JP5906013B2 - Evaluation and selection methods for bitterness inhibitors - Google Patents

Description

  The present invention relates to a method for evaluating and 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 (T2R), a bitter taste receptor expressed on the membrane surface of taste cells, mainly in the taste buds of the tongue. T2R is a kind of G protein-coupled receptor (GPCR), and 26 kinds of humans and 40 kinds of rats have been identified. T2R is known to couple with a gustducin classified as Gαi by binding a ligand, and to transmit a signal by causing an increase in intracellular calcium concentration (Non-patent Document 3). ). T2R is also expressed in AR42J (Non-patent document 4), STC-1 (Non-patent document 5) and HuTu-80 (Non-patent document 6), which are cells of the gastrointestinal tract other than the taste buds of the tongue. It has been reported that

  However, it has been reported that STC-1 cells do not respond to epicatechin (Patent Document 1), and no cell that has been confirmed to respond to catechin among cells expressing a bitter taste receptor has been found so far. Therefore, there has been no technique that can objectively evaluate substances that control catechin-derived bitterness.

JP-T-2005-522187

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) Wu SV et al., Physiol Genomics, 22, 139-149 (2005) Wu SV et al., Proc Natl Acad Sci USA, 99, 2392-2397 (2002) Rozengurt N, et al, Am J. Physiol Gastrointest Liver Physiol, 291, G792-802 (2006)

  The present invention relates to providing a method for simply evaluating and selecting a bitterness inhibitor that suppresses the bitter taste of catechins.

  As a result of searching for cells expressing a bitter taste receptor, the present inventor has responded to catechins in AR42J cells, which are rat pancreatic cancer cells, and the responsiveness well correlated with the bitter taste sensory evaluation in humans. It was found that by evaluating the inhibitory activity, a material that suppresses bitterness based on catechins can be easily screened.

That is, the present invention relates to the following 1) to 3).
1) A method for evaluating or selecting a bitterness-inhibiting substance for catechins, wherein the responsiveness of AR42J cells to catechins is evaluated.
2) A method for evaluating or selecting a bitterness-inhibiting substance for catechins, comprising the following steps (a), (b) and (c):
(A) contacting catechins and a test substance with AR42J cells;
(B) The activity of T2R when the catechins are contacted with cells in the presence of the test substance in step (a), and the T2R when the catechins are contacted with cells in the absence of the test substance. Comparing the activity,
(C) A step of selecting a substance that suppresses activation of T2R as a bitter taste inhibitor based on the comparison result of (b).
3) The method of 1) or 2) above, wherein the catechin to be contacted with a cell is epigallocatechin gallate (EGCg).

  According to the present invention, a bitterness-inhibiting substance for catechins can be evaluated or selected easily and objectively without depending on subjective sensory evaluation, and high-throughput screening can also be performed.

The graph which shows the response with respect to texans of AR42J cell. The graph which shows the correlation of the catechin response of AR42J cell and human sensory evaluation. The graph which shows the bitterness suppression effect (EGCg response suppression rate in AR42J cell) of (beta) -cyclodextrin ((beta) -CD).

Hereinafter, the method of the present invention will be described.
The method for evaluating or selecting a bitter taste inhibitor of the present invention uses AR42J cells, wherein a test substance that is a candidate substance for catechins and a bitter taste inhibitor is contacted to evaluate a substance that suppresses the cell response to catechins or To choose.
As shown in the examples described later, the rat pancreatic cancer AR42J cells were found to be responsive to catechins such as EGCg and found to correlate well with the bitterness score by human sensory evaluation. .
This result shows that a bitterness inhibitor can be evaluated or selected using the change (inhibition rate) of cell responsiveness to catechins of AR42J cells as an index.

Here, the “AR42J cell” is a cell derived from a rat pancreatic tumor, has a property of pancreatic progenitor cell, and is known as a model system for examining pancreatic β cell differentiation. In recent years, AR42J cells have expressed the bitter taste receptor T2R (Taste type 2 receptor) present in the taste buds of the tongue, and have been used as bitter substances such as cycloheximide (CYX), phenylthiocarbamide (PTC), and denatonium benzoate (DB). It has been reported that responsiveness is shown (Non-Patent Document 4).
It has been reported that T2R is expressed not only in AR42J cells but also in enteroendocrine cells STC-1 (Non-patent Document 5). However, it has been reported that STC-1 does not respond to epicatechin (Patent Document 1), and it is surprising that a response to catechin was observed in AR42J cells.

  In the present invention, as catechins, for example, catechin (C), gallocatechin (GC), catechin gallate (Cg), non-epimeric catechins such as gallocatechin gallate (GCg), epicatechin (EC), epigallocatechin ( EGC), epicatechin gallate (ECg), epigallocatechin gallate (EGCg) and the like epicatechins. Of these, epigallocatechin gallate (EGCg) is preferred.

