino acid number 310 in the amino acid sequence of human gustducin (SEQ ID NO: 6). Polypeptide G16gust44 (Ueda T, et al., J Neurosci, 23, 7376-7380 (2003)) which improves the responsiveness of the bitter taste receptor which consists of -354 amino acids expressed in the recombinant cell is mentioned. Human Gα16 is registered in GenBank as GI: 156104883, and human Gustducin is registered as GI: 156139155. The chimeric G protein is not particularly limited as long as it is a polypeptide that improves the responsiveness of a bitter taste receptor expressed in a recombinant cell, and includes, for example, a base sequence represented by SEQ ID NO: 7 in addition to G16gust44. G16gust36 etc. which are encoded by DNA and have the amino acid sequence shown by SEQ ID NO: 8 can be used (see Example 2).

In the method of the present invention, following the addition of the test substance and catechins, the activity of the bitter taste receptor for the test substance is measured. The measurement may be performed by any method known in the art as a method for measuring the activity of a bitter taste receptor, for example, a method for measuring the amount of intracellular calcium. For example, in HEK293T cells in which G16gust is expressed, a bitter taste receptor is known to increase intracellular calcium content by activating PLCβ2 in combination with G16gust when activated by a bitter substance. (Ueda T, et al., J Neurosci, 23, 7376-7380 (2003)). Therefore, the activity of the bitter taste receptor can be measured by using the amount of intracellular calcium after the addition of the bitterness substance as an index. As a method for measuring the amount of calcium, fluorescence intensity measurement using a calcium sensitive dye or the like can be mentioned. This method searches for a target substance by changing the fluorescence intensity (intracellular calcium concentration) when a test substance is contacted with a TAS2R14-expressing cell or TAS2R40 cell into which a calcium-sensitive dye has been introduced for a certain period of time. .
Examples of calcium-sensitive dyes include Fura-2, Fluo-3, and Fluo-4.

Next, the test substance is evaluated based on the measured activity of the bitter taste receptor, and the test substance that suppresses the receptor activity by the catechins is evaluated or selected as a bitter taste inhibiting substance for the catechins.
The test substance is evaluated based on, for example, the presence or absence of a significant difference. If inhibition of activity against catechins can be confirmed in the presence of the test substance in the absence of the test substance, the test substance can be determined (evaluated) as a bitterness-inhibiting substance against catechins. Preferably, a test substance showing an EGCg cell response inhibition rate of 50% or more can be used as a bitter taste inhibitor.
In the present invention, “selection” includes screening for a bitterness-inhibiting substance based on the above evaluation.

  In the method of the present invention, the test substance added to the bitter taste receptor is not particularly limited as long as it is a substance desired to be used as a bitterness inhibitor by catechins. The test substance may be a naturally occurring substance, a substance artificially synthesized by a chemical or biological method, etc., a new compound, a composition or a mixture. Also good. For example, nucleic acid, carbohydrate, lipid, protein, peptide, organic low molecular weight compound, compound library prepared using combinatorial chemistry technology, random peptide library prepared by solid phase synthesis or phage display method, or microorganism, Examples include natural components derived from animals and plants, marine organisms, and the like.

EXAMPLES Hereinafter, an Example is shown and this invention is demonstrated more concretely.
Example 1 Cloning of human bitter taste receptor gene
TAS2R14 and TAS2R40 were cloned by PCR using human genomic DNA female (Cat. No. G152A; Promega) as a template based on the sequence information registered in GenBank. Integrate into the pENTR ^TM / D-TOPO vector (invitrogen) according to the manual, and use the Not I and Asc I sites existing on the pENTR vector to incorporate Not I and Asc downstream of the Flag-Rho tag sequence on the pME18S vector Recombined to I site.

Example 2 Production of G16gust36 Chimeric Gene Human Gα16 and human Gustducin were cloned by PCR using the cDNA of human tongue (derived from a 26-year-old healthy person; Bio Chain) as a template. In accordance with the previous report (Ueda T, et al., J. Neurosci, 23, 7376-7380 (2003)), 36 amino acids on the C-chain end side of the amino acid sequence of Gα16 (SEQ ID NO: 5) are converted into amino acids of Gustducin (SEQ ID NO: 6) In order to prepare a gene (SEQ ID NO: 7) encoding a chimeric G protein substituted with amino acids 318 to 354 of the sequence, a primer corresponding to the binding site was designed and the chimeric gene was amplified by PCR. Thereafter, the amplified gene was incorporated into the pENTR ^™ / D-TOPO vector (invitrogen) according to the manufacturer's instructions and further recombined into the pcDNA3.2 / V5-DEST vector (invitrogen).

Example 3 Evaluation of TAS2R14 and TAS2R40 response to EGCg 1) Preparation of bitter taste receptor-expressing cells
HEK293T cells expressing TAS2R14 and TAS2R40, respectively, were prepared as follows. A reaction solution having the composition shown in Table 1 was prepared and allowed to stand in a clean bench for 20 minutes, and then added to each well of a 96-well plate (Becton Dickinson). Next, 100 μl of HEK293T (1.5 × 10 ⁵ cells / cm ² ) was seeded in each well, and cultured in an incubator maintained at 37 ° C. and 5% CO ₂ for 24 to 26 hours.
HEK293T cells can be obtained from RIKEN BioResource Center.

2) Measurement of changes in intracellular calcium concentration
The bitter taste receptor expressed in HEK293T cells increases the amount of intracellular calcium by coupling to G16gust36 and activating PLCβ2. To measure the bitter taste response in this study, we used a calcium imaging method in which changes in the amount of intracellular calcium were monitored as fluorescence values with the intracellular calcium-sensitive fluorescent indicator Fluo-4.
The medium was removed from the culture prepared in 1) above, and a Quencher solution (Calcium Kit; Dojin Chemical) containing intracellular calcium-sensitive fluorescent indicator prepared as shown in Table 2 was added to each well.

The cells were cultured in a CO ₂ incubator at 37 ° C. for 1 hour to incorporate Fluo-4 into the cells. After 1 hour, the fluorescence intensity at a detection wavelength of 510 nm at an excitation wavelength of 480 nm was measured every 2 seconds for 4 minutes using a high-throughput fluorescence plate reader (FDSS3000; Hamamatsu Photonics). 60 seconds after the start of measurement, 50 μl of a catechin solution or a solution obtained by mixing a test substance with a catechin solution was added, and the fluorescence intensity was measured to detect changes in intracellular calcium concentration. The measurement was performed at 37 ° C. The analysis was performed by calculating the fluorescence intensity ratio when the fluorescence intensity at the start of measurement was 1, and further setting the fluorescence intensity ratio when catechin was added as 0.

  The response intensity to EGCg in each well was calculated by the following equation.

(Equation 1)
EGCg response = (maximum fluorescence intensity ratio in TAS2R-expressing cells after addition of EGCg)-(maximum fluorescence intensity ratio in mock-expressing cells)

  The maximum fluorescence intensity ratio was defined as the maximum value of the fluorescence intensity ratio for 1 minute (60 to 120 seconds from the start of measurement) after addition of EGCg, and the data was shown as the average value of 4 wells.

  The results are shown in FIG. TAS2R14 and TAS2R40 each showed a strong response to EGCg. From this, it was shown that the bitter taste inhibitor for catechins can be selected using the bitter taste receptor TAS2R14 or TAS2R40.

Example 4 Correlation between catechin responsiveness and sensory evaluation in TAS2R14 expressing cells
Cell responses of various catechins in TAS2R14-expressing cells were measured by the same method as in Example 3 above.
Human sensory evaluation was performed using eight catechins (catechin (C), epicatechin (EC), gallocatechin (GC), epigallocatechin (EGC), catechin gallate (Cg), epicatechin gallate (ECg), gallocatechin gallate ( GCg) and epigallocatechin gallate (EGCg)) were evaluated by 5 panelists. Quinine sulfate was prepared in 10 stages with different bitterness intensity as shown in Table 3, and the bitterness score of quinine sulfate showing bitterness intensity equivalent to the bitterness intensity of various catechins (evaluation concentration: 2 mM) Judgment was made and the average value of 5 persons was obtained. Evaluation was performed by a method including 5 ml of a test sample in the mouth.

  As shown in FIG. 2, there was a correlation between the response of TAS2R14-expressing cells to various catechins and the bitterness score, suggesting that the response of TAS2R14-expressing cells to catechin reflects human bitterness. .

Example 5 Examination of EGCg Response Inhibitory Activity of TAS2R14-expressing Cells Using Existing Bitterness Inhibiting Substance β-Cyclodextrin (β-CD) Measurement was performed in the same manner as in Example 3 above. Β-CD was mixed at a final concentration of 0.5% by mass.
<Calculation method of bitterness suppression rate against EGCg>
The response intensity to EGCg in each well was calculated as follows.

(Equation 2)
EGCg response = (maximum fluorescence intensity ratio in TAS2R-expressing cells after addition of EGCg)-(maximum fluorescence intensity ratio in mock-expressing cells)

The maximum fluorescence intensity ratio was defined as the maximum value of the fluorescence intensity ratio for 1 minute (60 to 120 seconds from the start of measurement) after adding EGCg.
The EGCg response suppression effect (bitterness suppression rate) by β-CD is

(Equation 3)
Bitter taste inhibition rate (%) = [1-- (EGCg response in the presence of β-CD)
/ (EGCg response in the absence of β-CD)]] × 100

The data are shown as average values of 4 wells.

  The results are shown in FIG. Addition of β-CD, which is known to suppress catechin bitterness, suppressed the EGCg response of TAS2R14-expressing cells, and the bitterness suppression rate was 87%. From this, it was shown that by using TAS2R14-expressing cells, a material having a bitter taste-inhibiting action of catechin can be searched.

  In the process of developing a bitterness inhibitor for conventional catechins, it is necessary to select a bitterness-inhibiting substance by checking the bitterness-inhibiting effect of a vast number of substances one by one through sensory tests, etc. It took a lot of time and cost. According to the method of the present invention, it is possible to select a bitterness suppressing substance based on the activity of a bitter taste receptor that responds to catechins, and the efficiency of development of a bitterness inhibitor is greatly improved.

Claims (2)

A method for evaluating or selecting a catechin bitterness inhibitor,
Adding a catechin and a test substance to any one or more bitter taste receptors selected from the group consisting of TAS2R14 and TAS2R40;
Measuring the response of the receptor to the test substance and catechins;
Adding only the catechins without adding a test substance;
Measuring the response of the receptor to the catechins only; and comparing the response of the receptor to the test substance and catechins with the response to the catechins only. A step of evaluating or selecting the test substance as a bitterness inhibitor by catechins, if the response is less than the response to
Including a method.   2. The method of claim 1, wherein the bitter taste receptor is a bitter taste receptor on a cell that naturally expresses a bitter taste receptor or on a recombinant cell that has been genetically engineered to express a bitter taste receptor.

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