  The test substance in the screening method of the present invention may be any known compound and novel compound, for example, nucleic acid, carbohydrate, lipid, protein, peptide, organic low molecular weight compound, compound prepared using combinatorial chemistry technology Examples include libraries, random peptide libraries prepared by solid phase synthesis and phage display methods, or natural components derived from microorganisms, animals and plants, marine organisms, and the like.

Specifically, the method of the present invention includes, for example, the following steps (a), (b) and (c).
(A) Contacting catechins and test substance with AR42J cells (b) T2R activity when test substance is contacted with cells in the presence of test substance in step (a), and absence of test substance The step of comparing the activity of T2R when the catechins are brought into contact with the cells below (c) Based on the comparison result of (b) above, a substance that suppresses the activation of T2R is selected as a bitter taste inhibitor Process

In step (a), AR42J cells are placed under contact with catechins and a test substance.
AR42J cells can be obtained from ATCC (American Type Culture Collection).
The contact of the catechins and the test substance with the cells is carried out in a buffer solution after removing the culture medium, but a known one may be used.
For example, AR42J cells are preferably cultured in advance at a cell number of 10 ⁴ to 10 ⁵ cells / cm ² for ² to 5 days prior to contact with a test substance or the like. As the culture medium, a known medium suitable for culturing AR42J cells may be employed. An example is DMEM / F12 (1: 1) (invitrogen).
The contact conditions with the catechins and the test substance are preferably 37 ° C. and 1 to 5 minutes.

  In step (b), T2R activity in AR42J cells is first evaluated in the presence of the catechins and test substance. At the same time, the activity is compared with the activity in the absence of the test substance. Here, as an index for measuring the activity of T2R, intracellular calcium concentration, intracellular cAMP amount and the like can be mentioned.

  In step (c), the activity is compared based on, for example, the presence or absence of a significant difference. As a result of the evaluation, if the suppression of activity can be confirmed in the presence of the test substance relative to the absence of the test substance, the test substance can be determined as a bitterness-inhibiting substance against catechins. Preferably, a test substance showing an EGCg cell response inhibition rate of 40% or more can be used as a bitter taste inhibitor.

Below, the specific method in the case of measuring intracellular calcium concentration is illustrated.
That is, the method of the present invention includes the following steps (a ′), (b ′) and (c ′).
(A ′) contacting a catechin and a test substance with AR42J cells into which a calcium-sensitive dye has been introduced for a certain period of time;
(B ′) Fluorescence intensity (intracellular calcium concentration) when the catechins are contacted with AR42J cells in the presence of the test substance, and the catechin is contacted with the cells in the absence of the test substance A step of comparing the fluorescence intensity with
(C ′) A step of selecting a bitterness-inhibiting substance based on the comparison result of (b ′) above.

This method searches for a target substance based on a change in fluorescence intensity (intracellular calcium concentration) when a test substance is brought into contact with AR42J cells into which a calcium-sensitive dye has been introduced for a certain period of time.
Examples of calcium sensitive pigments include Fura-2, Fluo-3, and Fluo-4.

In the step (b ′), it is evaluated whether or not the fluorescence intensity (intracellular calcium concentration) in the AR42J cells varies in the presence of the test substance. This can be evaluated by comparing the measured fluorescence intensity (intracellular calcium concentration) with the fluorescence intensity (intracellular calcium concentration) in the absence of the test substance (only catechins are present).
Such fluorescence intensity can be measured using a method known per se. For example, it can be performed using a fluorescence intensity measurement plate reader or the like.
The cell response activity can be calculated as, for example, 340/380 nm (Ratio) by measuring fluorescence intensity at excitation wavelengths of 340 nm and 380 nm and a detection wavelength of 510 nm.

  In the step (c ′), the comparison of the fluorescence intensity is performed based on, for example, the presence or absence of a significant difference. As a result of the evaluation of the fluorescence intensity, a substance that suppresses the fluctuation range of the fluorescence intensity can be selected as a bitterness suppressing substance.

Example 1 Examination of AR42J cell catechin responsiveness Cells were seeded in 96-well plates (BD) at 6 × 10 ⁴ cells / cm ² and cultured for 3 days. After removing the culture solution, quencher solution containing intracellular calcium-sensitive fluorescent indicator (1 mM Fura 2-AM 50 μl, Quenching Buffer 5 ml, Hank's HEPES Buffer (10 ×) 0.5 ml (Calcium Kit II-Fura 2, triral; Dojin) Chemical) and 10 ml of Ringer solution were added and mixed with distilled water so that the total volume was 20 ml.) 200 μl was added to each well.
* Ringer liquid composition is as follows.
5 mM HEPES, 140 mM NaCl, 5.6 mM KCl, 2 mM Na pyruvate, 2 mM MgCl ₂ ,
1.5 mM EGTA, 9.4 mM Glucose, 1.25 mM KH ₂ PO ₄ , pH 7.4
After adding the quencher solution and incubating at 37 ° C for 1 hour, using a high-throughput fluorescent plate reader (FDSS3000; Functional Drug Screening System, Hamamatsu Photonics), the fluorescence intensity at an excitation wavelength of 340 nm and a detection wavelength of 510 nm at 380 nm is 4 minutes ( The change in intracellular calcium concentration was measured by measuring (recording every 2 seconds). 75 seconds after the start of measurement, 10 mM catechins (catechin (C), epicatechin (EC), gallocatechin (GC), epigallocatechin (EGC), catechin gallate (Cg), epicatechin gallate (ECg), gallocatechin gallate (GCg), epigallocatechin gallate (EGCg)) 50 μl (final concentration: 2 mM) was added to each cell. The measurement was performed at 37 ° C. The results are shown in FIG.
FIG. 1 shows that AR42J cells respond to various catechins.

Example 2 Correlation between catechin responsiveness and sensory evaluation in AR42J cells
The bitterness score of eight catechins (catechin, epicatechin, gallocatechin, epigallocatechin, catechin gallate, epicatechin gallate, gallocatechin gallate, epigallocatechin gallate) was evaluated by five panelists. Quinine sulfate was prepared in 10 stages with different bitterness intensity as shown in Table 1, and the bitterness score of quinine sulfate showing bitterness intensity equivalent to the bitterness intensity of various catechins (evaluation concentration: 2 mM) was determined as the bitterness score of the catechin. 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, since a correlation was observed between the response of AR42J cells to various catechins and the bitterness score, it was suggested that the response of AR42J cells to catechin reflects human bitterness.

Example 3 Examination of EGCg Response Inhibitory Activity of AR42J Cells Using Existing Bitter Taste Inhibiting Substance β-CD Cells were seeded in 96-well plates (BD) at 6 × 10 ⁴ cells / cm ² for 3 days. After removing the culture solution, quencher solution containing intracellular calcium-sensitive fluorescent indicator (1 mM Fura 2-AM 50 μl, Quenching Buffer 5 ml, Hank's HEPES Buffer (10 ×) 0.5 ml (Calcium Kit II-Fura 2, triral; Dojin) Chemical) and 10 ml of Ringer solution were added and mixed with distilled water so that the total volume was 20 ml.) 200 μl was added to each well.
* Ringer liquid composition is as follows.
5 mM HEPES, 140 mM NaCl, 5.6 mM KCl, 2 mM Na pyruvate, 2 mM MgCl ₂ ,
1.5 mM EGTA, 9.4 mM Glucose, 1.25 mM KH ₂ PO ₄ , pH 7.4

  After adding the quencher solution and incubating at 37 ° C for 1 hour, using a high-throughput fluorescent plate reader (FDSS3000; Functional Drug Screening System, Hamamatsu Photonics), the fluorescence intensity at an excitation wavelength of 340 nm and a detection wavelength of 510 nm at 380 nm is 4 minutes ( The change in intracellular calcium concentration was measured by measuring (recording every 2 seconds). 75 seconds after the start of measurement, 10 mM EGCg (final concentration: 2 mM) or 10 mM EGCg mixed with β-CD 0.05% (final concentration: 0.01 mass%) was added to the cells. The measurement was performed at 37 ° C.

<Calculation method of bitterness suppression rate against EGCg>
The change in intracellular calcium concentration was indicated by the ratio of the fluorescence intensity at 510 nm to the excitation wavelengths of 340 nm and 380 nm (Ratio _340/380 ).
The response intensity to EGCg in each well was calculated as follows.
(Equation 1)
EGCg cell response intensity = (Maximum ratio _340/380 after addition of EGCg)-(Minimum ratio _340/380 after addition of EGCg)
The EGCg response suppression effect (bitterness suppression rate) by each test substance is
(Equation 2)
Bitterness inhibition rate (%) = [1- (EGCg response intensity in the presence of test substance) / (EGCg response intensity in the absence of test substance)] × 100
The data are shown as the mean value of 3 wells ± standard deviation.

  The results are shown in FIG. The addition of β-CD, which is known to suppress catechin bitterness, suppressed the EGCg response of AR42J cells by about 90%. From this, it was shown that the material which has the bitter taste inhibitory effect of catechin can be searched by using AR42J cells.

Claims (2)

A method for evaluating or selecting a bitterness-inhibiting substance for catechins, comprising the following steps (a ′), (b ′) and (c ′):
  (A ′) contacting catechins and a test substance with AR42J cells into which a calcium-sensitive dye has been introduced,
  (B ′) Fluorescence intensity (intracellular calcium concentration) when the catechins are contacted with AR42J cells in the presence of the test substance, and the catechin is contacted with the cells in the absence of the test substance A step of comparing the fluorescence intensity with
  (C ′) A step of selecting a bitterness-inhibiting substance based on the comparison result of (b ′).   The method according to claim 1, wherein the catechin to be contacted with a cell is epigallocatechin gallate.

Images